IJS Perception of vertebrate volatiles in the tropical bont tick, Amblyomma variegatum Fabricius CHO ffiyr^j par Pascal Steullet V e Thèse présentée à la Faculté des Sciences de l'Université de Neuchâtel pour obtenir le grade de docteur es sciences 1993 Université de Neuchâtel Faculté des Sciences Perception of vertebrate volatiles in the tropical bont tick, Amblyomma variegatum Fabricius par Pascal Steullet Thèse présentée à la Faculté des Sciences de l'Université de Neuchâtel pour obtenir le grade de docteur es sciences 1993 IMPRIMATUR POUR LA THESE Perception of vertebrate volatiles in the .. .tropical, ..bant... txc^....Amblyomma-. .varxegatum... •Fabricius.................................................................................................. de M>nsieur...p.as.cal. .Steullet. UNIVERSITE DE NEUCHATEL FACULTÉ DES SCIENCES La Faculté des sciences de l'Université de Neuchâtel sur le rapport des membres du jury, MM. P. Guerin, P.A. Diehl, E. Stadler (Wädenswil) et R.A. Steinbrecht ..^^^^!^^-!!(^^^^!!^,....seeWiesen^.................................... autorise l'impression de la présente thèse. Neuchâtel, le .....2?._.noye^re__ 1993_ H.-h. Näofeli 1 ...La tique solitaire, concentrée et cachée dans son arbre, aveugle sourde et muette, tout occupée pendant des années, à flairer sur des lieues à la ronde le sang des animaux qui passent et qu'elle n'atteindra jamais par ses propres moyens........Mais la tique, butée, bornée et répugnante, reste embusquée, et vit, et attend. Attend jusqu'à ce qu'un hasard extrêmement improbable lui amène le sang juste sous son arbre, sous la forme d'un animal. Et c'est alors seulement qu'elle sort de sa réserve, se laisse tomber, se cramponne, mord et s'enfonce dans cette chair inconnue... ...Jl (Jean-Baptiste Grenouille) se mit à s'introduire nuitamment dans les étables, pour y envelopper pendant quelques heures des vaches, des chèvres ou des cochons avec des linges enduits de graisse, ou pour les emmailloter dans des bandages huileux. Ou bien il se glissait furtivement dans un enclos à brebis pour y tondre clandestinement un agneau, dont ensuite il lavait à l'esprit-de-vin la laine odorante....Les résultats ne furent d'abord guère satisfaisants. Car, à la différence d'objets dociles comme un bouton de porte ou une pierre, les animaux se montraient récalcitrants au prélèvement de leur odeur.... extrait de "Le Parfum" de Patrick Siiskind (traduit de l'allemand par Bernard Lortholary) édition Fayard (Le livre de poche) CONTENTS 2 CONTENTS I. INTRODUCTION 4 1.1. Systematic position of ticks and Amblyomma variegatum 4 1.2. Biology of Amblyomma variegatum 4 1.3. Host-seeking in Amblyomma variegatum 6 1.4. Olfactory sense organs in ticks and Amblyomma variegatum 8 1.5. Ultrastructure of olfactory sensilla in Amblyomma variegatum 10 1.5.1. Wall-pore single-walled A sensilla 10 1.5.2. Wall-pore single-walled B sensilla 11 1.5.3. Wall-pore double-walled A sensilla 11 1.5.4. Wall-pore double-walled B sensilla 11 1.5.5. Wall-pore double-walled C sensilla 11 1.6. Specificity of tick olfactory receptors 11 1.7. Outline of the present study 12 II. MATERIALS AND METHODS 13 2.1. Animals- 13 2.2. Electrophysiology 13 2.3. Stimulation 14 2.4. Gas chromatography-coupled electrophysiology 14 2.5. Gas chromatography-coupled mass spectrometry 17 2.6. Behavioural bioassays 17 2.6.1. Wind tunnel 17 2.6.2. Activation bioassay 17 2.6.3. Locomotion compensator (Kramer's sphere) 19 CONTENTS 3 RESULTS 21 3.1. Perception of breath components by the tropical bont tick, Amblyomma variegatum Fabricius (Ixodidae). I CO^receptors. J. Comp. Physiol. A (1992) 170:665-676 22 3.2. Perception of breath components by the tropical bont tick, Amblyomma variegatum Fabricius (Ixodidae). Il Sulfide receptors. J. Comp. Physiol. A (1992) 170.-677-685 34 3.3. Identification of vertebrate volatiles stimulating olfactory receptors on tarsus of the tick Amblyomma variegatum Fabricius (Ixodidae). I Receptors within the Halter's organ capsule. J.Comp. Physiol. A (in press) 43 3.4. Identification of vertebrate volatiles stimulating olfactory receptors on tarsus of the tick Amblyomma variegatum Fabricius (Ixodidae). II Receptors outside the Halter's organ capsule. J.Comp. Physiol. A (in press) 55 IV. GENERAL DISCUSSION 64 4.1. Location and number of tick olfactory sense organs 64 4.2. The capsule of Halter's organ, a complex olfactory sense organ 65 4.3. Specificity and sensitivity of the olfactory receptors 66 4.4. Correlation between receptor specificity and sensillum ultrastructure 69 4.5. Conservatism in the evolution of the tick olfactory system 71 4.6. Perception of vertebrate volatiles and host-finding 73 V. SUMMARY-RESUME 82 VI. REFERENCES 88 VIL REMERCIEMENTS 104 Vili. APPENDIX 106 INTRODUCTION 4 I. INTRODUCTION Whenever the word tick is pronounced, a host of fears, legends and questions about these blood-sucking animals invariably arise. " A dangerous insect that falls from trees onto humans and mammals to feed on blood " would likely be the erroneous definition of a tick given by many people. The usual ignorance about how ticks recognize and find a host contributes to the legends on these small ectoparasites. The present work on the chemical ecology of ticks, more precisely on host-odour detection in the tropical bont tick Amblyomma variegatum, consequently needs a short introduction on tick systematic, the biology of A variegatum, and different aspects of olfaction and the host-finding strategy in this tick species. 1.1. Systematic position of ticks and Amblyomma variegatum As members of the Order Acari ticks (900 sp.) are distinguishable from mites by stigmata located behind the fourth leg pair, but also have a highly expandible fused body (the idiosoma), and possess mouth parts adapted to haematophagy (the gnathosoma) equipped with 2 sharp chelicerae, 1 hypostome covered with recurved teeth, and 2 palps. These blood-sucking arthropods comprise 3 families: Nuttalliellidae, Argasidae (soft ticks), and Ixodidae (hard ticks) to which the genus Amblyomma belongs (Rg. 1.1.). The Ixodidae, subdivided into 5 subfamilies (Rg. 1.1.), are characterized by a sclerotized plate, which in the female and immature stages is limited to the anterior dorsal region, while in males it covers the entire back thus restricting male body extension during a blood meal. The subfamily Amblyomminae includes the genus Amblyomma (102 sp) which are considered primitive ticks because of their large size and their three-host life-cycle pattern (Sonenshine 1991). 1.2. Biology of Amblyomma variegatum A. variegatum is a large tick widely distributed in most tropical and subtropical regions of continental Africa, Madagascar, the South-West Arabic Peninsula, and the Cape Verde Islands (Hoogstraal 1956; Morel 1969). This species, also called the tropical bont tick, has been noted in the Caribbean Islands since the end of last century (Morel 1969) and threatens to colonize Central and South America. A. variegatum is a typical three-host tick with each stage (larva, nymph, adult) parasitizing mammals for a blood-meal of variable duration (larvae 5-8 days; nymphs 6-13 days; females 14-22 days) (Morel INTRODUCTION 5 1969). A blood meal is indeed a requirement for successful moulting of immatures, spermiogenesis and ovogenesis in adults, and for oviposition in females. Phylum: Arthropods Class: Chelicerata Sub-class: Arachnida Order: Acari Sub-order: Metastigmata = ticks Family: Ixodidae (hard ticks), NuttaEliellidae, Argasidae (soft ticks) Amblyomminae Haemaphysalinae Hyatomminae Aponomma Amblyomma Haemaphysalis Hyabmma Rhiptcephalinae Ixodinae Dermacentor Cosmiomma Nosomma Rhipicephalus Anomabhytnalaya Rhipicentor Boophilus Margaropus Ixodes Fig. 1.1. Systematic position of ticks (ca. 900 sp) and the genus Amblyomma (102 sp). Immature stages of the tropical bont tick parasitize a wide range of reptiles, birds, and mammals (Matthysse and Colbo 1987), but Barré (1989) noticed a preference for ungulates. Adults for their part feed principally on large ruminants (Morel 1969; Matthysse and Colbo 1987; Barré 1989). Although large wild mammals such as buffaloes and antelopes are thought to be the natural hosts, domestic animals (cattle, sheep, goats) are also strongly infested in many areas (Hoogstraal 1956; Matthysse and Colbo 1987). Aeschlimann (1967) suggested that the distribution of this tick species has been expanded by the development of cattle ranching. A varìegatum, a vector of various pathogens such as Cowdria ruminantium (heart-water), Coxiella burnetii (Q- fever) and Dermatophilus congolensis (dermatosis), also causes anaemia and skin damage (Aeschlimann 1976, Barré 1989). It has thus become a serious life-stock pest responsible for much animal weakness and illness. INTRODUCTION 6 1.3. Host-seeking In Amblyomma variegatum Parasitizing vertebrates is a very tough task for all tick life-stages, since the potential host is much bigger and more mobile. Thus, specific physiological capabilities and adequate host-seeking strategies are the keys to successful completion of the life-cycle. Tremendous resistance to desiccation and starvation allow ticks to survive for months during non-parasitic phases. While some tick species live within nests or burrows and thus maintain a close relation with their host, others, such as A variegatum, quest along pathways or areas frequented by their hosts. Consequently, the latter need well-adapted host-seeking strategies and efficient sense organs to optimize chances of meeting a host. Waladde and Rice (1982) reviewed the different senses involved during the successive behavioural steps of host-seeking, from tick arousal to its attachment on a host. In this context, olfaction is considered a major sense. Transported by wind and air turbulence, odour molecules can be detected over great distances. Odour quality as well as concentration constitute potential sources of information, i.e. estimation of the distance to a host, host recognition, host location. Immature A. variegatum quest on vegetation and grasp any mammal which passes nearby (Barré 1989). Mechanical disturbance causes significant activation of immature stages (Barré 1989). By contrast, adult A. variegatum, which are hunter ticks (Morel 1969; Barré 1989), rest in the litter zone until a host in the close vicinity activates them (Rg. 1.2.). Excited ticks then start to walk about on the ground in order to locate the vertebrate, orient to it and grasp it, and then find a suitable feeding-site (Rg. 1.2.). Activation of resting adults is mainly mediated by host odour such as CO2, while vibrations, temperature, infrared radiation, and visual cues do not play a primordial role during the arousal steps of host-finding (Stämpfii 1987; Steullet unpublished; Poffet unpublished; Kaltenrieder et al. 1989; Yunker and Norval 1991). On the other hand, odours along with vertebrate-emitted infrared radiation (Poffet, unpublished), and visual cues (Kaltenrieder et al. 1989; Kaltenrieder 1990) intervene in host location. Males are first to infest mammals at the end of the dry season (Morel 1969), and females appear a few days later on the infested hosts (Hoogstraal 1956; Morel 1969; see Rg. 1.2.). Others have observed that attached males (the primary colonizers) of different species of Amblyomma attract conspecific females and other males (the secondary colonizers) to the same feeding-site (Gladney et al. 1974; Rechav et al. 1977; Norval and Rechav 1979). Schoeni et al. (1984) and Apps et al. (1988) identified the components of INTRODUCTION 7 A uninfested host Host location / AV / Contact with the host PIONEERCOLONIZERS ïï Arousal "resting V male AAP Attachment Sexual maturation Emission of pheromone and feeding Feeding-site selection B infested host AAP Host location onj SECONDARY COLONIZERS Contact with the host Attraction to mature male resting female Attachment and feeding AAP: aggregation-attachment pheromone Fig. 1,2. Diagrammatic illustration of host-finding behaviour in adult A. variegatum. INTRODUCTION 8 the aggregation-attachment pheromone released by fed male A variegatum and A. hebraeum. Diehl et al. (1991) found that the amount of pheromone emitted by a male of both species is considerable. Thus, a male A. variegatum can secrete in one hour more than 1 ug of 2-nitrophenol, one of the major component of the aggregation-attachment pheromone. The combination of host odours and the aggregation-attachment pheromone leads other members of at least some species of Amblyomma to a suitable host on which blood-feeding and mating can be accomplished (Rg. 1.2.). Nevertheless, for pioneer Amblyomma males, host odours are the only olfactory cues available during host-seeking. Studies on the sensitivity and the specificity of olfactory sense organs in A. variegatum should thus provide information on host-odour cues employed by this parasite. 1.4. Olfactory sense organs in ticks and Amblyomma variegatum With the exception of special olfactory organs such as the sensilla placodea in honeybees (Boeckh et al. 1965), olfactory sense organs in arthropods are normally innervated setae with pore-walls. The latter favour diffusion of molecules from the air to the dendritic membrane of the olfactory receptor cell(s) within the sensillum via the receptor lymph. Sensillum ultrastructure and morphology reviewed by Altner and Prillinger (1980), and Zacharuk (1985), and olfactory transduction processes such as odour molecule diffusion, odour molecule-receptor affinity, enzymatic cascade, electrical events, adaptation and specificity of olfactory receptors have been most extensively investigated in insects (reviews by Kaissling 1986; Kaissling et al. 1987; de Kramer and Hemberger 1987; Vogt et al. 1987a and b; Lerner et al. 1990; Stengl et al. 1992). However, only some works on the ultrastructure of olfactory sensilla and only a few studies on receptor specificity have been published in ticks. Odour-stimulated ticks often raise the first pair of legs in the air as insects do with their antennae. This behaviour soon suggested to acarologists that the first leg pair bears crucial sense organs. Haller (1881) first described a capsule equipped with numerous setae which he suspected to be a hearing organ on the tarsus of the first leg pair. However, further behavioural experiments in which different parts of tick appendages were amputated or masked revealed that the tarsus of the first leg pair was in fact the main site of odour perception (Hindle and Merriman 1912, Lees 1948). The morphology and ultrastructure of tarsal sense organs, and principally the Haller's organ in ticks have been intensively studied (i.e. Schulze 1941; Leonovitch 1979, 1980; INTRODUCTION 9 Fig. 1.3. Right tarsus of the first leg pair of an adult A. uariegatum and location of the wall-pore sensilla with their respective names (according to the nomenclature proposed by Hess and Vlimant, 1982). A brief description of the morphological properties of each olfactory sensillum is given in the text.  detailed diagram of the anterior pit (lateral view) and the capsule (dorsal view) of Haller's organ is also provided. The arrow indicates the slit-like opening of the capsule. The seven wall-pore single walled sensilla (s) and cuticular pleomorphs (p) enclosed in the capsule are represented under the capsule opening. Roshdy and Axtell 1972; Roshdy et a!. 1972; Foeiix and Axtell 1972; Waiadde 1976,1977). Haller's organ which contains almost half of all olfactory receptors of the tarsus (Hess and Vlimant 1986) has two dense groups of olfactory sensilla, the anterior pit and the capsule (Fig. 1.3.). Hess and Vlimant (1982, 1983, and 1986) described 5 different types of olfactory sensilla on the tarsus of A. variegatura, but none elsewhere (either on the other legs, mouthparts, or on INTRODUCTION 10 the idiosoma, Vlimant, personal communication). Three types of olfactory sensilla (wall-pore single-walled A and B; wall-pore double-walled C) are restricted to Haller's organ, whereas two other types (wall-pore double-walled A and B) are located on other parts of the tarsus. Adults and nymphs have 19 olfactory sensilla sharing a total of 68-94 suspected olfactory receptors per tarsus, while larvae possess only 14 olfactory sensilla with 57-77 olfactory receptors (Hess and Vlimant 1986). Furthermore, the number and location of tarsal sensilla is highly conserved between Ixodidae. However, primitive ticks possess slightly more olfactory sensilla, i.e. 20 in Ixodes ricinus, 19 in A. variegatum, and 17 in Dermacentor marginatus compared to 14 in Boophilus microplus (Hess and Vlimant 1986). Nevertheless, the total number of olfactory receptors is remarkably similar for the same life-stages of different tick species. Reduction of sensillum number results from the fusion of sensilla (Hess and Vlimant 1982). Figure 1.3. shows the position of each olfactory sensillum on the tarsus of an adult A. variegatum and a brief description of these sensilla is provided in the following section (for further details, see Hess and Vlimant 1982, and 1986). 1.5. Ultrastructure of olfactory sensilla in Amblyomma variegatum 1.5.1. Wall-pore single-walled A sensilla (L. N. A: 2V These sensilla basiconica (50-60 u,m long) have relatively thick walls (0.6-1 u,m thick) with a large number of plugged pores (0.1 urn diameter). One of the two present, the Dl. 1 sensillum, is located on the dorsal side distalIy from the anterior pit of Haller's organ (Fig. 1.3.) and includes 5 neurones with branched dendrites. The other, the Dl 1.1 sensillum, belongs to the anterior pit (Fig. 1.3.) and is innervated by 3 sets of neurones (2x5 + 1 x 4) with branched dendrites. The latter is thought to result from the fusion of 3 separate sensilla. Indeed, each set of neurones possesses a thecogen cell and a trichogen cell, while the tormogen cells form a common outer receptor lymph cavity (Hess and Vlimant 1982). Interestingly, this fusion is not complete in the related species A americanum where two wall-pore single-walled A sensilla are found in the anterior pit, one with two sets of neurones and the other with only one set of neurones (Foelix and Axtell 1971). 1 number of sensilla per tarsus in larvae (L), nymphs (N). and adults (A) INTRODUCTION 11 1.5.2. Wall-pore single-walled B sensilia (L: 4/ N. A: 7) Wall-pore single-walled B sensilia, occasionally bifid, are deeply enclosed in a cavity, the capsule of Haller's organ (Rg. 1.2). Projecting from the floor or from the proximal or lateral cuticular walls of the capsule towards the slit opening, these sensilia have thin walls (0.08-0.14 jam tick) with wide plugged pores (0.1-0.16 urn) and are innervated by 3 to 5 neurones with dendrites showing various degrees of branching. 1.5.3. Wall-pore double-walled A sensilia (L: 2/ N. A: 4) Called DIV, 1, 2, 3, and 4 respectively, these sensilia trichodea (35-40 \i m long) are located very close together on the dorsal side of the tarsus, praxi mally from the capsule (Fig. 1.3.). They are innervated by 1 or 2 neurones with unbranched dendrites and have grooved walls. Radial canals lead from the central cavity of the sensillum to narrow pores, situated in the longitudinal grooves. 1.5.4. Wall-pore double-walled B sensilia (L. N. A: A) These sensilia chaetica resemble the wall-pore double-walled A type but have two neurones forming a tubular body (mechanosensory units) and 4 to 7 neurones with unbranched dendrites. One of these sensilia (Dlll.2) is located proximally from the capsule, another (LAI 1.1) is inserted on the lateral side, and the other two (Vl 1.1 and 4) are found on the ventral side of the tarsus (Fig. 1.3.). 1.5.5. Wall-pore double-walled C sensilia (L. N. A: 21 Both wall-pore double-walled C sensilia basiconica are confined to the anterior pit (Fig. 1.3.) and have walls with longitudinal but interrupted grooves. Three neurones with unbranched dendrites innervate these sensilia. 1.6. Specificity of tick olfactory receptors Despite several studies, our current knowledge regarding specificity of tick olfactory receptors is rather limited and fragmented. Only a few receptors have been properly characterized. Receptors sensitive to 2,6-dichlorophenol, a tick pheromone component, have been identified in wall-pore single-walled A sensilia of A americanum (Haggart and Davis 1981), and A variegatum (Waladde 1982; Schoeni 1987), and in the wall-pore single-walled C sensillum of I. ricinus (Thonney 1987). Wall-pore single-walled A sensilia of A variegatum also contain receptors which respond to 2-nitrophenol (Schoeni 1987) or to short-chain fatty acids (Hess and Vlimant 1980). Other receptors confined to the anterior pit are excited by NH3 in Rhipicephalus sanguineus (Haggart and INTRODUCTION 12 Davis 1980), by breath and cattle wash in B. microplus (Waladde and Rice 1982). Furthermore, methylsalicylate, an aggregation-attachment pheromone component in A variegatum (Schoeni et al. 1984), stimulates a receptor in the capsule of Haller's organ of A variegatum (Hess and Vlimant 1986), but also in I. ricinus (Guerin, unpublished) and B. microplus (de Bruyne, unpublished). Other capsule receptors were found to respond to breath and CO2 in B, microplus (Waladde and Rice 1982), while Sinitsina (1974) obtained responses to mouse odour, pentanoic acid, breath, but not to CO2 in capsular receptors of Hyalomma asiaticum. Holsher et al. (1980) also reported presence of CO2- excited receptor(s) with a tungsten electrode inserted proximalIy in the capsule of three tick species. Finally Haggart and Davis (1980) found a receptor sensitive to high concentrations of NH3 in a wall-pore double-walled A sensillum of R. sanguineus. The latter is the only report of a response to an olfactory stimulant by receptors in wall-pore double-walled sensilla of ticks. 1.7. Outline of the present study Although breath, mouse odour, and the pelage wash of cattle can stimulate receptors of some tick species, CO2, NH3, and short-chain fatty acids are currently the only vertebrate-associated volatiles known to stimulate tick olfactory receptors. Considering the total number of olfactory receptors present on the tarsus (68-94 in A. variegatum), it would be very surprising if no other host-odour compounds could be detected. Although Haller's organ is generally considered as the main site of host-odour detection, the exact location and number of host-odour receptors are not known. The present work aims to systematically investigate the specificity of olfactory receptors of A. variegatum, an ideal tick model for such a study. The morphology and ultrastructure of olfactory receptors in this tick species are well known (Hess and Vlimant 1982, 1983, and 1986), the size of A variegatum (up to 1 cm) renders electrophysiological preparation relatively easy, and olfaction plays a primary role in host-finding for this hunter tick. This study should permit identification of different classes of vertebrate-associated volatiles perceived by A. variegatum and allow us to understand how finely tuned the olfactory system of A. variegatum is to the odour bouquet of various vertebrates. Finally, the host- odour stimulants identified will be considered as behavioural cues in host- finding. METHODS 13 II. METHODS In this chapter, only the main principles of the Methods used are described. Further details are provided in the corresponding publications presented in chapter 3. 2.1. Animals Male of A. variegatura were principally used because of their pioneer role in parasiting hosts. Rearing conditions are described in chapter 3.1.. 2.2. Electrophysiology (chapters 3.1., 3.2., 3.3., 3.4.) Since Monta and Yamashita's pioneering work on Bombyx (1961), classic electrophysiology methods have been used in arthropods to pick up such electrical events as receptor potentials and action potentials generated at the level of olfactory receptors for the study of their specificity and sensitivity. In the present work, electrical activity of tick olfactory receptors has mostly been captured by contacting the cut tip or the pored wall of a sensillum with a glass electrode. A tungsten electrode inserted into the receptor lymph at the base of a sensillum was also employed for specific purposes in this study (see chapter 3.1.). Glass electrodes show several advantages over tungsten electrodes. A recording glass electrode permits recording of shifts in potential and action potentials associated with receptor cells, whereas a tungsten electrode will cut off lower frequencies (slow potential shifts). Furthermore, receptor cells can be more easily destroyed by tungsten than by glass electrodes Typically, glass electrodes are glass capillaries filled with an electrolyte solution which is electrically connected via Ag/AgCI wires to the amplification circuit. Small voltage changes between the reference glass electrode (ground) inserted in the tick haemolymph and the recording electrode in contact with the sensillum were thus amplified (10 to 100Ox) via a 1012 Ohm high input impedance preamplifier and an AC/DC amplifier. Signals were then displayed on an oscilloscope, printed on paper, stored on video tapes, or fed to a computer for further analysis (Fig. 2.1.). In many cases, analysis of spike shapes and amplitudes (extracellular recordings of action potentials generated by each receptor cell in response to stimulation with volatiles) permits characterization of the sensitivity and specificity of each responding receptor cell of a sensillum. Indeed, spike shapes and amplitudes generated by the separate receptor cells in the same sensillum often differ. Specific passive properties (impedance and capacitance) of the electrical circuitry within the METHODS 14 sensillum (through receptor cells and accessory cells) may indeed contribute to the shape and amplitude of the extracellularly recorded spikes (de Kramer 1985; Rumbo 1989). 2.3. Stimulation (chapters 3.1., 3.2., 3.3., 3.4.) Air from a cartridge containing volatiles was briefly swept via electrically controlled valves into a charcoal-filtered air stream leading to the tick preparation. Shifts in potential and spike frequency changes of the olfactory receptors resulting from stimulation were analysed. The specificity of the receptors was tested with a series of well-known vertebrate-associated volatiles representing different- chemical classes (0(¾, CH4, NH3, acetone, 4- heptanone, y-butyrolactone, y-valerolactone, pentanol, 1-octen-3-olf 4- methylphenol, hexanal, propanoic acid, butanoic acid, 2-methylpropanoic acid, 3-methylbutanoic acid, pentanoic acid, heptanoic acid, L-lactic acid, ), with some tick pheromone components (nonanoic acid, 2,6-dichlorophenol, 2- nitrophenol, methylsalicylate), some other volatiles (3-pentanone, 6- caprolactone, 1-octene, octylamine, hexyl acetate) and with vertebrate odours (human breath, human axillary secretions, bovine breath, bovine skin wash, odours collected around steers and rabbits). Vertebrate-associated volatiles were collected on a charcoal trap (for breath), on the porous polymer adsorbent Porapak Q (for breath and odours of steers and rabbits, see Rg. 2.2.), or in a cold trap (for headspace over cotton pads used to wash bovine skin, see Fig. 2.3.) and extracted with solvent for use as host-odour stimuli. Breath as well as cotton pads soaked with human axillary secretions were also introduced into the stimulus cartridge and used directly as stimuli. 2.4. Gas chromatography-coupled electrophysiology (chapters 3.2., 3.3., 3.4.) Whenever a host-odour extract stimulated receptor(s), the next step consisted of identifying the stimulant(s) among the numerous constituents of the extract. These volatiles were separated by gas chromatography on a high- resolution capillary column coupled to an electrophysiological preparation of a tick olfactory sensillum, thus permitting observation of the effect of each component of the extract on the activity of the receptors (Wadhams 1982). Figure 2.4. provides a diagram of the set-up. METHODS 15 Fig. 2.1. Diagram of the electrophysiology set-up used to study olfactory receptors of A. variegatum. (0): reference glass electrode impaled in the coxa of one of the first legs. (1): Recording glass electrode in contact with the cut tip of the sensillum. The electrode is electrically connected via an Ag/AgCl wire to a high input impedance preamplifier (symbolized by a triangle). (2): Universal AC/DC amplifier with separate outputs for the AC and DC parts of the recorded signal. (3): Oscilloscope to visualize the AC and DC signals. (4): Loudspeaker to render spikes audible in the AC signal. (5): PCM-I Digital- VCR instrumentation recorder adaptator for analogue/digital or digital/analogue conversion of the AC and DC signals. (6): Video tape recorder used to store digitized signals and to play them back for further analysis via digital/analogue conversion in (5). (7): "Go-Box" which contains the circuitry necessary to provide the trigger to start digitization on the computer of the analogue AC signal played back from the video tape recorder via (5). (8): IBM-compatible computer equipped with an analogue/digital board to digitize the analogue AC signal and with software (SAPID tools) to visualize and analyse the digitized signal, (9): Laser printer to print signals and analyses. (10): Recorder to print signal. (11): Stimulus delivery controlled by solenoid valves. The stimulation period is indicated by a pulse signal which can be stored on an audio channel of (6) and used subsequently as a trigger signal, i.e. in (7) or in (10). (12): Microphone used to add comments stored on the second audio channel of (6). (13): Loudspeaker to listen to comments during play-back. Bold circles: inputs to each instrument; open circles: outputs of each instrument; bold triangles: trigger inputs. METHODS 16 1 odour collection 2 extraction 3 extract concentration CH2Cl2 air from the room wHh cattle V N2 h v concentrated extract extract Fig. 2.2. Odour collection using adsorbent material (charcoal or Porapak Q). This method was used to collect volatiles in the air of rooms with either cattle or rabbits. 1 odour collection N2 70 'CJ cotton pad rubbed on bovine skin L flowmeter -7O0C 2 solvent extraction CH2Ci2V cold trap W acetone + dry-ice \ extract extract V liquid (organic phase) -1O0C O 1S N2 Ny^ (water phasePy/ TJ phase separation extract concentration Fig. 2.3. Odour collection using a cold trap. This method was used to collect volatiles from cotton pads rubbed on bovine skin (skin wash). METHODS 17 2.5. Gas chromatography-coupled mass spectrometry (chapters 3.3., 3.4.) Chemical analysis of the active component(s) of an extract, located by gas chromatography-coupled electrophysiology, was then tackled with gas chromatography-coupled mass spectrometry. After separation of the extract components by gas chromatography, the mass spectrum of the active component(s) (located on the chromatogram with reference to its Kovat's index1) was then analysed. Identification of the unknown was based on the match of its mass spectrum with that of a known product stored in a computer- based library. The mass spectrum and the calculated Kovat's index of the unknown were then compared with those of the library-proposed synthetic analogue, injected under the same conditions. Finally, identification was confirmed by testing the electrophysiological activity of the synthetic substance on the relevant receptor. 2.6. Behavioural bioassays (chapters 3.1,3.2., 3.3., and discussion) Behavioural bioassays were designed to investigate some identified olfactory stimulants, namely breath stimulants (CO2, and H2S), as behavioural cues for adult A variegatum. 2.6.1. Wind tunnel (chapter 3.1.) Resting adult A variegatum were placed in a wind tunnel and then stimulated with CO2 as described in chapter 3.1.. Activation as well as positive anemotaxis were observed and quantified as described in chapter 3.1.. 2.6.2. Activation bioassav (chapter 3.2.1 Resting adult A. variegatum were enclosed in glass flasks continuously swept with charcoal-filtered and humidified air. Every 24 hours, ticks are confronted with short stimulations of two olfactory stimulants identified by electrophysiology, i.e. CO2, H2S, and mixture of both. The tick's degree of 'The Kovat's index indicates the retention characteristic of a product on a column with respect to n- alkanes. It is expressed as following; /= 100[ n{(log V* - log V0V(IOg Ve+11 - log Ve)! + c] where Vx is the retention time of the unknown compound, Ve is the retention time of the alkane elu ting before the unknown, V0+11 is the retention time of the alkane eluting after the unknown, c is the carbon number of the alkane eluting before the unknown, n refers to the difference in the number of carbon atoms for the two n-alkanes used as reference. METHODS 18 Fig. 2.4. Diagram of the gas chromatography-coupled electrophysiology set-up (GC-EL). (0): Reference glass electrode impaled in the coxa of one of the first legs. (1): Recording glass electrode in contact with the cut tip of the sensillum. The electrode is electrically connected via an Ag/AgCl wire to a high input impedance preamplifier (symbolized by a triangle). (2): Universal AC/DC amplifier with separate outputs for the AC and DC components of the recorded signal. (3): Oscilloscope to visualize AC and DC signals. (4): Window discriminator apparatus and frequency/voltage converter to sort spikes from background noise of the AC signal and to transform the frequency of the spikes into a DC voltage. (5): Loudspeaker to render spikes audible in the AC signal. (G): Chart recorder which printed the DC signal, the frequency to voltage converted signal, and the response of the chemical detector ( 17) on separate channels. (7): PCM-1 Digital-VCR instrumentation recorder adaptator for analogue/digital or digital/analogue conversion of the AC signal. (8): Video tape recorder used to store the AC signal and to play it back for further analysis via digital/analogue conversion in (7). (9): "Go-Box" which contains the circuitry necessary to provide the trigger to start digitization on the computer of the analogue AC signal played back from the video tape recorder. (10): IBM-compatible computer equipped with an analogue/digital board to digitize analogue AC signal and software (SAPID tools) to visualize and analyse the digitized signal. (11): Laser printer to print signals and analyses. (12): Microphone used to add comments stored on an audio channel of (8). (13): Loudspeaker to listen to comments during play-back. (14): Pulse signal stored on the second audio channel of (8) which is used to mark stimulation period and to subsequently trigger via (9) the digitization of the analogue AC signal in the computer. (15): High-resolution gas capillary column enclosed in the oven of a gas Chromatograph. (16): Splitter delivering the column effluent {thin arrow) to the chemical detector (17) and the electrophysiological preparation via the heated transfer line (18) of the Chromatograph. (19): Water-jacketed glass tube to maintain the humidified main air-stream (large arrow) flowing over the preparation at a constant temperature. Bold circles: inputs to each instrument; open circles: outputs of each instrument; bold triangles: trigger inputs. METHODS 19 arousal is evaluated immediately after each stimulation as described in chapter 3.2.. 2.6.3. Locomotion compensator (Kramer's sphere! (chapter 4.6.1 Preliminary experiments on the triggering and maintenance of orientation in excited male A. variegatura by CO2, H2S, and mixture of both were undertaken. Only the most relevant results of these behavioural bioassays are given in chapter 4.6. and briefly discussed. As it is not described elsewhere, details of the method and procedure employed for these preliminary experiments are provided here. A breath-excited tick was placed on the top of a sphere (50 cm diameter) which rotated so that the animal was always kept at the sphere north pole. A light beam perpendicularly illuminated the sphere pole (field diameter: 3 cm). A small retroflective foil (n0 7610, 3M, Switzerland), glued to the dorsal side of the tick, reflected light back to a sensor which continuously evaluated the deviation of the reflective foil from the sphere pole. According to these deviation evaluations, signals were sent to two servomotors placed orthogonally on the equator of the sphere to recenter the reflective foil and hence the tick (Kramer 1976, Rickli et al. 1992). Displacement of the sphere was registered by two incremental pulse generators with a resolution of 0.1 mm. The X and Y coordinates of the tick's position were sampled at 0.1 s intervals and fed into a computer for track analysis. The sphere was enclosed in a styropore chamber to isolate the experimental area from the open laboratory. An upright aluminium cylinder (12 cm diameter, 11 cm high) with an upwind and a downwind aperture surrounded the north pole of the sphere to render the environment near the tick uniform. Experiments were accomplished during the last 6 hours of light of the daily cycle (LD 12:12) with unfed 6-7 month old males. Breath-excited individuals were placed on the sphere and allowed to walk for 2 min. Animals which regularly stopped were discarded. Walking responses were then recorded for 90 consecutive s divided into 30 s prestimulation control followed by 30 s of stimulation and a further 30 s poststimulation. The tick behaviour was also recorded on video for further analysis with a CCD camera focused on the tick. A humidified charcoal-filtered air stream was directed at the north pole of the sphere in a 3.6 cm diameter water-jacketed stainless steel tube to provide a constant air flow of 0.1 m/s at 25 ± 1°C and 70 ± 5% RH. Stimuli carried by solenoid-activated air flow(s) were added to the main air stream at 14 cm from the outlet of the tube situated at 3 cm from where the tick walked. H2S METHODS 20 stimulation was provided by air (120 ml/min) which passed through a 50-ml gas-wash flask with 1 ml of an aqueous solution of Na2S (1-100 mg/ml). CO2 stimulation was made with various additions of a flow from a 5% C02/95% N2 gas cylinder which passed through a 50-ml gas-wash flask with 1 ml distilled water added to provide concentration of 0.15-0.6% COg at the north pole of the sphere. CO2/H2S stimulation was made with synthetic 5% CC>2/95% N2 from a gas cylinder mixed with H2S vapours from the flask described above. Polluted air was evacuated via a funnel placed 20 cm downwind from where the tick walked and cleaned over charcoal. RESULTS 21 III. RESULTS 3.1. Perception of breath components by the tropical bont tick, Amblyomma variegatum Fabricius (Ixodidae). I C02-receptors. J. Comp. Physiol. A (1992) 170:665-676 3.2. Perception of breath components by the tropical bont tick, Amblyomma variegatum Fabricius (Ixodidae). Il Sulfide receptors. J. Comp. Physiol. A (1992) 170:677-685 3.3. Identification of vertebrate volatiles stimulating olfactory receptors on tarsus of the tick Amblyomma variegatum Fabricius (Ixodidae). I Receptors within the Haller's organ capsule. J.Comp. Physiol. A (in press) 3.4. Identification of vertebrate volatiles stimulating olfactory receptors on tarsus of the tick Amblyomma variegatum Fabricius (Ixodidae). Il Receptors outside the Haller's organ capsule. J.Comp. Physiol. A (in press) RESULTS - CHAPTER 3.1. 22 J Comp Physiol A (1992) 170:665-676 Journal of Comparative SET Physiology A SSSt © Springet-Verlag 1992 Perception of breath components by the tropical bont tick. Amblyomma variegatum Fabricius (Ixodidae) L C02-excited and C02-inhibited receptors Pascal Steullet and Patrick M. Guerin Institute of Zoology, University of Neuchâtel, Chantemerle 22, CH-2007 Neuchâtel, Switzerland Accepted March 23, 1992 Summary. Wall-pore olfactory sensilla located in the capsule of Haller's organ on the tarsus of Amblyomma variegatum ticks bear cells responding to vertebrate breath: one of these sensilla contains a C02-excited re- ceptor and a second sensillum has a CO2- inhibited recep- tor. Each of these antagonistic C02-receptors) which display typical phasic-tonic responses, monitors a dif- ferent CO concentration range. The C02-inhibited re- ceptor is very sensitive to small concentration changes between 0 and ca. 0.2¾, but variations of 0.01% around ambient (ca. 0.04%) induce the strongest frequency mod- ulation of this receptor. An increase of just 0.001-0.002% (10-20 ppm) above a zero C02-level already inhibits this receptor. By contrast, the C02-excited receptor is not so sensitive to small CO2 shifts around ambient, but best monitors changes in CO2 concentrations above 0.1%. This receptor is characterized by a steep dose-response curve and a fast inactivation even at high CO2- concentrations (>2%). In a wind-tunnel, Amblyomma variegatum is activated from the resting state and at- tracted by CO2 concentrations of 0.04 to ca. 1%, which corresponds to the sensitivity range of its C02-receptors. The task of perceiving the whole concentration range to which this tick is attracted would thus appear to be divided between two receptors, one sensitive to small changes around ambient and the other sensitive to the higher concentrations normally encountered when ap- proaching a vertebrate host. Key words: Tick - C02-excited receptor- C02-inhibited receptor - Haller's organ - Host finding Introduction The CO2 contained in vertebrate breath is an activating stimulus or attractant for most blood-sucking arth- ropods (e.g. mosquitoes: Gillies and Wilkes 1968; tsetse Correspondence to: P. SlcuHel flies: Turner 1971; Stomoxys calcitrarti.• Warnes and Finlayson 1985; Simulidae: Fallis and Raybould 1975; Tabanidae: French and Kline 1989; Reduvidae: Bernard 1974; Siphonaptera: Osbrink and Rust 1985). Some C02-sensitive receptors have been described on palps of mosquitoes (Kellogg 1970) and on antennae of tsetse flies (Bogner 1989). Ticks also respond strongly to breath and CO2, and Garcia (1962) has shown that CO2 attracts many different tick species. Since that first report several authors have devised CO2 baited traps for field sampling (e.g. Garcia 1965; Wilson et al. 1972; Gray 1985; Gu- glielmone et al. 1985; Norval et al. 1987, 1988), or have studied the effects of CO2 on tick behaviour in the lab- oratory (Nevill 1964; Sauer et al. 1974). In spite of the above, our knowledge of breath and CO2 perception in ticks is fragmentary. Breath- stimulated ticks lift their first pair of legs in the air to sample the surroundings as insects do with their anten- nae. From these observations, and various behavioural experiments where different parts of tick appendages were amputated or masked (Hindley and Merriman 1912; Lees 1948), we know of the primordial role of the tarsus of leg pair 1 for host odour perception. A large number of ultrastructural studies have described dif- ferent kinds of olfactory sensilla located on the tarsus of the first leg pair (reviews: Waladde and Rice 1982; Hess and Vlimant 1986), and the Haller's organ situated on the dorsal side of the tarsus (Fig. IA and B) bears a significant proportion of all tick olfactory sensilla. Thus, among the 19 tarsal olfactory sensilla of Amblyomma variegatum, 3 belong to the anterior pit of Haller's organ and 7 to the capsule of Haller's organ. Nevertheless, few investigations on physiological and functional charac- teristics of the tarsal olfactory sensilla have been under- taken in relation to host odour perception. Sinitsina (1974) using electrophysiological methods found olfac- tory cells responding to breath, mice odours, and n-vale- ric acid in the capsule of the Haller's organ in Hyalomma asiaticum, but he failed to account for a C02-receptor. On the other hand, Waladde and Rice (1982) mentioned the presence in Boophilus microplus of cells responding to RESULTS-CHAPTER 3.1. 23 666 P. Steullet and P.M. Guerin: Perceplion of breath components by Ambtyomma I breath and cow wash in the anterior pit of Haller's organ, and changes in the activity of cells from the capsule when stimulated with either breath or CO2. But these studies were mainly qualitative and apparently involved few recordings. The small number of physiological studies on olfactory sensilla of ticks may be ascribed to their limited accessibility, especially for those located inside the cap- sule, as well as the added complication of the high num- ber of cells in many of these sensilla. Several questions remain unresolved. Do any of these olfactory cells re- spond to breath? Where are they located? What are the stimuli contained in breath which induce the response? Based on complete ultrastructural studies on all tarsal sensilla of Amblyomma variegatum (Hess and Vlimant 1982, 1983, 1986), we have systematically searched for tarsal olfactory sensilla responsive to breath and its com- ponents. Materials and methods Tick rearing Experiments were mainly undertaken with unfed Amblyomma va- riegatum males but unfed females were also used for some record- ings. Originating from West Africa (Adiopodoumé, Ivory Coast), ticks were reared at the Agricultural Research Centre of Ciba-Geigy Ltd. (St-Aubin, Switzerland). AU stages (immatures and adults) were fed on Simmcntal calves at 22 to 24 0C. Ticks were kept under constant darkness at 28 °C/8O-909É RH except during moulting when conditions were 29 0C and 90¾ RH. Finally, adults were maintained in this laboratory in an environmental cabinet under the following conditions: 10 h of darkness at 18 0C/ 95« RH and 10 h of light at 25 °C/85% RH separated by 2 h "dusk" and "dawn" transition periods. Light and scanning electron microscopy Scanning electron microscope examination was made on ticks which were killed and fixed in 80% ethanol for several days, cleaned with ether/chloroform in a soxhlet extractor for 12 h, dehydrated in acetone, and critical point dried in CO2 with a Balzers CPD device. The mounted specimens were gold sputtered in a Balzers sputtering apparatus, and then observed in a Philips 500 PSEM. Light microscopy examination was made on sections of cut tarsi which were fixed in 2% glutaraldehyde (Sabatini et al. 1963), post- fixed in 2% OsO4 (Palade I952), dehydrated in acetone, and em- bedded in SPURR. Embedded tarsi were cut, at the level of the capsule of the Haller's organ, in either transversal or sagittal sec- tions of 0.5 u.m and observed under a light microscope (Vanox-S, Olympus, Japan) after toluidine blue staining. Tick preparation The tick was immobilized on a perpex holder on double-sided sticky tape. Pedal nerves were destroyed by pinching coxa of the forelegs with fine forceps ; this prevented muscle activity during electrophysi- ological recordings. To make proper recordings from the 7 olfactory sensilla located in the capsule of Haller's organ (an olfactory pit some 80 urn deep and 60 urn wide), dissection was needed to improve their accessibility as the opening of this capsule is just a narrow slit of ca. 5 urn wide and ca. 50 urn long across the tarsus (Fig. IB). The cuticular roof was removed (Fig. IC) with a piece of razor blade in a holder (John Weiss & Son LTD., England) mount- ed on a Leitz micromanipulator under an Olympus SZH stereomi- croscope at a magnification 192 x (working distance: 48.5 mm). Electrophysiology In order to improve contact, the tips of sensilla not located in the capsule were cut with the flame-pulled tip of a glass rod (1.5 mm dia.) oscillating in the ultrasound frequency range (ca. 120 kHz) as induced by a piezoelectric transducer disk (n0 4322 020 177721, Philips, The Netherlands) (Gödde 1989). The recording glass elec- trode filled with 0.2 M KCl was brought into contact with the cut tip of the sensillum with a Leitz micromanipulator, and the refer- ence glass electrode, filled with 0.2 M NaCl, was inserted in the coxa of one of the anterior legs. Electrical activity of capsular sensilla was also recorded with glass electrodes gently introduced into the dis- sected capsule until cell activity was captured. These recording electrodes also contained 1% polyvinylpyrrolidone K90 (Fluka, Switzerland), in order to prevent electrolyte flowing from the tip. Indeed, tips sometimes broke when they touched cuticular plec- morphs located between sensilla (Fig. IC). Nevertheless, it was still possible to record properly with broken tips of up to ca. 5 um. With some experience it was possible to recognize patterns typical of different sensilla according to 1) electrode position, orientation and depth inside the capsule, as the relative position of each sensillum in the cavity was indeed very consistent between individuals, 2) typical spontaneous activity of its cells, 3) spike shapes, and 4) behaviour of these cells to various stimuli. Tungsten electrodes were also used in some cases when the preparation was exposed lo a dry air stream. Recorded signals were fed via a IO12 Cl input impedance pream- plifier into a universal AC/DC amplifier (UN-03, Syntech, The Netherlands) and registered on video tapes via a PCM-I Digital VCR-instrumentation recorder adaptator (Medical System Corp. Grecnvale, USA) onto a video cassette recorder (Grundig VS540 Monolith, Germany) (Gödde I985). Records were visualized either by playing them back onto a paper recorder (Graphtec WR7600, Japan) used in memory mode, or by using the plot or the view option of the spike analysis programme SAPID (Smith et al. 1990). For the latter, the recordings were fed into a 386 IBM compatible computer (Mandax) via the DAS 16 analogue/digital plug-in board (MctraBytc Corporation, USA) at a digitizing rate of 10 kHz. Discrimination for (he activated cells according to their amplitudes, shapes, and spike frequencies was made by eye. This simple method was found to be the most appropriate one for the multicellular responses evoked by breath in these sensilla. SAPID was quite inadequate to properly analyse these multicellular responses be- cause of the large number of overlapping spikes and, moreover, because of the change in amplitude and/or shape of some spikes. The length of spike trains employed for determining activity will be indicated for each case in Results. Nevertheless, for experiments with long CO1 stimulation, the clear nature of the response of CO1-CXCiICd or COz-inhibited recep- tors in their respective sensilla allowed us to sort spikes of CO1- receptors with a window discriminator (model 121, W-P Instru- ments Inc., USA), whose frequency was converted into a DC vol- tage by a frequency-voltage converter (time constant: I s) in the UN-03 amplifier. Visualisation of the window discriminator upper and lower levels on the oscilloscope (Tektronix 5112, USA) allowed us to sort spikes properly for unambiguous discrimination for those of a C02-receptor from others in a record. The firing rate of other cells was rather low, thus inducing few or no overlapping spikes. Nevertheless, some rare spikes, not typical of C02-receptors, were occasionally counted. But the error was estimated at less than 556. Stimulation Tarsal sensilla of ticks frequently contain receptors for different modalities, i.e. apart from chcmorcccptors they may also support thermoreceptors and hygroreceptors other than those reported by Hess and Loftus (1984). In order to discriminate for responses induced primarily by the chemicals being tested here, it was neces- sary to maintain the sensillum. as far as was practically possible, in RESULTS-CHAPTER 3.1. 24 Hg. I. A Tarsus of the foreleg of a Iemale Anihlyoiiinia varivqatitm. The white arrow shows Nailer's organ on the dorsal side with olfacior) sensilla and the opening of the capsule; scale bar: 200 pm. B Mailer's organ with the group of anterior pit sensilla (/) and opening of the capsule [J). scale: 30 um. arrow proximal end of the iarSUS. C" Capsule of I lallei s organ w uh culiculai roof removed revealing two wafl-pore capsular sensilla white arrows) andcuticu- lar pleomorphe <*). The 3 dashed white arrow s indicate approximate electrode positions and angles of approach towards sensilla with breath-sensitive cells : a lor one inhibited by CO2. h l'or one excited by CO2. f for one insensitive to CO2 (described as a Il ,S-sensilive cell m Steatiti and Guelfa 1992): scale bar: 20 urn. Nodi arrow proximal end of the tarsus. D Longitudinal section through the capsule showing a sensillum (hlaik arrow ) ; the base of a second sensillum is also visible (while arrow i ; *: culiculai pleomorph. scale bar: M) um. A1 B, and C are scanning electron micrographs. I) is a light microscope view RESULTS - CHAPTER 3.1. 25 668 P. Steullet and P.M. Guerin: Perception of breath components by Amblyomma I a controlled air stream. For that purpose, air scrubbed in charcoal and silicagel, and humidified to 80% RH, at 22 ± I *C in a water bath, was continuously blown at 10 ml/s through a glass tube onto the tarsus. The outlet of the tube (3 mm i.d.) was 5 mm from the tarsus, providing an air speed at the level of the preparation of about 1.5 m/s. The tip of a 5-mI polypropylene syringe containing the odour (breath, CO2, or other volatiles) was introduced through a septum-covered hole in the tube, 3 cm or 25 cm from its outlet, depending of the experiment (see Results). A charcoal-filtered air pulse, delivered by a solenoid valve, was administered via a stopper at the back of the syringe, so that 2 ml of the syringe content was injected in 1 s into the glass tube. To prevent changes in air flow during stimulation, a charcoal-filtered air flow of 2 ml/s was de- livered via another solenoid valve through a blank syringe into the glass tube, and at the same distance from the preparation, during stimulus off. Stimulations followed at 3 min intervals. CO2. A range of concentrations of CO2 were produced by mixing the manometer-controlled outflows from 100% CO2 or 5% CO2/ 95% O1 gas cylinders in fixed proportions to pure N2. A 5-ml syringe with a rubber stopper in place of the plunger was filled with precise concentrations of CO2, as confirmed with an IR-gas analyser (Binosl, Leybold-Heraeus, FRG). The lip of the syringe was then introduced into the glass stimulus-delivery-tube and 2 ml of its content flushed over the preparation as described above. As the flow rate of the main humidified air flow was 10 ml/s, CO2 concentration of the stimulus pulse was diluted 6 times in its passage to the preparation to provide a range of concentrations from ca. 0.04% to 5% CO2. In longer experiments with continuous or pulsed CO2 stimulation, a mixture of 5% C02/95% O2 from a gas cylinder was injected directly into the glass stimulus-delivery-tube. Various concentrations of CO2 were obtained by regulation of a voltage- pressure converter which controlled the flow rate of the C02/02 mixture into either the continuous humidified air stream of ca. 0.04% CO2 or into a dry synthetic air stream of 20% O2/80% N2 which was free OfCO2. In order to prevent changes in air speed at the level of the tarsus, two solenoid valves operated alternatively, permitting delivery of either the C02/02 mixture or an equivalent charcoal-filtered air stream into the continuous air flow. Breath. Human breath was blown into the barrel of a 5-ml syringe used as stimulus cartridge and delivered to the preparation as de- scribed above. The CO1 concentration of breath was likewise mea- sured with the IR-CO2 analyser. Taking into account a dilution factor of 6 in the delivery tube, the estimated concentration at the level of the tarsus was therefore ca. 0.6%. Other volatiles tested. The following volatiles were also tested: methane, ammonia, acetone, 3-pentanone, 4-heptanone, g-buty- rolactone, g-valerolactone, g-caprolactone, hexanal, pentanol, 1-oc- ten-3-ol, 1-octene, propionic acid, n-butyric acid, ìso-butyric acid, n-valcric acid, iso-valcric acid, heptanoic acid, L-lacttc acid (all vertebrate-associated volatiles), nonanoic acid, 2-nitrophenoI, 2,6- dichlorophenol, methylsalicylatc (tick phcromonc components), dichloromethane and distilled H2O (solvent blanks). The purity of these products, except for the first two gases, was >99% as in- dicated by GC. Ten ul of a 10"3 or 10"2 M stimulus solution in dichloromethane (Merck analytical grade) or in distilled H2O was deposited on a piece of filter paper and enclosed in a stoppered 5-ml syringe (stimulus cartridge) after evaporation of the organic solvent. Three min later, 2 ml of the syringe content was evacuated into the delivery lube as described above. Methane was taken from the mains. A stock solution of 35% NH4OH diluted 10 or 100 limes was used as ammonia stimulus. Spatial field of perception of the capsule of Holler's organ Stimulation of the C02-excited receptor found in one of the cap- sular sensilla was made from different points in space around the tarsus to determine whether preferential directions exist in the capsule's field of perception. For this purpose, female ticks, which are slightly bigger than males, were mounted on a pointed perpex holder allowing stimulation from almost any direction. A tungsten electrode, chosen to economise space in the capsular slit, was gently introduced through the slit-like opening of an undissected capsule until good contact was made with the sensillum bearing the CO2- excited receptor. The recording electrode did not occupy more than 10% of the slit opening, and thus had a minimal effect on the way air could enter the capsule. The reference tungsten electrode was fixed in the coxa of one of the anterior legs. CO2 stimulation was administered in I s into the delivery stream (40 cm/s) blowing from the directions indicated in Fig. 7 onto the tarsus, which was placed at 15 mm from the orifice of the stimulus-delivery-tube. Wind-tunnel experiments Behavioural experiments were made in a wind-tunnel (111 cm long, 45 cm wide, and 28 cm high) at 22 ±2 0C and ca. 45% RH. Room air was blown down the tunnel by a fan through a charcoal filter (Therma, Switzerland) and a glass-fibre net (1.4 mm mesh). Five resting males or females, with legs folded under the body, were introduced 80 cm downwind from the stimulus source. Ticks were discarded unless they remained immobile during the 2 min prior to stimulation. The stimulus source was a humidified synthetic air flow bearing various CO2 concentrations which was introduced via a nozzle centrally at floor level in the upwind part of the tunnel. This stimulus was immediately carried by the wind flowing down the tunnel at 20 cm/s to the resting ticks. Laminarity of lhe flow, wind speed, and plume characteristics at the floor were defined with cigarette smoke. The stimulus was diluted 25 to 30 times as mea- sured by CO2 indicator tubes ±5-10% error (Dräger, Germany), to give a mean concentration from ambient control of ca. 0.04% to 0.35% CO2 over the resting ticks at the highest level tested. The mean CO1 concentration within 8 cm of the source ranged from ambient control to 1.1 % at the highest level tested. Nevertheless, it is important to mention that the plume, as indicated by cigarette smoke, had a disrupted structure with some twofold differences in concentration around the mean concentration. Ticks were observed during 5 min of stimulation. Individuals initiating locomotion as well as those walking upwind to within 8 cm of the source were counted. Experiments were replicated 20 times for each CO2 con- centration and each sex. Results Breath only elicited responses in sensory cells of 3 of the wall-pore-single-walled sensilla according to Altner's et al. classification (1977) in the capsule of Haller's organ. Two of the 7 capsular sensilla as well as some pleo- morphs are shown in Fig. IC, along with the approxi- mate orientation of the recording electrodes used to cap- ture electrical activity of cells which were responsive to breath. The activity pattern of cells recorded from each of these 3 electrode orientations was distinctive and con- sistent between ticks, and between the left and the right tarsus of the same individual in terms of the number of cells, the spike amplitudes and the spike shapes. Other precise orientations of electrode insertion permitted cap- ture of other patterns of olfactory cell activity which were altered by other stimuli such as methylsalicylate (Hess and Vlimant 1986) or by vertebrate body odours (Steul- let, unpublished). Indeed, the different orientations of the electrodes to where cell activities were recorded corre- sponded to the sensilla locations within the capsule as RESULTS - CHAPTER 3.1- 26 P. Steullel and P.M. Guerin: Perception of breath components by Amblyomma I 669 observed by microscopy. The pattern of cell activity was the same whether the tip of the capillary remained un- broken (tip diameter < I ^m) or broke on contact (diam- eter up to 5 urn). This result indicated that simple contact of the electrolyte with the wall of the sensi Hum sufficed for a good recording. The walls of these sensilla are indeed very thin (0.08-0.14 urn) with large and numerous pores (0.1-0.16 urn dia.) (Hess and VIimant 1982). Re- cordings generally displayed activity of 3 to 5 cells, an observation which correlated well with ultrastnictural studies showing that capsular sensilla in A. variegation contain 3 to 5 sensory cells (ibid.). This and the fact that the sensilla were never seen to touch one another in sections under high magnification lead to suggest that the spikes observed in a given recording were picked up from a single sensillum. The fact that electrophysiological ac- tivity of cells responding to breath was captured with the electrode inserted in 3 different orientations proximally in the capsule suggests that 3 different sensilla were im- plicated. Each type displayed a characteristic multicellular response. CO2 excited a cell in one of these sensilla (Fig. 2A), and inhibited a cell in another sensillum (Fig. 6), whereas cells of the third type of breath sensillum did not respond to CO2. In the latter, one cell is described as being a sulfide-sensitive cell (Steullet and Guerin 1992). Despite the difficulty associated with working blindly within the capsule, many reproducible recordings were obtained by judicious placement of the electrode. This permitted re- cordings from 65 breath sensilla bearing the C02-excited cell, 17 breath sensilla with the C02-inhibited cell, and 37 breath sensilla with no C02-receptor, in different ticks. C02-excited receptor Figure 2A illustrates the cell response of one breath sensillum bearing a C02-excited receptor to increasing CO2 concentrations and human breath, all injected 25 cm from the outlet of the stimulus-delivery-tube and subse- quently diluted in the humidified air stream. Detailed sections of some of these responses are given in Fig. 3. As the stimulus onset was not sharp, the phasic portion of the response was not so pronounced and the maximum frequency occurred between 200 and 600 ms after a gradual increase in spike frequency. With the absence of a strong phasic part in the response, it was easier to categorize the spikes visually (Fig. 3). The firing rate of the spike numbered 1 (C02-excited receptor) induced by human breath diluted in clean air was quite similar to that induced by the equivalent CO2 concentration (Fig. 3). This suggests that breath contains nothing else capa- ble of modifying the response of this receptor. Moreover, none of the volatiles, listed in Materials and methods, elicited a response from this receptor. C02 0.04 % ambient concentration C02 0.10 % ^1 *,----------Kt C02 0.68 % t?k ^t^\\\\m^ M- ¦t ' "t" 4t C02 2.16 % \\\\W\ \\\\^'f Hl'Mltlll)^ A-f human breath 1:6 ^ f-J j !¦ ¦ |rtl .{I, (i H^fejH^^Mti^^W^^'^^'^'^i^M^^ *' ' ¦ '¦ B ^ ¦ H' HH"" ¦ IM 'Nlll'HIIIHHlH'N'HHl lamp OFF 24 0C ^ lamp ON 29 0C rhh M^h1' 4 *M'l' 'f[ 1I I*1!*1,-'Kh *|M HMMH' I lamp ON 29 0C lamp OFF 24 0C Fig. 2. A Representative responses of a capsular sensillum bearing a CO1- excited receptor of a male A. variega- tion (small biphasic spike I with bold arrow) to increasing CO2 concentra- tions and diluted human breath. Stimuli were injected into the stim- ulus-delivery-tube 25 cm from its out- let. so that the stimulus onset at the preparation was not very sharp, and cell frequencies increased gradually within the first 200 ms of the re- sponse. The preparation was main- tained in a humidified air stream at ca. 0.04% CO2. Four spike types are indicated (see text and Fig. 3); num- bering of spike types same for Figs. 2 and 3. Spike 4 (asterisk) is a sulfide- receptor (according to Steullet and Guerin 1992). Spike 3 (white arrow) is a cell inhibited by increasing COj levels, but activated by breath. Its response seemed to be associated more with temperature changes as in- dicated by its response to turning off and on the microscope lamp (B). Thus, a T° increase activated it, whereas a corresponding T° decrease slightly diminished its activity. T° changes were measured with a therm- istor put at the place of the prepara- tion. In A, horizontal bar, 1 s stim- ulation; vertical bar, 1 mV. In B, horizontal bar, i s; vertical bar, I mV RESULTS - CHAPTER 3-1. 27 670 P. Steullet and P.M. Guerin: Perception or breath components by Ambtyomma I A C02 0.68% ' i i t 1 1 1 1 Ï 1 1 1 1 1 1 1 1 1. * .1 1 1 ) U^UiU^AlUUV 180 Fig. 3A, B. Detailed sections from the recordings illustrated in Fig. 2 to show how the 4 spike categories were discriminated visu- ally. A Recording from the first 750 ms of stimulation with 0.68% CO2, and B recording from the first 750 ms of stimulation with human breath diluted 1:6 in air (ca. 0.6% CO2). Spike 1, activated either by breath or CO2, is the COj-excited receptor. Spike 2 is another cell with a persistently low firing rate. Spike 3 may be a thermoreceptor, responding to a slight increase in T0 during stim- ulation with human breath. Spike 4. which changed its sign during stimulation with breath, is a sulfide-reccptor responding to H2S (described in Steullet and Guerin 1992). Spike numbers underlined are overlapping events. Numbering of spike types as in Fig. 2. Horizontal bars 100 ms; vertical bars 1 mV o.I ! CO2 concentration % 10 Fig. 4. Dose-response curve of the C02-excited receptor of male A. variegatum established from the first 160 ms of the response (phasic part). Preparations were maintained in a humidified air stream at ca. 0.04% CO2 into which CO2 stimuli were injected 3 cm from the outlet of the tube to the preparation. Data (mean ± SD) have been obtained with 4 C02-excited receptors, all from different males. Abscissa: estimated concentrations of CO2 arriving at preparations Figure 4 shows the dose-response relation established with C02-excited receptors from 4 different males which were stimulated with increasing CO2 concentrations in- jected 3 cm from the outlet of the stimulus-delivery-tube (sharp stimulus onset). The response magnitude was de- termined from the first 160 ms of stimulation (phasic portion). The possibility of overlapping spikes was also considered in the determination of the response intensity. The C02-excited receptor responded to a concentration range covering some 2 to 3 log orders of magnitude. At ambient concentration of ca. 0.04%, activity was weak at a mean of 4.4 impulses/s, and gradually increased with higher concentrations up to about 140 impulses/s for 5% CO2. The relation between the CO2 dose and the tonic Fig. SA-C. Amblyomma variegatum C02-excited receptor response un- der continuous and repetitive 1 s stimulation: A 0.7%, B 1.2%, and C 1.8% CO2. Horizontal bars : stim- ulus duration. The preparation was maintained in a humidified air stream at 0.04% CO2 into which stimuli were injected. These re- sponse profiles were obtained by frequency to voltage conversion of the AC signal after sorting spikes of the C02-cxcitcd receptor from others with a window discriminator (see Materials and methods). Re- sponses were reproducible RESULTS - CHAPTER 3.1. 28 P. Steulict and P.M. Gucrin: Perception of breath components by Amblyomma I 671 portion of the response, established with firing rates ob- served after 2 min of exposure to a given CO2 concentra- tion, shows that the C02-excited receptor coded rather well constant levels of CO2 higher than ca. 0.1 % (Fig. 8). With repetitive 1 s pulses of CO2, or with continuous stimulations of more than 10 s, strong adaptation only occurred at levels as high as 1.8¾ CO2 (Fig, 5). Even then, however, receptor inactivation was remarkably fast, returning abruptly to its former level of activity after stimulation off (Fig. 2A). At high CO2 concentrations the spike amplitude of the C02-excited receptor sometimes diminished as shown in Fig. 2A, but this did not occur systematically. No difference between C02-excited recep- tors of males and females was recorded. The sensillum housing the C02-excited receptor also had a cell which was inhibited on stimulation with CO2 in our set-up (cell numbered 3 in Figs. 2A, and 3). Nevertheless, this response was not due primarily to CO2, since human breath slightly activated this cell. Thermosensitivity may be responsible since a slight in- crease in T° resulting from switching on the microscope lamp stimulated this cell, whereas switch-off caused some slight inhibition (Fig. 2B). CO2 stimulation did cause a decrease in T* (<0.5 0C), and breath stimulation an increase in T° (< 1 0C) in our set-up. CO ^inhibited receptor This type of receptor was inhibited by an increase in CO2 concentration and activated by a decrease. Complete Fig. 6. Representative responses of a capsular scnstllum of a male A. variegatura bearing a CO2-Inhibited receptor (bold arrow) to increasing CO2 concentrations and diluted human breath. The preparation was maintained in a humidified air stream at ca. 0.04% inhibition of this receptor (bold arrow in Fig. 6) was achieved by short 1 s stimulation with diluted breath or with the relatively high concentrations of CO2, i.e. greater than 0.1%, and reactivation of the receptor was clearly delayed after stimulus off. This post-stimulus in- hibition was more pronounced with increasing CO2 con- centrations and lasted, in any case, significantly longer than the complete decline of the C02-excited receptor. Thus, 1 s stimulation with 0.68% induced a post-stimulus inhibition of 1203 ±283 ms (mean ± SD) in 6 CO2- inhibited receptors, whereas the complete post-stimulus decline of 9 CÒ2-excited receptors with the same stimulus was reached after 313±126 ms. Reactivation following inhibition due to 1 s CO2 stimulation was typified by a burst in spike activity which was stronger the higher the CO2 concentration employed. The frequency of the reac- tivation, based on the first 400 ms of the response was 22 ±8 impulses/s (mean ± SD, n = 3) after a 0.1% CO2 stimulus, 30±14 impulses/s after Oj68% (n = 6), and 35± 14 impulses/s after 2.16% (n = 2), whereas activity at ambient was 17±9 impulses/s (n= 18). As expected, the COj-inhibited receptor was also affected by human breath in the same way as with an equivalent concentra- tion of CO2. Diluted breath containing ca. 0.6% CO2 elicited inhibition lasting 1170 ±448 ms (mean ± SD, n=6) from stimulation off and a reactivation frequency of 25 ± 11 impulses/s comparable with 1203 + 283 ms (mean +SD, n = 6) post-stimulus inhibition and a reac- tivation burst of 30+14 impulses/s for an equivalent CO2 concentration. Nevertheless, as Fig. 7 clearly shows, the same CO2 drop could induce a very different reactiva- CO1 into which stimuli were injected 25 cm from the outlet of the tube. White arrow: receptor activated by an unknown breath com- ponent with a latency of ca. 300 ms. Horizontal bar 1 s stimulation ; vertical scale 1 mV C02 0.04 % ambient concentration * mmm mm C02 0.10 % / iWflW'fl>^frWq>|Mp^'^ffrtWi**^'W mm C02 0.68 % ||M^^ C02 2.16 % *44* pT' WTrlrtM *(h»¥tWtfi^"'*','*W* frfw'tt 4^^^^^^41^*^ fWMH^ M4 H * human breath 1:6 ^ 1^^4^41^ I RESULTS - CHAPTER 3.1. 29 672 P. Steullet and P.M. Guerin: Perception of breath components by Amblyomma I 20-r « « 1OJ 3 E - Ol 1 min *$ * .5 Cl S ° 1 2 3 4 5 6 7 "¦ ¦ 'iM'i I H\ I" Ii I....." ' 0.04% ^ 0.19% ^ 'i I).....I" 0.04% B r 'f-i-'ti1 0.04% O- 0.19% 0.19% I "Il...... l'I......!»""If '>"< 0.19% '" 1I" "I11 'Ji......^1''1 ' *......^1I f.......'1^1HI I11111 Il ^ 0.19% ¦44 0.38% 0.19% 0.19% W------H------1 „, iiii|.mThNIIHI|HU1IM l|ll Il 0.19% 0.04% 1 s Fig. 7A1B. Adaptation of a C02-inhibitcd receptor (boldarrow) of a male A. variegation at different CO2 concentrations. A Upper trace: response profile of the C02-inhibited receptor, obtained by frequency to voltage conversion of the AC signal after sorting spikes of the C02-inhibited receptor from others with a window discrimi- nator (sec Materials and methods); lower trace: CO2 concentra- tions (either 0.04, 0.19, or 0.38¾) delivered to the C02-inhtbited receptor. B Detail of the spike pattern underlying the response profile in A, where numbers on the left of each record refer to the sections numbered in A. White arrow up, increase in CO2 concentra- tion; white arrow down, decrease in CO2, concentration. Responses were reproducible tion burst in this type of receptor, depending on the CO2 conditions pertaining before the drop, i.e. the longer the receptor was inhibited by exposure to a given CO2 con- centration then the stronger the reactivation. When the . C02-inhibited receptor was submitted to long stimula- tion with CO2 above ambient as in Fig. 7A, it was first completely inhibited but adapted within minutes to another frequency level. These observations provided evidence for the phasic-tonic characteristic of the CO2- inhibited receptor. The tonic responses of both the C02-inhibited and the C02-excited receptors after 2 min of exposure to stable concentrations of CO2 ranging from 0% to 5% are shown in Fig. 8. The tonic response of the C02-inhibited recep- tor changes most with concentration between 0% and 0.2% CO2, i.e. the C02-inhibited receptor codes best small shifts in concentration around ambient (Fig. 9). In experiments where the CO2 level was changed approxi- mately every 5 s, it was clear that the firing rate of the C02-inhibited receptor was most affected by shifts of 0.01 to 0.02% CO2 around the 0-05% level (Fig. 9A), than o o.oi o.t i to CO2 concentration % FIg. 8. Dose versus tonic response relationship for both thé CO2- exciled (solid circles) and the C02-inhibited receptors (open c'tr~ des) of male A. variegatum. The tonic activity was calculated from a 4000 ms spike train after 2 min of exposure of the receptor to the different CO2 concentrations, obtained by adding different amounts of 5% C02/95% O2 into a dry synthetic air flow of 20 % O2/80% N2 to achieve a CO2 range of0% to 5%. Data points were estab- lished with, respectively, 3 C02-excitcd and 3 C02-inhibited recep- tors, all from different ticks. Trend Unes connect mean values RESULTS - CHAPTER 3.1. 30 P. Stcullet and P.M. Guerin: Perception of breath components by Amblyomma I 673 «SO «40 o «30 I20 " 10 ^ .07 g M O 03 D °-J\A^-^v^ 50 40 30 20 10 .07 .DS .03 10 S 10 S Fig. 9A-D. Modulation of the spike frequency of both a C02-inhibited receptor (A and B) and a C02-excited receptor (C and D) of a male A. va- riegatum by small changes in CO2 concentration around 0.05% (A and C) and around 0.12¾ (B and D) in a dry synthetic air stream. Changes in CO1 concentration were obtained by varying the manometer-controlled flow from a gas tank con- taining 5¾ C02/95% O2 which was added into the dry synthetic air stream of 20% O2/80% N1. Response profiles (upper trace in each case) were obtained by frequency to voltage conversion of the AC signal after sorting spikes of either the COj-excited or the C02-inhibitcd receptor from others with a window discriminator (see Ma- terials and methods). The tower trace in each case is the representation of the stepwise changes made to the CO2 concentration in time (range 0.03 to 0.07% in A and C, and 0.07 to 0.18¾ in B and D). Time scale on the horizontal axis IO s/ div. Responses were reproducible A B .--°-*.. ...a.. >-"'" Is ""¦¦¦*? C 1* •0— r ,/ ,«w?. t V, lateral posterior--------(T""* anterior/ k.. \ J t *'' "T" r" Fig. I0A, B. Field of perception of the C02-excited receptor in the capsule of Haller's organ established by the re- sponse of the sensilla housing this re- ceptor in 6 female A. variegatum to a CO1 stimulus coming from different di- rections around the tarsus. Responses (mean ± SD) are given as a percentage of the response to stimulus delivered perpendicularly to the dorsal surface of the tarsus (star). Reference response shown at apex of octagon. A Re- sponses to stimulation at various direc- tions perpendicular to the longitudinal axis of the tarsus. B Responses to stim- ulation at various directions on the longitudinal axis of the tarsus. No stimulation was possible from the pos- terior-ventral direction, where the body of the tick was fixed by even bigger shifts around 0.12% (Fig. 9B). Even an increase of just 0.001-0.002% CO2 (i.e. 10-20 ppm) above 0% already elicited a visible decrease in spike activity of the CÓ2-inhibited receptor. By contrast, the C02-excited receptor coded poorly small shifts around ambient (Fig. 9C, D), but became much more efficient for greater shifts around 0.12% (Figs. 5, 8). None of the other volatiles listed in Materials and methods influenced activity in the C02-inhibited recepì tor. Nevertheless, this receptor was affected by large T* and humidity changes. A decrease in RH of some 50% or an increase in T° of 5 0C slightly stimulated this receptor, while it was mildly inhibited by a large jump in RH of 50% or a T decrease of 5 0C. However, the response to stimulation with either breath or CO2 was not due to T or humidity shifts, as both breath (T increase of < 1 0C, and RH increase of ca. 2%) and CO2 (T decrease of <0.5 0C, and RH decrease of ca. 10%) both inhibited the receptor. Moreover, large T" shifts only elicited slight modulation of the activity of this receptor when it was exposed to CO2 concentrations near ambient (ca. 0.04%), whereas the same T shifts did not influence spike frequency in a C02-free atmosphere. Be- cause of the extreme sensibility of the C02-inhibited receptors to small changes of concentration around am- bient, the apparent responses of this receptor to T or RH shifts are probably due to the influence of these parameters on the CO2 content of the air. Human breath also activated another cell of the breath sensillum bearing the C02-inhibited receptor, but the responsible stimulus is still unknown (white arrow in Fig. 6). Spatial field of perception of the capsule The direction from which wind carrying the CO2 stimu- lus arrived, as tested in 6 different females, did not have RESULTS - CHAPTER 3.1. 31 674 P. Sleullet and P.M. Guerin: Perception of breath components by Amblyomma I mean CO2 concentration % over resting ticks 80 cm downwind O) C O Ï V) U O CL O 100 p 90 - 80 - 70 - 60 - 50 - 40 • 30 - 20 - to - 0 - 0.04 ____L_ 0.12 0.20 _____1__ ' 0.1S 0.3S B •o C O O O O a. 0 0.01 0.1 1 10 CO2 concentration % at the source Flg. IIA, B. Behavioural response of male (solid circles) and female (open circles) A. variegatum to various CO2 concentrations in a wind-tunnel. A Locomotor stimulant effect of various CO2 levels as measured by proportion of resting ticks induced to commence walking. Lower abscissa the CO2 concentration measured at the source, upper abscissa the mean CO2 concentration as it passed over resting ticks 80 cm downwind from the source. Above 3056, the locomotor effect of the stimulus is significant (P < 0.05, the exact mean CO2 concentration % at 8 cm from the source 0.04 0.35 0.60 0.01 0.1 1 10 CO2 concentration % at the source method for 2x2 tables). B Attraction of CO2 as measured by proportion of ticks walking upwind to within 8 cm of the source. Lower abscissa CO2 concentrations at the source, upper abscissa mean CO2 concentration at 8 cm from the source. Above 10%, attraction is significant (P<0.05, the exact method for 2 x 2 tables). Between 90 and 100 males and females were tested at each CO2 concentration any influence on the response of the C02-excited receptor enclosed in the undissected capsule (Fig. 10). Indeed, the intensity of the response to stimulation from various directions did not differ from the response to the same stimulus directed perpendicularly to the dorsal side of the capsule, taken here as a reference. Thus, despite en- closure of the olfactory scnsilla in a capsule on the tarsus with a narrow slit as an opening to the exterior, A. variegatum is able to detect CO2 effectively, regardless of the direction from which air currents approach it. Wind-tunnel experiments CO2 is a locomotor stimulant and attractant for both sexes of A. variegatum, although males respond better than females. Wind-tunnel experiments show that this tick species'responded best to a very narrow range of CO2 concentrations (Fig. 1IA, B). Humidified air with a mean concentration of 0.15% CO2 passing over the resting ticks was the best locomotor stimulant (Fig. 1 IA). These activated ticks were attracted to within 8 cm of the source when mean CO2 concentrations were between 0.45% and 0l6%, whereas a mean of ca. 1.1% CO2 within 8 cm of the source was no longer attractive (Fig. IIB). Discussion CO2 acts as ja locomotor stimulant and an attractant for A. variegatum in the wind-tunnel. A source of 3.5 to 5% CO2, diluted by almost a factor of 10 at 8 cm from the source, and 25 to 30 times at 80 cm downwind where ticks were resting, was most attractive. This suggests that the best attractant in the field for this species, which normally lies in wait in the litter zone, would be a respir- ing host reposing or grazing a few meters away. Indeed, field experiments with the related species, Amblyomma kebraeum, have shown that this tick is attracted over a range of a few meters to cattle or sheep (Norval et al. 1987). The upper limit of some 1% CO2 which still at- tracted A. variegatum to the source in the wind-tunnel, but above which level they were repelled, is not surpris- ing. In this laboratory we have frequently observed that expiring directly onto an individual of this species which is running toward the observer results in repulsion. CO2 levels which cause activation and attraction of male and female A. variegatum in the wind-tunnel correspond to the discriminative range of C02-receptors as revealed by the electrophysiology. Nevertheless, CO2 is a better loco- motor stimulant for males than for females. This dif- ference in behaviour should most probably be ascribed to some internal control at the level of the CNS as no differences have been found between the sexes at the level of the C02-receptors. Despite of the crucial importance CO2 may play in host-finding behaviour of this species, it is surprising that the tick possesses on the tarsus of its forelegs just one C02-excited and one C02-inhibited receptor in the cap- sule of the Haller's organ. Stämpfli (1987) showed that A. variegatum no longer responded to CO2 after removal or masking of the tarsus of each anterior leg. As adults of A. variegatum are often obliged to wait for months under occasionally quite adverse environmental con- RESULTS-CHAPTER 3.1. 32 P. Steultet and P.M. Gucrin: Perception of breath components by Amblyomma I 675 ditions before being provided the chance to find a suit- able host, the few C02-receptors need then to remain entirely functional during a long period, ever alert to any abrupt change in the CO2 level of its environment. It seems therefore quite normal that these cells should be well protected from any physical damage or desiccation inside the capsule of Haller's organ. Local high humidity around sensilla within the capsule permits thinner walls with larger! pores than in sensilla not located within a cavity, factors which consequently increase molecular diffusion through the sensillar wall. In Lepidoptera (Bogner et al. 1986;Bogner 1990), CO2 receptors are also more or less enclosed. Enclosure could furthermore im- prove CO2 perception since a higher local RH around the sensillum could enhance better adsorbtton of CO2 or its derivatives into the sensillar lymph. The rigid architec- ture of the capsule does not restrict the passage of vola- tiles, at least highly diffusable CO2 molecules, to the capsular sensilla, nor is a specific orientation of the tarsus vis à vis the wind direction at all critical. Local air tur- bulence around the tarsus, combined with molecular diffusion is probably sufficient to permit permeation of CO2 and other volatiles into the capsule. The main characteristics of the C02-excited receptor of A. variegatum are: 1) a phasic-tonic response, 2) a steep dose-response curve covering 2-3 log orders of magnitude from ambient to ca. 5% CO2, 3) adaptation occurring only at concentrations as high as 1.8%, and 4) a fast inactivation process after stimulus off even at the highest concentrations tested (5%). The steepness of the dose-response relationship is not very different from that established for the C02-receptors of Aedes aegypti (Kel- logg 1970), Glossina pallidipes (Bogner 1989), Lucilia cuprina (Stange 1974) and for the electroantennogram re- sponse of Stomoxys calcitrans to CO2 (Warnes and Fin- layson 1986). The C02-excited receptor of A. variegatum seems, nevertheless, less sensitive to small changes in CO2 concentration around ambient than those described for haematophàgous Diptera and especially mosquitoes (Kellogg 1970). However, as in A. variegatum, a fast inactivation process characterizes all of these C02-recep- tors (Kellogg 1970). A C02-irihibited receptor has not, to our knowledge, been reported previously. This cell is extremely sensitive to very small changes in CO2 levels around ambient concentrations, and the tonic part of the response is most affected by small changes in concentration of between 0% and ca. 0.2%. Shifts of 0.01 to 0.02% CO2 around am- bient are nicely monitored by the phasic part of the response of this receptor. Stimulation with 0.001-0.002% CO2 (10-20 ppm) in a C02-free atmosphere already clearly diminishes the spike frequency of the CO2- inhibited receptor. This extreme sensitivity explains why large T0 or RH shifts, which can certainly modify the CO2 content of ambient air, can induce small changes in the activity of this receptor. This phenomenon has al- ready been observed for the very sensitive COz-receptors of Lepidoptera (Bogner 1990). At concentrations up to ca. 0.2%, the C02-inhibited receptor follows changes in the order of 0.01% CO2 by modulation of spike fre- quency. At higher levels, the receptor is so strongly in- hibited that it can no longer monitor abrupt changes in concentrations. Our experiments have shown that the length of post-stimulus inhibition is concentration de- pendent. Furthermore, the reactivation burst after stim- ulus off is not only concentration dependent but also related to the length of inhibition by CO2. On the other hand, the C02-excited receptor responds weakly to small changes in CO2 concentration near ambient, but perfect- ly codes for concentration changes higher than ca. Ö. 1 %. In addition, inactivation of the C02-excited receptor even after stimulation with high concentrations OfCO2 is very fast, much faster than the post-stimulus recovery of the C02-inhibited receptor for the same CO2 stimulus. Consequently, A. variegatum possesses a CO2 percep- tion system operating with divaricate C02-excited and C02-inhibited receptors, each working most efficiently in a different but complimentary concentration range. This evidently permits the parasite to perceive the whole range OfCO2 concentrations to which it is confronted in host- finding, i.e. from ambient concentrations to the 5% level of vertebrate breath. However, since the C02-excited receptor operates through the whole range, from ambient to at least 5%, it can not evidently show at the same time a very high resolving power for small changes of con- centration around ambient. The C02-inhibited receptor compensates amply for this by being very sensitive to small CO2 shifts around ambient. The task of perceiving the whole CO2 range to which the tick is confronted is thus divided between the two receptors. The CO2 levels in the litter zone where A. variegatum lies in wait for its host vary most probably between 0,03 % to ca. 0.09% (cf. Holsher et al. 1980), well within the range of the C02-inhibited receptor. The latter may thus be used to detect small alterations in CO2 concentration and alert a resting tick to the presence of a host nearby. Then the C02-excited receptor could take over at the higher concentrations encountered during orientation to the host. Acknowledgements. We are indebted to the Hasselblad, Roche, Sandoz, and the Swiss National Science Foundation (Grant Nos. 3.609-0.87 and 31-28684.90), the Ciba-Geigy-Jubilaeums-Stiftung, and the Swiss Office for Education and Science for funding studies on tick sensory physiology at Neuchâtel. We thank Misters Bouv- ard, Rohrer and Cesari of CIBA-GEIGY Ltd., St. Aubin, Switzer- land for supplying us with adult ticks. We are grateful to Michèle Vlimant for her help and advice with the microscopy, and to Dr. Tilman Haug (Regensburg University) for his useful discussions regarding clcctrophysiology. This paper is part of the Ph.D. thesis of Pascal Steullet at the University of Neuchâtel. References Altner H, Sass H, Altner I (1977) Relationship between structure and function of antennal chemo-, hygro, and thermoreceptive sensilla in Periplaneta americana. 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J Med Entomol 12:349-351 French FE, Kline DL (1989) l-octen-3-ol, an effective attractant for Tabanidae (Diptera). J Med Entomol 26:459-461 Garcia R (1962) Carbon dioxide as an attractant for certain ticks (Acarina, Argasidae and Ixodidae). Ann Entomol Soc Am 14:605-606 Garcia R (1965) Collection of Dermacentor andersoni (Stiles) with carbon dioxide and its application in studies of Colorado tick virus. Am J Trop Med Hyg 14:1090-1093 Gillies MT, Wilkes TJ (1968) A comparison of the range of attrac- tion of animal baits and of carbon dioxide for some West African mosquitoes. Bull Entomol Res 59:441-456 Godde J (1985) Low cost storing of two electrical biosignals from DC to 20 kHz at more than 80 dB dynamic range. Pflügers Arch 403:324-327 Gödde J (1989) Vibrating glass stylets: tools for precise microsur- gery on cuticular structures. J Neurosci Methods 29:77-89 Gray JS (1985) A carbon dioxide trap for prolonged sampling of Ixodes ricinus L. populations. Exp Appi Acarol 1:35-44 Guglielmone AA, Moorhouse DE, WoIfG (1985) Attraction to carbon dioxide of unfed stages of Amblyomma triguttatum triguttatum Koch, under field conditions. Acarologia 26:123-129 Hess E, Loftus R (1984) Warm and cold receptors of two scnsilla on the foreleg tarsi of the tropical bont tick Amblyomma va- riegatum. J Comp Physiol A 155:187-195 Hess E, Vlimant M (1982) The tarsal sensory system of Amblyomma variegatum Fabricius (Ixodidae, Metastriata). I. Wall pore and terminal pore scnsilla. Rev Suisse Zool 89:713-729 Hess E, Vlimant M (1983) The tarsal sensory system of Amblyomma variegatum Fabricius (Ixodidae, Metastriata). III. Mapping of sensory hairs and evolution of the relative importance of sensory modalities during post-embryonic development. Rev Suisse Zool 90:887-897 Hess E, Vlimant M (1986) Leg sense organs of ticks. In: Sauer JR, Hair JA (eds) Morphology, physiology, and behavioural biol- ogy of ticks. Ellis Horwood, Chicester, pp 361-390 Hindley E, Merriman G (1912) The sensory perception of Argas persicus (Oken). Parasitology 5:203-216 Holsher KH, Gearhart HL, Barker RW (1980) Electrophysiological responses of three tick species to carbon dioxide in the laborato- ry and field. Ann Entomol Soc Am 73:288-292 Kellogg FE (1970) Water vapour and carbon dioxide receptors in Aedes aegypti. J Insect Physiol 16:99-108 Lees AD (1948) The sensory physiology of the sheep tick Ixodes ricinus. J Exp Biol 25:145-207 Nevill EM (1964) The role of carbon dioxide as a stimulant and attractant to the sand tampan Ornithodoros savigny (Audpuin). Onderstepoort J Vet Res 31:59-68 Norval RAI, Yunker CE1 Butler JF (1987) Field sampling of unfed adults of Amblyomma hebraeum Koch. Exp Appi Acarol 3:213-217 Norval RAI, Yunker CE, Gibson JD, Deem SLD (1988) Field sampling of unfed nymphs of Amblyomma hebraeum. Exp Appi Acarol 4:173-177 Osbrink WLA, Rust MK (1985) Cat flea (Siphonaptcra: Pulicidae): Factors influencing host-finding behavior in the laboratory. Ann Entomol Soc Am 78:29-34 Palade GE ( 1952) A study of fixation for electron microscopy. J Exp Med 95:285 Sabatini DD, Bcnsch KG, Barmett RJ (1963) Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J Cell Biol 17:19-58 Sauer JR, Hair JA, Houts MS (1974) Chemo-attraction in the lone star tick (Acarina: Ixodidae). 2. Responses to various con- centrations of CO2. Ann Entomol Soc Am 67:150-152 Sinitsina EE (1974) Electrophysiological reactions of the neurons of the Haller's organ to the odour stimuli in the tick Hyalomma asiaticum. Parazitologiya 8:223-226 Smith JJB, Mitchell BK, Rolseth BM, Whitehead AT, Alben PJ (1990) SAPID tools: microcomputer programs for analysis of multi-unit nerve recordings. Chem Senses 15:253-270 Stampili N (1987) Etude des facteurs intervenant dans la phase initiale du comportement de recherche de l'hôte, chez la tique, Amblyomma variegatum, Fabricius 1794 (Acarina: Ixodidae). Thèse Université de Neuchâtel Stange G (1974) Linear relation between stimulus concentration and primary transduction process in insect COz-rcccptors. In: Denton DA, Coghan JP (eds) Olfaction and Taste V. Pergamon Press, London, pp 207-211 Steullet P, Guerin PM (1992) Perception of breath components by the tropical bont tick Amblyomma variegatum Fabricius (Ixod- idae). II. Sulfide-reccptors. J Comp Physiol A 170:677-685 Turner DA (1971) Olfactory perception of live hosts and carbon dioxide by the tsetse fly Glossina morsitans Orientalis Vander- plank. Bull Entomol Res 61:75-96 Waladde SM, Rice MJ (1982) The sensory basis of tick feeding behaviour. In: Obenchain FD, Galun R (eds) Physiology of ticks. Pergamon Press, Oxford New York Toronto, pp 71-118 Warncs ML, Finlayson LH (1985) Responses of the stable fly, Stomoxys cakitrans (L.) (Diptera : Muscidae) to carbon dioxide and host odours. 2. Orientation. Bull Entomol Res 75:717-727 Wames ML, Finlayson LH (1986) Electroantennogram responses of the stable fly, Stomoxys cakitrans. to carbon dioxide and other odours. Physiol Entomol 11:469-473 Wilson JG, Kinzcr DR, Sauer JR, Hair JA (1972) Chcmoattraction of the lone star tick (Acarina, Ixodidae). I. Response of different developmental stages to carbon dioxide administered via traps. J Med Entomol 9:245-252 RESULTS - CHAPTER 3.2. 34 J Comp Physiol A (1992) 170:677-685 Journal of Comparative SyS* Physiology A £Ä£ © Springer-Verlag 1992 Perception of breath components by the tropical bont tick, Amblyomma variegation Fabricius (Ixodidae) II. Sulfide-receptors Pascal Steullet and Patrick M. Guerin Institulc of Zoology, University of Neuchâtcl, Chantemerle 22, CH-2007 Neuchâtel, Switzerland Accepted March 23, 1992 Summary. Wall-pore sensilla in the capsule of Haller's organ on foreleg tarsi of the tick, Amblyomma variega- tum, show multicellular responses upon stimulation with human and bovine breath. Filtering breath through char- coal removes the stimulant for some of these receptors. Analysis by gas chromatography coupled with olfactory sensillum electrophysiological recordings indicates that an ethanol extract of the breath components trapped on charcoal contains a major stimulant eluting at the same retention time as H2S. Two types of H2S-sensitive recep- tors have been identified. They are housed in separate sensilla, and are called sulfide-receptor 1 and 2. Although, both receptor types are characterized by a high sensitivity to H2S with an estimated threshold of ca. 0.1 ppb and a response range covering 5-6 log orders of magnitude, their overall response to sulfides and mercap- tans is nevertheless dissimilar. The type 1 receptor fires slightly more upon stimulations with H2S than type 2, whereas ethylmercaptan induces a stronger response from type 2, and dimethyl sulfide activates only recep- tor 2. In a bioassay, H2S tested at concentrations of ca. 0.02 ppm and 1 ppm equally arouses 60% of resting ticks. Two-thirds of these ticks quest the air with their first pair of legs, and the remainder start active search. By con- trast, H2S at ca. I ppm in a mixture with CO2 severely diminishes the locomotor stimulating effect of CO2. Key words: Tick - Host-finding - Vertebrate breath - Hydrogen sulfide - Sulfide-receptors Introduction Several investigators have demonstrated that volatiles of vertebrate origin, other than CO2, act as cues for host- finding by haematophagous anthropods. Despite this, chemical analysis of breath components has been made only in a few cases; l-octen-3-ol and acetone in cow breath are used for host-location in tsetse flies (Hall et al. 1984; Vale and Hall 1985). Furthermore, octenol and/or acetone improve attractiveness of CO2 in other blood- sucking Diptera (Cuticidae: Kline et al. 1990; Stomoxys calcitrans: Warnes and Finlayson 1985a, b; Tabanidae: French and Kline 1989). It is still not clear whether breath components other than CO2 elicit a response in ticks, although human subjects and CO2 traps attract roughly the same numbers of Amblyomma americanum in the field (Mount and Dunn 1983). Besides, l-octen-3- ol and acetone do not attract Amblyomma hebraeum (Norval et al. 1987). However, there may be other verte- brate associated volatiles which are relevant for Ambly- omma sp. Indeed, studies in this laboratory on all wall- pore sensilla on the tarsus of the foreleg of Amblyomma variegatum have revealed that some olfactory cells in Haller's organ respond to components of vertebrate odour (Steullet, unpublished). Three other sensilla in the capsule of Haller's organ also bear cells which are stim- ulated by breath (multicellular responses). One of the activated cells is a COj-excited receptor, another a CO2- inhibited receptor (Steullet and Guerin 1992), and the response of two other cells to sulfur components of breath is described here. Materials and methods Ticks and electrophysiology Experiments were undertaken for the most part with unfed Ambly- omma variegatum males, nevertheless, some unfed females were also used for comparison. Rearing methods, preparation of ticks, elec- trophysiological set-up, as well as stimulus-delivery system are already described in Steullet and Guerin (1992). i Stimulants Correspondence lo: P. Steullet Human breath. Human breath was blown via the needle connexion into the barrel of a 5-ml polypropylene syringe whose other end RESULTS - CHAPTER 3.2. 35 678 P. Stcullct and P.M. Guerin: Perception of breath components by Amblyomma II. bore a rubber stopper, and was immediately used as stimulus. Breath was collected as a rule in the morning. Bovine breath. A personal sampling pump (SKC Inc., USA) sucked air into a Tediar sampling bag (SFCC, USA) at a rate of 250 ml/min via a teflon lube placed in the mouth of a 200 kg Simmental steer held in a rearing pen at the Agricultural Research Station of Ciba- Geigy (St-Aubin, Switzerland). Breath was thus transported to the laboratory and used for stimulation 1-2 h later by venting from the Tediar bag into the barrel of 5-ml syringes which were immediately used as stimulus cartridges. Porapak conditioning and filtering human breath. Porapak Q, a porous polymer which selectively desorbs water while retaining a large spectrum of volatiles was used to collect breath-borne odours. Conditioning of Porapak Q (50-80 mesh, Milipore Corporation, USA) was carried out by: 1) heating under N2 (11/min) at 200 0C for 24 h, 2) extraction with dichloromethane (Merck, analytical grade) in a Soxhlet extractor for 24 h, and 3) drying under N2 (1 1/min) at 110 0C for 2 h. About 600 mg of conditioned Porapak Q was then packed into the barrel of a Pasteur pipette (70 mm long, 5 mm i.d.) and human breath was blown directly through this adsorbant trap for 30 s. A portion of the filtered breath was in- troduced in the barrel of a 5-ml syringe to be used as stimulus. Filtering human breath through charcoal. Commercially available charcoal air-sampling tubes (coconut-base 50/100 mg in a 6 mm OD x 70 mm long trap, SKC1 USA) were also used to collect breath-borne volatiles. Charcoal traps a wide range of volatiles like Porapak Q, but its adsorbtion capacity for small molecular weight compounds is higher. Human breath was blown through the trap for 30 s and a portion of the filtered breath was entrained in a 5-ml syringe to be used as stimulus. Hydrogen sulfide. Two methods of generating H2S vapours were employed. A certified H2S-permeation tube {Dynacal, VICI Met- Fig. 1. Representative responses of the Amblyomma variegatum breath sensillum 2 to human breath, Porapak-filtered human breath, charcoal-filtered human breath, and CO2. Filtered and unfiltered breath was diluted by a factor of 6 in the air stream conveying the stimulus to the preparation to give, for unfiltered breath, an equivalent of 0.68% CO1. Stimuli were introduced, in this ronics, USA) liberating H2S at 546 ng/min ± 2% at 30 0C was used. Different concentrations of H2S were obtained by mixing flows of charcoal-filtered air with that passing from a 500-ml gas-wash bottle containing the H2S-permeation tube held at 30 0C in a constant VC bath. Precise concentrations were introduced into the stimulus car- tridge to cover 4 log orders of magnitude. Considering the dilution which occurred in the slimulus-delivery-tube, H2S concentrations at the level of the preparation (5 mm from outlet of tube) ranged from ca. 0.Û03 ppm to ca. 2.6 ppm. Continuous or pulsed stimula- tions with H2S were made by passing charcoal-filtered air through the 500-ml flask containing the H2S-permea lion tube, and then directly into the main air stream flowing over the preparation. Air flows were controlled by voltage-pressure converters and the dura- lion of stimulation by solenoid valves. The T° increase (ca. 1 0C) which ensued in the main air stream during a stimulation had no effect on the sulfide-receptor response. The second method employed to generate H2S was the use of aqueous solutions OfNa2S. This method was particularly useful for providing H2S doses higher than that obtainable from the permea- tion tube. Ten pi aliquots of various Na2S solutions (10"s mg/10 pi to 10"! mg/10 pi H2O) were applied to filter paper strips which were then enclosed in the 5-ml syringe. After allowing 3 min for evapora- tion of H2S vapour, 2 ml of the stimulus cartridge volume were injected in 1 s into the main air stream flowing over the preparation. The quantity of H2S leaving the lower Na2S concentrations was calibrated by comparing the sulfide-receptor responses tó these solutions with those obtained with the reference H2S values from the certified permeation tube. Other sulfides. A certified ethylmercaptan permeation tube (452 ng/min at 30 0C, Dynacal, VICI Metronics, USA) was em- ployed in the same way as the H2S-permeation tube to provide a graded concentration series of ethylmercaptan. Dimethyl sulfide (>99% GC, FIuka, Switzerland) was successively diluted in paraffin oil to provide 10"' to 10-4 molar solutions. Ten pi of thesje solu- tions were applied to filter paper strips, enclosed in the barrel of a 5-ml syringe, and used as stimulus source. case, at 25 cm from the outlet of the stimulus-delivery-tube. Bold arrow, sulfide-receptor 2 (cell 4 in Fig. 2), which displays a negative going spike at the beginning of a stimulation, but then becomes biphasic. White arrow, C02-excited receptor (cell 1 in Fig. 2). Horizontal bar 1 s stimulation; vertical bar 1 mV human breath (1:6) tflp^^^ Porapak Q filtered human breath (1:6) charcoal filtered human breath (1:6) ||Tiit'imllill^ f-4f^^*-jW4 C 02 0.68% IWlillillllilll ¦ iln i ,-V.4-V RESULTS - CHAPTER 3.2. 36 P. Steullet and P.M. Guerin: Perception of breath components by Amblyomma II. 679 Other volatiles tested. The following volatiles were also tested to screen sulfide-receptor specificity: methane, CO2, ammonia, ac- etone, 3-pentanone, 4-heplanone, hexanal, pcntanol, l-octen-3-ol, 1-oclen, propionic acid, n-butyric acid, isobutyric acid, n-valcric acid, iso-valcric acid, heptanoic acid, L-lactic acid, y-butyrolactone, y-valerolactone, y-caprolactone (all vertebrate odours), nonanoic acid, 2-nitrophenol, 2,6-dich loro phenol, methylsalicylate (tick pheromone components), dichloromethane and distilled water (sol- vents). Chemical purity of trade products was > 99% as indicated by GC. Chemicals were dissolved in analytical grade CH2Cl2 or in distilled water, depending on their nature, at IO-3 and 10~2 M, and prepared for stimulation as for dimethyl sulfide (above). Methane from the mains was entrained in the barrel of a 5-ml syringe for stimulation; for CO2 stimulation see Steullet and Guerin (1992). A concentrated aqueous solution of 35% NH4OH was diluted 10 and 100 times in distilled H2O and used as the ammonia stimulus source. Coupled gas chromatography - single unit recording Human breath was blown through a coconut-charcoal trap for 15 min. The trap was then eluted with either dichloromethane or ethanol (Merck, analytical grade). From the first drop of the eluate, 1.5 ul was immediately injected on-co!umn onto a DBWAX fused silica capillary gas chromatography column (30 m, 0.32 mm i.d., 1 1 V. 1 1 human breath 1 1 1 1 1 1 1 1 J \^A^J{\rJf^ B charcoal-filtered human breath 1 1 1 1 1? 1 1 l|3 ] ?j 1 JJ 1 ? 1 13 1 ? * ? 1 a? 1 ? 1 3 1 3 « 3 1 » 1 3 I3 13 Ip 3l 3] 3 1 3 S1 3 3 1. 31 Fig. 2A, B. Detail of recordings from breath sensillum 2 illustrated in Fig. I. A Response to human breath. B Response to human breath filtered through charcoal. Spike discrimination was made by eye. Note that unfiltcred human breath elicits higher firing rates than charcoal-filtered human breath in cell I (C02-excited receptor, white arrow in Fig. I) and cell 4 (sulfide-receptor 2, bold arrow in Fig. I). Note also that spike 4 has a strong negative phase at the lower frequency in B, but becomes biphasic at the higher frequency in A. Cell 3, in this particular case, is slightly more excited in B than in A, but no statistical difference exists as a rule (Fig. 3). Cell 2 fires at A very low frequency. It is absent from this example. Enumera- tion corresponds to cell numbers in Fig. 3, and underlined spikes are overlapping events. Horizontal bars ÌO0 ms; vertical bars I mV 0.25 urn film thickness, G&W Scientific, USA) with H2 (0.5 m/s) as carrier gas; oven temperature: 30 0C for 5 min, then programmed at IO cC/min to 230 0C. The column effluent was divided with a glass Y-piece splitter in the ratio 2:1 over, respectively, the flame ionisation detector (FID) and the biological detector. The latter consisted of an electrophysiological preparation of a capsular sen- sillum known to have cells responding to breath. An air stream, maintained at ca. 80% RH and 22 ± I "C in a water-jacketed tube (3 mm i.d.), swept a third of the column effluent from the heated (250 0C) transfer line of the Chromatograph to the tick preparation at a speed of 1.5 m/s. The outlet of the tube was 5 mm from the tick tarsus. Column effluent was thus simultaneously monitored by both the FID detector and sensillum in order to locate any electrophysiol- ogically active component in (he extract (Wadhams I982). A 1.5 pi aliquot of the first solvent drop eluting from an unused charcoal trap served as the blank control. A standard H1S stimulus was obtained by injection of 10 pi of headspace from an aqueous solu- tion OfNa2S (1 g/IO ml) moderately acidified with few drops of I N HCI. All spikes were sorted from noise with a discriminator level incorporated in the amplifier (UN-03 Syntech, The Netherlands) and the frequency was converted into a voltage. This converted signal, the receptor potential and FID responses were simultaneous- ly printed on a chart recorder and stored on video tapes (Steullet and Guerin 1992). Play back of parts of the recording where an increase in spike frequency occurred was necessary to identify the responding ccll(s). Discrimination for different spike types was made by eye (as described in Steullet and Guerin 1992). Identifica- tion of a stimulant in the extract was based on 1) the response of the biological detector to both a component of the extract and the standard eluting at the same retention time on the capillarycolumn, and 2) on the type of cell activated. Behavioral bioassay A behavioral bioassay based on the activity level of ticks was developed. During first steps in host-searching by adult A. variega- tum, 3 distinct phases are observed: 1) resting, where fully inactive ticks keep their legs folded under their body; 2) questing, where 80 70 60 50 OT J« 40 3 CL c 30 20 to 0 O O O OQO O cell! cell2 cell3 cell4 Fig. 3. Responses of cells of breath sensillum 2 in the capsule of Haller's organ of 6 different male A. uariegatum to human breath, charcoal-filtered human breath, and to CO2 at a concentration equivalent to the level in unfiltered breath. Stimuli were introduced into the main air stream at 25 cm from the outlet of the stimulus- deli very-tube. Cell frequencies were determined on spike trains of 1 s from the beginning of the response. For each cell, blocks labelled a and b are significantly different from one another (i><0.05) (Wilcoxon's paired comparison test). Bars associated with each block arc standard deviations. Sec also Fig. 2. ¦ carbon dioxide (breath cone), ¦ human breath, D charcoal-filtered human breath RESULTS - CHAPTER 3.2. 37 680 P. Stcullet and P.M. Guerin: Perception of breath components by Ambtyomma II. v-h-T^swamj. ß : ^ajL-w^^^^ kL ______ -*---------1 D ^--------1 L~y>TYyrr-ri*-~*V*^^ ,vaJ^^**^^^ spike frequency KN 30 30 160 TX Flg. 4A-E. Analysis of human breath by gas chromatography cou- pled with breath sehsillum 2 electrophysiology recordings from the capsule of Haller's organ of a male A. variegatum. A, B, and C spike activity of breath sensillum 2 (upper trace, frequency to voltage converted signal) recorded simultaneously with chromatograra (lower trace obtained with a flame ionisation detector, FID). A Breath components adsorbed on charcoal and extracted with etha- nol, B blank control, i.e. an ethanol extract of an unused charcoal trap, and C 10 ul of the headspace over an acidified aqueous solution of Na2S which generates H2S. Note that the breath com- ponent, which causes an increase in spike frequency of the breath sensillum in A, clutes at the same retention time as H2S (negative FID response) in C, where a corresponding increase in spike fre- quency is also observable. Column: DBWAX (J & W Scientific, USA) high resolution fused silica capillary column (30 m, 0.32 mm i.d., 0.25 urn film thickness) was temperature programmed after 5 min at 30 CC at 10 °C/min to 230 0C. Temperature scale same for A, B, and C D Expanded trace of the breath sensillum 2 response, before and during elution of the active breath component in A. E Expanded trace of the breath sensillum 2 response, before and during elution of H2S in C. For both D and E, horizontal bar is 1 s; vertical bar 1 mV. Bold arrow: sulfide-rcceptor 2 (cell 4 in Figs. 2 and 3). Note how the spike of this receptor changes sign on stimula- tion in D, and remained biphasic after strong stimulation. This explains why in E (experiment made after D) the sulfide-receptor 2 is already biphasic even before arrival of H1S at the sensilluni ticks raise at least one of the forelegs ; 3) walking, where licks finally rise to their feet and begin locomotion. Groups of 25 male A. va- riegatum (7-8 months old, all fed on the same steers) were placed in 100-ml glass gas-wash bottles. A charcoal-filtered and humidified air stream (8096 RH, 23 ±2 "C, 3.3 ml/s) passed continuously through each bottle. Blanks or stimuli, contained in 10-ml syringes, were injected at 0.8 ml/s for 10 s and thus diluted by a factor of 5 in the humidified air stream before entering the bottle at 2 cm from the floor, where most of ticks lay. Cigarette smoke indicated that the stimuli were distributed throughout the bottle for ca. 40 s before being flushed out. Only one stimulus was tested per day on the same group of ticks. Ticks had thus a day to return to the resting position (most do so < I h after stimulation). On successive days, a series of stimulations was applied to each group of ticks in a different sequence, to avoid any influence of the order in which the stimuli were tested. The number of resting, questing, and walking ticks was recorded just before and after stimulation. Data concerning the same stimulus were pooled and compared by Chi-square with data for other stimuli. The following stimuli enclosed in 10-ml syringes were tested: 1) H1S produced by 10 ul of an aqueous solution of Na2S at either 10"4 or 10~3 mg/10 ul deposited on a filter paper strip and enclosed in the syringe in an atmosphere of either N2, 5¾ CO21Or 1% CO1; 2) dimethyl sulfide (IO ul of a 10"* M solution in paraffin oil) enclosed in a syringe with N2; 3) 5% CO2; 4) 1% CO2; 5) human breath collected in the morning; N2 (blank control). Results Breath-sensitive cells Three functionally different types of sensilla, whose cell activity was captured with different recording electrode RESULTS - CHAPTER 3.2. 38 P. Stcullct and P.M. Guerin: Perception of breath components by Amblyomma II. 681 human breath (1:6) hmm^J^j^^ Porapak Q filtered human breath (1:6) , 44#4fU4(44^ charcoal filtered human breath (1:6) Fig. 5. Representative responses of sulfide-receptor 1 (bold arrow) of breath sensillum 1 in the capsule of Haller's organ of a male A. variegation to human breath, Porapak-filtered human breath, and charcoal-filtered human breath. Filtered and unfiltered breath samples were diluted by a factor of 6 in the air stream conveying the stimulus to the preparation. Stimuli were introduced at 25 cm from the outlet of the stimulus-delivery-tube. Horizontal bar 1 s stimulation; vertical bar 1 mV orientations within the capsule, carry cells responding to breath (Steullet and Guerin 1992). For convenience, these sensilla types are termed here breath sensillum 1, 2, and 3. As previously demonstrated, breath sensillum 2 has a COa-excited receptor and breath sensillum 3 a C02-inhibited receptor (Steullet and Guerin 1992). Re- sults described in this paper deal with 2 other cells, respec- tively found in breath sensilla 1 and 2, which were ac- tivated by human and bovine breath. These results are based on 37 recordings from breath sensillum type 1 and 65 recordings from breath sensillum type 2 from different ticks. For practical reasons, most experiments were un- dertaken with human breath. Responses of these cells to breath strongly decreased over the working day, suggest- ing the active component(s) in human breath was much more prevalent in the morning. Sulfide-sensitive cell in breath sensillum 2. In breath sen- sillum 2, Porapak-filtered breath elicited a multicellular response, involving 4 different cells, which was hardly different from that produced on stimulation with human breath. By contrast, the pattern of cell activity induced by charcoal-filtered breath was much simpler (Fig. 1 and details in Fig. 2). Recordings from sensilla of 6 different males indicated that cells 1 and 4 of this sensillum (Figs. 1, 2, and 3) were significantly less activated with char- coal-filtered breath than with unfiltered breath (P < 0.05, Wilcoxon's paired comparison test), whereas no dif- ference in the response of cell 3 was observed (Fig. 3). Cell 1 is the C02-excited receptor described in Steullet and Guerin (1992). A slight retention OfCO2 on charcoal is responsible for the weaker response of this cell to charcoal-filtered breath. On the other hand, the breath component activating cell 4 was not adsorbed by Por- apak, but was retained on charcoal. But even on the latter, some breakthrough occurred as evidenced by the fact that air displaced from the charcoal trap, through which breath had been blown for a few minutes, excited cell 4. This suggested that the active breath component was very volatile. A dichloromethane extract of breath components ad- sorbed on charcoal did not stimulate cell 4, but an etha- nol extract did so strongly. In coupled gas chromato- graphy and breath sensillum 2 recordings, one early eluting component of the latter extract elicited a response as evidenced by an increase in cellular activity (Fig. 4A) and a clear receptor potential. This active component had a retention time of 1 min, eluting before the solvent. A careful examination of the associated spikes establish- ed that this response was due to a strong activation of cell 4 (bold arrow in Fig. 4D), characterized by a quite small spike amplitude. An extract of an unused charcoal trap did not stimulate any cell of breath sensillum 2 (Fig. 4B). The retention time of the active breath component matched that OfH2S, which also activated cell 4 (Fig. 4C, bold arrow in Fig. 4E). Figure 6 illustrates the response of this sulfide-receptor to increasing concentrations of H2S. Sulfide-sensitive cell in breath sensillum 1. Unfiltered or Porapak-filtered breath was equally effective as a stimu- lant for a cell in breath sensillum I, whereas charcoal- filtered breath failed to stimulate the same cell in this sensillum (bold arrow in Fig. 5). However, this cell re- sponded strongly to an ethanol extract of the charcoal trap. Like cell 4 of breath sensillum 2, this cell also responded selectively to H2S. Properties and specificity of the two sulfide-sensitive cells Both the activated cell of breath sensillum 1 (bold arrow in Fig. 5) and cell 4 of breath sensillum 2 (bold arrow in Fig. 1) are sulfide-sensitive. Nevertheless, they differ in important respects and will be termed sulfide-receptor RESULTS-CHAPTER 3.2. 39 682 P. Stcullet and P.M. Guerin: Perception of brcaih components by Amblyomma II. *3 H2S 0.003 ppm ^e3 H2S 0.03 ppm J .^J4t»u*"l»-*'4^**[4uUi^ H2S 0.3 ppm ^ i.A \a{ ». ^.1^^14^-1^***!*^ #3 H2S 2.6 ppm ?4 n—I—i—h-H—r-t ......**^^iinuu^i(uit;^L^^m^kamtuthvw^4^;m;a4W^uiutw^u^u-uu^uiu^^4U^*' A -f »)• A A iA A A> 'a * f Û. *.' & * h -Tja). sHJPt K4 A. / l_ ¦ . 1 t 160 UO 120 <2 100 « (i) ^o 80 3 Q. 40 20 I- oL-V^ -5 -4 -3 -2 -1 Na2S amounts per cartridge log [mgl Fig. 7. Relationship between dose OfH2S and the phasic part of the response of the sulfide-receptor 2 of male A. variegation obtained by two methods of stimulation: a) graded stimulus onset was ob- tained by introducing the H2S vapour 25 cm from the outlet of the stimulus-delivcry-tube to the preparation, b) much sharper stimulus onset was obtained by introducing H2S vapour 3 cm from the outlet of the stimulus-delivery-tube. Response magnitude was determined for the period when spike frequency was maximal: for curve a for 400 ms after 200 ms from the beginning of the response, and for b for the first 200 ms of the response. In curve a, hollow triangles (n= 12) refer to responses obtained with aqueous solutions OfNa1S as stimulus source (bottom abscissa), and hollow circles (n=4) arc responses obtained with the certified H2S-penneation tube which provided a maximal concentration of 3 ppm (top abscissa). Aque- ous solutions of Na2S as stimulus source were used for curve b (filled triangles) (n=4). The amounts of H2S produced by the aqueous solutions of Na2S was calibrated with the responses of the same receptors to H2S from the certified permeation tube. Star (lower left) is the response to H2S free air (n=12). Trend lines connect mean values rate of the type I was significantly higher than that of the type 2 over the concentration range tested, suggesting that type 1 is more sensitive (Table 1). On the other hand, ethylmercaptan as well as dimethyl sulfide elicited a sig- nificantly stronger response in the type 2 than in the type 1 over the range of concentrations tested; the firing rate of the type 1 was not modified significantly by increasing dimethyl sulfide concentration (Table 1). None of the other synthetic volatiles listed in Materials and methods activated either of these two sulfide-receptors. Although, the number of animals tested were sufficient to detect only large differences in sulfide-receptor responses be- tween males and females, these did not appear. Behavioral bioassay Either 0.02 ppm or 1 ppm H2S blown into the bottle containing the ticks aroused ca. 60% from rest, ap- proximately 40% raised only their forelegs, and ca. 20% began walking (Table 2). A slightly weaker behavioral response, though not significantly different, was recorded for 10~2 M dimethyl sulfide (Table 2). Behavior triggered by 104 or 2000 ppm CO2 (1 % or 0.2%, respectively) in this bioassay was different: although about 60% of ticks were likewise aroused from rest with CO2, only ca. 10% remained with raised forelegs, but > 50% started to walk Table 2. Behavioral response of A. variegation males to CO2, H2S, mixtures of these two volatiles, dimethyl sulfide, and human breath (sampled early in.the morning). Activity states observed (numbers of resting, questing, and walking ticks) following each stimulus are compared pairwise, and are assigned different letters when signifi- cantly different (P < 0.05, Chi-square test). Data are presented here in percentages to make results more comprehensible. H3S was generated by aqueous solutions of Na2S at either IO"4 or 10~3 mg/10 ul, generating vapours in the stimulus cartridge correspond- ing to ca. 0.1 ppm and ca. 5 ppm H2S, respectively. Before arriving in the bottle containing the ticks, stimuli were diluted by a factor of 5 in the main humidified air stream Stimulus in the cartridge n % ticks % licks % ticks activated questing walking Blank 300 18 17 1 a CO1 5% 200 63 7 56 b CO2 1% 100 62 10 52 be CO2 5%+ H1S 0.1 ppm 50 72 10 62 be C025% + H2S5ppm 50 58 18 40 cd CO2 196 + H2S 0.1 ppm 50 50 22 28 de CO2 1% + H2S 5 ppm 50 78 52 26 f H2S 0.1 ppm 100 61 41 20 efg H2S 5 ppm 100 53 38 15 eg Dimethyl sulfide IO"2 M 50 48 38 10 g Human breath 50 66 54 12 ig Fig. 8. Representative response of a sulfide- receptor 2 in the capsule of Haller's organ of a male A. variegation to continuous (left) and repeated stimulation (right) with ca. 0.1 ppm H2S. The traces are the frequency to voltage converted response of the sulfide-receptor which could be sorted with a window discrim- inator from other spikes because of its small amplitude. Horizontal bar, stimulation. Re- sponses illustrated here arc typical for both types of sulfide-receptors present in the cap- sule, and were highly reproducible RESULTS - CHAPTER 3.2. 41 684 P. Steullet and P.M. Gucrin: Perception of breath components by Amblyomma IL (Table 2). In mixtures, CO2 and H2S appeared to act antagonistically on A. variegatum behavior. Depending upon the relative concentrations of CO2 and H2S in the mixture, activated ticks tended to either raise their forelegs or begin walking. With CO2 at 10* ppm and H2S at ca. 0.02 ppm in the mixture, ticks tended to respond as for stimulation with CO2 alone. By contrast, with a mixtures of CO2 at 2000 ppm and H2S at ca. 1 or even as low as 0.02 ppm, ticks responded as for stimulation with H2S alone (Table 2). Finally, human breath collect- ed early in the morning, when it normally strongly ac- tivated sulfide-sensitive cells, evoked a similar response to that of H2S alone, or to that of a CO2 and H2S mixture with a relatively high concentration of H2S (Table 2). Discussion Sulfide-sensitive cells responding to breath have been found in two wall-pore sensilla located in the capsule of Haller's organ of adult A. variegatum. Both cells are very sensitive to H2S, but the type 1 is slightly more sensitive. The H2S dose-response covers a wide range of concentra- tions, from < 0.1 ppb (estimated threshold) to > 10 ppm. The phasic part of the response to H2S is significantly different depending on whether the stimulus is in- troduced at 25 cm or 3 cm from the outlet of the stimulus- delivery-tube and proves, as expected, to depend on the sharpness of the stimulus onset. The log/log H2S dose and phasic part of the response relation does not have a steep slope: 0.21 with a sharp stimulus onset and 0.11 with a graded stimulus onset. Interestingly, the subjective H2S dose-response relation for humans also has a similar slope (0.11), and the absolute threshold is ca. 10"3 ppm (Lindvall 1977). This may suggest equivalent primary transduction processes for H2S perception in both ticks and humans, an equally high sensitivity most probably imposed by the extreme toxicity of H2S for cucaryotypes. The concentration of sulfides in human breath, produced in situ by anaerobic bacteria living on decom- posing food remains and saliva in the buccal cavity, is highly variable during the day, ranging from ca. 0.007 ppm to 0.7 ppm for H2S. Peak levels depend on prolonged periods of reduced saliva flow and abstinence from food or liquid uptake (Tonzetich 1977). Thus, sul- fur compound concentrations are generally much higher early in the morning than later in the day. This cor- roborates our findings that human breath collected from the mouth in the morning stimulated the sulfide-sensitive cells much more than the same collected after lunch. Ruminants, the commonest hosts of adult A. variegatum, produce large quantities of H2S, with concentrations up to about 100 ppm in the rumen (Hungate 1966). The amount of H2S expelled from the rumen will then vary according to the frequency of eructation events, which is in turn linked to the digestion state. The tick can most probably detect H2S liberated by an eructating host from significant distances as sul fide-receptors are still sensitive to H2S levels 106 times lower than that found in the rumen. Concentrations OfH2S expired from vertebrates cover more than 3-4 log orders of magnitude, a range within which the su I fide-receptors of A. variegatum can easily discriminate (<0.1 ppb to > 10 ppm). This contrasts with the narrow sensitivity range of the C02-excited receptor which can only discriminate over 2-3 log con- centrations OfCO2, or just 1-2 log concentrations for the C02-inhibited receptor (Steullet and Guerin 1992). Moreover, sulfide-receptors are comparatively much more sensitive than the C02-receptors (estimated thresh- old for the sulfide-receptors: <0.I ppb H2S, against ca. 10-20 ppm CO2 for the C02-inhibited receptor, and ca. 50-100 ppm CO2 for the C02-excited receptor). Thus, both the C02-receptors and the sulfide-receptors are adapted for the perception of CO2 and H2S levels given off by vertebrates. Stimulation with low molecular weight sulfur com- pounds reveals that the specificity of the two sulfide- receptors differs. The type I responds significantly stron- ger to H2S, but significantly weaker to ethylmercaptan than the type 2. On the other hand, dimethyl sulfide elicited a strong response in the type 2, but not at all in the type 1. Thus, replacement of one or both of the hydrogens on the sulfur atom by another group (e.g. methyl- or ethyl-) alters perception by sulfide-receptor 1. Although many sulfur compounds other than H2S have been identified in human breath such as ethyl-3-mercap- toproprionate, methyl-n-propyl sulfide, n-hexylmercap- tan, dimethyl sulfide, methyl sulfide, and di-tert-butyl- disulfide (Krotoszynski et al. 1977; Tonzetich 1977), no further active breath component other than H2S was found for either of the sulfide-receptors during gas chro- matography coupled with breath sensillum recordings. Concentrations of sulfides other than H2S (e.g. dimethyl sulfide) in the breath extracts tested here might have been too low to be detected. Nevertheless, presence of two types of receptors which differ in their specificity suggests that the tick may discriminate for various sulfur compounds. These products abound not only in vertebrate breath but also in general vertebrate body odours (O'Connell et al. 1979; Natynczuk et al. 1989), and a response of these sulfide-receptors to cotton pads impregnated with hum- an axillary secretions was recorded (Steullet, unpublish- ed). Such receptors could provide the tick with specific information about hosts and possibly predilection sites. Little is known about sulfur compound perception in arthropods. Some carrion beetles (Necrophorus sp.) bear carrion sensilla excited by a wide variety of volatiles including H2S and butylmercaptan (Waldow 1973). On the other hand, some phytophagous insects are attracted by disulfides as well as by thiosulfinates and thiosul- fonates (Al Rouz and Thibout 1988; Auger et al. 1989a, b). However, H2S has never figured to date among the host-finding cues listed for haematophagous arthropods. Glossina pallidìpes showed no electroantennogram re- sponse to H2S in this laboratory, but recordings in the capsule of Haller's organ of the camel tick, Hyalomma dromedarii, provided preliminary evidence for the presence of at least one sulfide-receptor. Kneidel (1984) observed that some American dog ticks, Dermacentor variabilis, were attracted by carrion. This behavior could possibly be mediated by sulfides and mercaptans. In A. variegatum, H2S as well as dimethyl sulfide seem to act in the arousal phase of host-finding, i.e. ticks raise RESULTS - CHAPTER 3.2. 42 P. Stcultct and P.M. Guerin: Perception of breath components by Ambtyomma II. 685 their forelegs in the air but few start walking. Thus, unlike CO2, H2S cannot be considered as a strong loco- motor stimulant. In fact, H2S and CO2 act antagonisti- cally with regard to activation of A. variegatum. Arousal depends greatly on the relative concentration of H2S in the mixture. A high amount of the latter diminishes the locomotor stimulant effect of CO2, whereas a smaller quantity of H2S does not very much alter the response of A. variegatum to CO2. Human breath sampled early in the morning strongly stimulates sulfide-receptors, but does not initiate the same level of locomotion as an equivalent concentration of CO2 alone. Sulfides as well as other components of vertebrate body odour perceived by the tick (as indicated by gas chromatography coupled to recordings from other olfactory sensilla, Steullet, in preparation) could subsequently intervene as cues bes- towing a certain host specificity on A. variegatum. Acknowledgements. We are indebted to the Hasselblad, Roche, Sandoz, the Swiss National Science Foundation (Grants Nos. 3.609-0.87 and 31-28684.90), the Ciba-Geigy-Jubilaeums-Stiftung, and the Swiss Office for Education and Science for funding studies on lick sensory physiology at Neuchâtel. We thank Misters Bouv- ard, Rohrer and Cesari of CIBA-GEIGY Ltd., St. Aubin, Switzer- land for supplying us with adult ticks. This paper is part of the Ph.D. thesis of Pascal Steullet at the University of Neuchâtel. References Al Roux H, Thibout E (1988) Analyse en olfactomètre de l'attrac- tion des larves A" Acrolepiopsis assecteîh par des substances allélochimiques. Entomol Exp Appi 47:231-237 Auger J, Lecomte C, Paris J, Thibout E (1989a) Identification of leek-moth and dîamondback-moth frass volatiles that stimulate parasitoid, Diadromus puichellus. J Chem Eco! 15:1391-1398 Auger J, Lecomte C, Thibout E (1989b) Leek odor analysis by gas chromatography and identification of the most active substance for the leek moth, Acrolepiopsis assectella. J Chem Ecol IS:1847-1854 French FE, Kline DL (1989) l-octen-3-ol, an effective attractant for Tabanidae (Diptera). J Med Entomol 26:459-461 Hall DR, Beevor PS, Cork A, Nesbitt BF, Vale GA (1984) 1-octen- 3-ol, a potent olfactory stimulant and attractant for tsetse is- olated from cattle odours. Insect Sci Applic 5:335-339 Hungate RE (1966) The rumen and its microbes. Academic Press, New York Kline DL, Takken W, Wood JR, Carlson DA (1990) Field studies on the potential of butanone, carbon dioxide, honey extract, I-octen-3-ol, L-lactic acid and phenols as attractants for mos- quitoes. Med Vet Entomol 4:383-391 Kneidel KA (1984) Carrion as an attractant to the American dog tick, Dermacentor variabilis (Say). J NY Entomol Soc 92:405-406 Krotoszynski B, Gabriel G, O'Nci! J, Claudio MPA (1977) Charac- terization of human expired air: A promising investigative and diagnostic technique. J Chrom Sci 15:239-244 Lindvall T (1977) Perception of composite odorous air polluants. In: Le Magnen J, Macleod P (eds) Olfaction and taste VI. Information Retricvial, London Washington, pp 449-458 Mount GA, Dunn JE (1983) Economic thresholds for Lone Star Ticks (Acari: Ixodidac) in recreational areas based on a rela- tionship between CO2 and human subject sampling. J Econ Entomol 76:327-329 Natynczuk S, Bradshaw JWS, Macdonald DW (1989) Chemical constituents of the anal sacs of domestic dogs. Biochcm Syst Ecol 17:83-87 Norval RAI, YunkcrCE, Butler JF (1987) Field sampling of unfed adults of Amblyomma hebraeum Koch. Exp Appi Acarol 3:213-217 O'Connell RJ, Singer AG, Pfaffmann C, Agosta WC (1979) Phero- mones of hamster vaginal discharge. Attraction to femtogram amounts of dimethyl disulfide and to mixtures of volatile com- ponents. J Chem Ecol 5:575-585 Steullet P, Guerin PM (1992) Perception of breath components by the tropical bont tick Amblyomma variegatum Fabricius (Ixodi- dae). I. Œ^-excited and CO2- inhibited receptors. J Comp Physiol A 170:665-676 Tonzetich J (1977) Production and origin of oral malodor: a review of mechanisms and methods of analysis. J Periodontology 48:13-20 Vale GA, Hall DR (1985) The role of l-octen-3-ol, acetone and carbon dioxide in the attraction of tsetse flies, Glossina spp. (Diptera: GIossinidae) to ox odour. Bull Entomol Res 75:209-217 Wadhams LJ (1982) Coupled gas chromatography-single cell re- cording: a new technique for use in the analysis of insect phero- mones. Z Naturforsch 37C: 947-952 Waldow U (1973) Elektrophysiologie eines neuen Aasgeruchrezep- tors und seine Bedeutung für das Verhalten des Totengräbers (Necrophorus). J Comp Physiol 83:415-424 Warnes ML, Finlayson LH (1985a) Responses of the stable fly, Stomoxys calcitrans (L) (Diptera Muscidae) to carbon dioxide and host odours 1. Activation. Bull Entomol Res 75:519-527 Warnes ML, Finlayson LH (1985b) Responses of the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae) to carbon dioxide and host odours. 2. Orientation. Bull Entomol Res 75:717-727 RESULTS - CHAPTER 3.3. 43 J Comp Physiol A (1993) || ||| IU Journal of Comparative Physiology A © Springer-Verlag 1993 Identification of vertebrate volatiles stimulating olfactory receptors on tarsus I of the tick Amblyomma variegatum Fabricius (Ixodidae) I. Receptors within the Haller's organ capsule P. Steullet, P.M. Guerin Institute of Zoology, University of NeuchäteL Chantemerle 22, CH-2000 NeuchäteL Switzerland Accepted: 30 July 1993 Abstract Gas chromatography-coupled electrophysio- logical recordings (GC-EL) from olfactory sensilla within the capsule of Haller's organ of the tick Amblyomma var- iegatum indicate the presence of a number of stimulants in rabbit and bovine odours, and in steer skin wash. Some of these stimulants were fully identified by gas chromatography-mass spectrometry analysis and by matching electrophysiological activity of synthetic ana- logues as: I) hexanal, 2-heptenal, nonanal, furfural, benz- aldehyde, and 2-hydroxybenzaIdehyde (in all extracts); 2) heptanal, 2-, 3-, and 4-methylbenzaldehyde, and y- valerolactone (only in bovine and rabbit odour). Careful examination of the electrophysiological responses permit characterization of 6 receptor types: 1) a benzaldehyde receptor, 2) a 2-hydroxybenzaldehyde receptor, 3) three types of receptors responding differently to aliphatic aldehydes, and 4) a lactone receptor. Key words: Tick - Haller's organ - Olfactory receptors - Benzaldehyde - 2-HydroxybenzaIdehyde - Aliphatic aldehydes - v-Valerolactone Introduction Adults of the tropical bont tick, Amblyomma variegatum (Acari. Ixodidae), lie in wait in the litter zone for hosts such as domestic and wild bovidae. The presence of a vertebrate in the vicinity arouses adults of this tick spe- cies to initiate active search on the ground in order to locate the host. At the end of the dry season, males of A. variegatum are first to find a suitable host, and feed for few days before emitting an aggregation-attachment pheromone (Schoeni et al. 1984), which in turn enhances attractivity of the host for conspecifics (Norval et al. Abbreviations: GC-EL gas chromatography-coupled electrophysi- ological recording; GC-MS, gas chromatography-coupled mass spectrometry Correspondence to: P.M. Guerin 1989; Barré et al. 1991). This favours meeting of the sexes on the host. While the aggregation-attachment pheromone together with host odour seems crucial for host-seeking and attachment by females (Barré I989; Barré et al. 1991), host odour alone is important for infes- tation of the host by pioneer males. This paper deals with olfactory receptors housed in wall-pore single-walled sensilla within the capsule of Haller's organ on the tarsus of the leg pair I, considered to contain some of the main host-odour receptors in ticks. This supposition was confirmed by the behavioural bioassay of Lees (1948) on Ixodes ricinus, and in electro- physiology experiments in which mouse odour was used to stimulate capsule receptors in Hyalomma asiaticum (Sinitsina 1974). In addition, breath components CO2 and H2S have been clearly identified as olfactory stimulants for receptors in the capsule of Haller's organ of A. varie- gatum (Steullet and Guerin 1992a, b). The present study aims to extend our knowledge on other olfactory recep- tors (specificity spectrum) responding to host odour with- in wall-pore single-walled sensilla of the Haller's organ in this tick species. Gas chromatography-coupled electro- physiology recordings of host-odour receptors are then employed to isolate active constituents in vertebrate odour concentrates. Materials and methods Tick rearing. A. variegatum, originating from the Ivory Coast (Adiopodoumé), have been reared since 1981 at the Agricultural Research Centre of Ciba-Geigy Ltd. (SL Aubin, Switzerland). All stages (immature and adutt) are fed on Simmental calves at 22 to 24°C and then kept under constant darkness during moult at 28°C/ 80-90% RH. Unfed males foreseen for these experiments were maintained in an environmental cabinet: 1Oh light at 25°C/85% RH, 1Oh darkness al 18°C/95% RH separated by 2 h dusk and dawn periods. Electro physiology. Unfed male A. variegatum (under 7 months old) were immobilized on a perspex holder with double-sided sticky tape. Pedal nerves of the anterior leg pair were destroyed by pinch- ing the coxa with fine forceps to prevent muscle activity during RESULTS - CHAPTER 3.3. 44 electrophysiological recordings. The narrow opening of lhe capsule (a slit across dorsal side of the tarsus 50 urn long and 5 urn wide) was enlarged to provide better access to the 7 wall-pore sensilla within by using a piece of razor blade mounted on a Leitz microma- nipulator. Recordings from olfactory receptors were accomplished with glass electrodes connected to a high-input impedance preamplifier and an AC/DC amplifier (UN-03, Syntech, The Netherlands). The reference electrode, filled with 0.2 M NaQ, was inserted into the coxa of one of the anterior legs, whereas the recording electrode (tip diameter <5 urn), filled with 0.2 M KCl and 1% polyvinylpyrro- lidon 90 K (FIuka, Switzerland), was mounted on a Leitz microma- nipulator and gently introduced into the dissected capsule until cell activity was captured. Contact between the electrode tip and the pore-wall of a sensillum was sufficient to capture cell activity. Recordings from different sensilla within the capsule were made by varying the orientation of the recording electrode in the capsule, Cell activity could thus be consistently recorded at 6 distinct loca- tions (Fig. 1). AC and DC signals were stored on video tapes as in Steullet and Guerin (1992a). AC signals were also fed into a IBM- compatible computer and visually analysed using the view option of the spike analysis programme SAPID (Smith et al. 1990), and dis- played on paper with a laser printer. Stimulus delivery. Air scrubbed through charcoal and silicagel, and humidified to 80% RH at 220C ± I0C passed continuously at 40 cm/s over the preparation from a 8 mm diameter glass tube, the outlet of which was about 10 mm from the tarsus. Stimulation was achieved by applying a charcoal-filtered air stream to a 5-ml polypropylene syringe containing the stimulus. A solenoid valve permitted displacement of 2 ml of the syringe content in 1 s into the humidified air stream through a septum-covered hole in the glass tube at 3 cm from its outlet. To prevent changes in air flow during stimulation, a solenoid-controlled charcoal-filtered air flow (2 ml/s) was delivered continuously through a blank syringe into the humid- ified air stream during stimulus off. Stimulations followed at 3 min intervals. Different concentrates of host odours and the following synthet- ic chemicals were at first used to study the specificity of receptors located in the different parts of the capsule: ammonia (3.5% and 0.35% NH4OH in distilled H2O), acetone (10_i M and lö"2 M in distilled H2O), 3-penlanone, 4-heptanone, y-butyrolactone, y- valerolactone, 6-caprolactone, pentanol, l-octen-3-ol, propanoic acid, 2-methylpropanoic acid, butanoic acid, 3-methylbutanoic acid, pentanoic acid, heptanoic acid, L-Iactic acid, and 4- methylphenol (all vertebrate-associated volatiles): nonanoic acid, 2-nitrophenol, 2,6-dichlorophenol and methylsalicylate (tick pheromone components); and 1-octene, octylamine, hexyl acetate (others); dichloromelhane and distilled H1O (solvent blanks). Ex- cept for ammonia and acetone, all these chemicals ( > 98% pure as indicated by GC) were dissolved in dichloromethane (Merck, ana- lytical grade) and tested at 10"J and 10-2 M dilutions (levels normal- ly evoking clear responses in most responsive receptors). When a receptor responded to a tested chemical, graded dilutions from 10_i to 10,; M were delivered to the preparation to determine a dose-re- sponse curve. A 10 pi aliquot of the stimulus solution was deposited on a piece of filter paper and placed in the stimulus cartridge after evaporation of the organic solvent. Separate cartridges were em- ployed for each stimulus and each concentration. Each cartridge was only used once. Three min were arbitrarily allowed for stimulus evaporation inside the syringes prior to delivering the volatile to the preparation. CH4 (neat from the mains) and CO2 (from a gas cylin- der of 5% COj/95% O1) were also tested; stimulus syringes were then directly filled with these gases. Host-odour stimuii. Human breath, human axillary secretion, and extracts of bovine and rabbit odours were employed as host-odour stimuli. Human breath was blown into a 5-ml syringe used as stim- ulus cartridge (for further details, see Steullet and Guerin 1992b). Human axillary secretion was collected with a dry-acetone-washed cotton pad (7x7 cm) rubbed on the axillary area of a 28 year-old P. Steullet, P.M. Guerin: Olfactory receptors of Amblyomma. I male and then enclosed in a stimulus cartridge. The axillary region was not treated with deodorants or perfumes, and was not washed for 24 h prior to secretion collection. The stimulus blank consisted of a dry-acetone-washed cotton pad. Air from a metallic cage containing a single tick-naive rabbit (New Zealand), a white strain sometimes used in this laboratory to feed A. variegatum, was pumped for 24 h at 500 ml/min through ca. 600 mg of conditioned Porapak Q (60-80 mesh) packed in a glass tube 7 cm long x 4 nun diameter (Steullet and Guerin 1992b). The cage was located in an animal room with 20 other rabbits of the same, strain. Volatiles were desorbed with 3 ml dichloromethane (Merck, analytical grade) and the extract was then slowly concen- trated under N2 to ca. 50 pi. One or 10 pi of the concentrated extract was enclosed on filter paper in the stimulus cartridge. Air from adjacent rooms without animals (blank control) was also collected as described above on Porapak and analysed by GC-MS. Air from a 30 m1 stall occupied by 2 tick-naive Simmental steers (about 200 kg each), a race frequently used to rear A. variegatum, was pumped for 24 h at 500 ml/min through 600 mg of conditioned Porapak Q. Solvent desorbtion and concentration were achieved as for rabbit odour and 1 or 10 pi of the concentrated extract was used as stimulus. Extract of air from a washed Hall unoccupied for a month was used as a blank control and analysed by GC-MS. Col- lection of rabbit and bovine odour was undertaken on several occa- sions with different rabbits and steer. The concentrated extracts smelled very similar to the natural odours. Different body parts (head, shoulder, side, dewlap, chest, belly, legs, armpit, and perianal area) of two tick-naive Simmental steers were rubbed with acetone-washed cotton pads (7x7 cm) soaked with dichloromethane (analytical grade). Gloves were used for this operation. The cotton pads were placed in a 500-ml gas-wash bottle, held at 7O0C, through which N2 passed for 1 h at 100 ml/min. Re- leased volatiles were held up in a cold trap (4 mm diameter, 20 cm long glass U-tube steeped in a dry ice/acetone mixture) in a Dewar flask. Dichloromethane (2 ml, analytical grade) was used to extract trapped volatiles, water was removed by lowering the extract to — 100C and removing the solvent from the ice. The extract was subsequently concentrated under N2 to ca. 50 pi One or 10 ul of the concentrate was then used as stimulus. Gas chromatography-coupUd electrophysiological recordings (GC- EL). Olfactory receptors, characterized as responding to vertebrate odours, were subsequently employed to locate active produces) among the many constituents of odour extracts by GC-EL. Compo- nents of an active extract (bovine or rabbit odours collected on Porapak, skin wash of steer) were separated on a high-resolution capillary gas chromatography column (Chromatograph: Carlo Èrba Instruments HRGC 5160 with an on-column injector; fused-silica column: 30 m DBWAX, internal diameter 0.32 mm or 0.25 mm, 0.25 pm film thickness, G&W Scientific, USA; carrier gas: H1 at 0.5 m/s at 400C; temperature programmed : 600C for 5 min, 8°C/min to 2300C, and held for 10 min). The column effluent was split (glass Y-splitter), 2/3 being sent to the flame ionisation detector (FlD) and 1/3 (longer arm) to an electrophysiological preparation with recep- torfs) sensitive to host odour {biological detector). An air stream (11/min), maintained at ca. 80% RH and 22 ± 1°C in a 7 mm diameter glass water-jacketed tube, swept one third of the column efTlucnt to the tick preparation 30 cm away from a heated transfer line (2500C) in the wall of the Chromatograph. The outlet of the glass tube (reduced outlet of 3 mm diameter) was 5 mm from the tick tarsus where the air speed was 1.5 m/s. Column effluent was thus simultaneously monitored by the FID and the activity of the receptors recorded to locate possible active component(s) of the extracts being analysed (Wadhams 1982). AU spikes from what usually amounted to multicellular record- ings (AC signal) were sorted from background noise with a level discriminator incorporated in the UN-03 amplifier, and the sum of the frequencies of all firing cells was continuously converted to a voltage (time constant of the frequency to voltage converter: 1 s). This signal was printed on a multichannel chart-recorder simulta- neous with the FID response. An electrophysiological response was _________RESULTS ¦ CHAPTER 3.3.____________45 F. Steullet, P.M. Guerin: Olfactory receptors of Amblyomma. I indicated by a sudden change in the overall activity of the olfactory cells recorded. Time delay (about 3 s) between the FID response and the biological response, due to the added travel time of substances in the longer arm of the splitter and delivery tube to the electro- physiological preparation, was estimated by recording activity of an easily accessible 2-ni(rophenol-sensitive receptor located in a sensil- Iurn on the anterior pit of Haller's organ during elution of 10 ng 2-nitrophenol (Diehl el al. 1991). A difference in the delay between the FID response and the receptor response was never observed between the synthetic compounds tested. Variation in the response latency of the different receptors studied was negligible compared to the passage lime of the substances to the preparation. The Kovat's index for each active component detected was calculated with refer- ence to alkancs (C10 to C10) injected under the same GC conditions. Gas chromatography-coupled mass spectrometry (GC-MS). Extracts analysed by GC-EL to locate active constituents were subsequently concentrated about 5 times and analysed on the same GC phase by GC-MS (Hewlett Packard 5890 series I I Chromatograph - mass selective detector 597IA) to identify the active products. One ul of extract was injected on-column to the DBWAX capillary column (30 m, 0.25 mm internal diameter, 0.25 urn film thickness, G&W Scientific, USA) connected via 1 m of deactivated fused-stlica capil- lary to the MS (ionisation chamber temperature 1800C; ionisation energy 70 eV). Helium was used as carrier gas under constant pres- sure (velocity 0.3 m/s at 400C) and separation was achieved with the same temperature programme as in GC-EL. Active components of the extracts located by GC-EL were relocated in GC-MS from the calculated Kovat's index, and by comparison of the chromatogram profiles obtained in GC-EL and GC-MS. Identification of the active components. Identification of an electro- physiologically active peak in an extract was first based on the match of its mass spectrum with that of a known product stored in a computer-based library of the GC-MS. The mass spectrum and the calculated Kovat's index of the extract-unknown were then compared with those of the library-proposed synthetic analogue injected under the same conditions. Biologica] activity of the identi- fied product was subsequently tested with the synthetic analogue by electro physiology experiments on the olfactory receptor concerned. Full identification based on the mass spectrum was not always feasible because of the small amount of compound present and/or because of coeluting products* which obscured the spectrum. In some extracts, compounds either suspected or clearly identified in other extracts, such as methylbenzaldehyde isomers and v-valero- Iactone, were nevertheless detected by searching at the retention times of the volatiles in question for some of their characteristic fragment ions whose mass to charge ratios (M/Z) were 65, 91, 119, and 120 for methylbenzaldehyde isomers and 56, 85, and 100 for y-valerolactone. The following synthetic chemicals were employed as standards in GC-MS and electrophysiology: hexanal, heptanal, nonanal, (E)-2-heptenal, furfural, benzaldchyde, 2-methylbenzalde- hyde, 3-melhylbcnzaldehyde, 4-methylbenzaldehyde, 2-hydroxy- benzaldehyde, and y -valerolactone. In addition, (E)-2-hexenal, 3- hydroxybenzaldehyde, 4-hydroxybenzaldehyde, 1-phenyl« hanone and cyclohexanone were used because of their relatcdness to sus- pected active products. Dilutions of these chemicals (2 98% purity) from 10-i to 10~2 M in dichloromethane were tested on the recep- tors concerned as described above. Blank extracts (air from rooms without rabbits or steer) were also analysed by GC-MS to check for the possible presence of stimuli previously identified in bovine and/or rabbit odour extracts. For this purpose, 2-bromophenol (1.64 ng) was added as internal standard to 1.5 ml rabbit, bovine, and blank extracts before concen- tration. Using the single ion monitoring facility (SIM) of the mass selective detector, the presence of a stimulus was searched for, at the retention time of the synthetic analogue, by one of its characteristic fragment ions with a mass to charge ratio (M/Z) of 72 for hexanal, 70 for heptanal. 83 for (E)-2-heptenal, 98 for nonanal, 96 for furfural, 106 for benzaldchyde, 119 for methylbenzaldehyde isomers, and 122 for 2-hvdroxybenzaldehyde. Quantification was achieved by peak integration for the characteristic fragment ion chosen. Abundance of each stimulus was then normalized with reference to the amount of 2-bromophenol calculated from the peak area of one of its char- acteristic fragment ions (M/Z = 172 corresponding to M+-I). Olfactory receptor characterization. After identification of the host- odour stimulants, attention was focused on the olfactory receptors concerned. Dose-response curves based on the first 200 ms of the response to synthetic stimulants and thus the specificity spectrum of the responsive receptors were studied. To discriminate between dif- ferent receptor alls of similar spike shape and amplitude in multi- cellular recordings, double successive stimulation was sometimes necessary. This consisted of delivering an active compound A for a few s to the preparation interrupted for 1 s by stimulation with active compound B, and vice versa. No change in response, decrease or increase in firing of cellfs), cessation of firing in cell(s) or excita- tion of new cellfs) during the transition from compound A to B indicates if the same cell is responsive to the two substances or if different cells are excited. Possible cross-adaptation between recep- tor cells was not examined. Results Activity of olfactory receptors was captured at 6 distinct locations in the capsule of Haller's organ as revealed by hundreds of recordings. Results presented here deal with receptors recorded from within regions I and VI of the capsule (Fig. 1). Whereas one receptor in region I was Fig. 1. A Distal end of the tarsus of the leg I of adult A. oariegatum with the slit opening (arrow) to olfactory sensilla within the capsule of Haller's organ. B Diagrammatic view of the lay-out of olfactory sensilla in the capsule as shown by microscopic studies. The capsule is divided here into 6 regions (1-VI) from which olfactory responses were obtained. An outline of the slit opening is surimposed (see arrow). D distal; E external; I internal; pi pleomorph (for more details, see also Steullet and Guerin 1992a) RESULTS - CHAPTER 3.3. 46 P. Steullet, P.M. Gucrin: Olfactory receptors of Amblyomma. I rabbit odour Fig. 2A-E. Responses recorded from olfactory sensilla in region VI (Fig. 1) of the capsule of Hatler's organ in male A. variegatum where several receptors are excited by vertebrate odours. In preparation one: responses to rabbit odour (A), steer odour (B) both collected on Porapak Q, and to a wash of conditioned but unused Porapak Q (blank odour) (Q. The response to stimulation with the blank odour was much lower than that due to either bovine or rabbit odour. In preparation two: responses to human armpit secretion collected on cotton (D), and to cotton alone (blank) (E). A slight inhibition occurs during stimulation with the blank. Horizontal bars 1 s stimulation; vertical bars I mV 13b iob tit¥*Wiï¥^ 6 8 10 elutlon time [min] 12 sensitive to y-valerolactone, a compound detected in traces in bovine and rabbit odours, the other receptors stimulated by bovine and rabbit odours collected on Po- rapak (Fig. 2A-C) were all found in the proximal region of the capsule (region VI in Fig. 1). Receptors within the latter region were also regularly excited by human axil- lary secretion (Fig. 2D, and E), but did not respond clear- ly to either human breath or to physiological relevant levels of the synthetic chemicals cited in Materials and methods under stimulus delivery. Identification of some vertebrate volatiles stimulating olfactory receptors in region VI of the capsule Gas Chromatograph y-co u pi cd electrophysiological recordings (GC-EL) revealed that several components of bovine and/or rabbit odours stimulated olfactory recep- tors in region VI of the capsule (Table I and Figs. 3, 4, and 5). Gas chromatography-coupled mass spectrometry (GC-MS) subsequently permitted identification of some of these active volatiles, often in minor amounts in both bovine and rabbit odours collected on Porapak. These volatiles were hexanal, hep tan a I1. 2-heptcnal, nonanal, furfural, benzaldehyde, 2-, 3-, and 4-methylbenzaIdehyde, and 2-hydroxybenzaldehyde (Table I). Hexanal, 2-hepte- nal, nonanal, furfural, benzaldehyde, and 2-hydroxybenz- Fîg. 3A, B. Analysis of a bovine odour, col- lected on Porapak, by gas chromatography- coupled electrophysiology of olfactory recep- tors within region VI of the capsule of Haller's organ in male A. variegatum. The bottom trace is the flame ionisation detector response (FID). The upper traces A and B represent the summed frequency of all firing cells (frequency to voltage converted signal) in two different recordings; hollow vertical bar is 50 impulses/s. Difference in response to the odour constituents between A and B was due to the different population of olfac- tory receptors captured in each case, al- though the recording electrode was placed in the same region of the capsule for both analyses. Two olfactory sensilla occur in this region of the capsule (Fig. 1). GC-MS analy- ses of the active peaks indicated that re- sponses were recorded in both A and B for benzaldehyde (13), 3-methylbenzaldehydc (16), and the unknown peak 19, but only in A for 4-methylbenzaldehyde (17) and 2-hy- droxybenzaldehyde (18). Receptors respond- ing to (E)-2-heptenal (6) and the unknown peaks 2, 5, 7, and 14 were present in B but not in A. Other minor responses recorded once or only very occasionally were not ac- counted for. Active constituents are num- bered as in Table I. The FlD trace indicated 0.5 to 1 ng for benzaldehyde (13) in the odour extract injected. Spike trains generat- ed in B during elution of (E)-2-heptenat (6b), benzaldehyde (13b), and the unknown peak 19b arc given at the top of the figure. Hollow horizontal bars depict the approximate time taken by the products to elute from the GC column; solid horizontal bar Is; solid vertical bar 1 mV 16; Î 1-8 -19 14 16 RESULTS - CHAPTER 3.3. 47 P. Steullet, P.M. Guerin: Olfactory receptors of Amblyomma. I Table 1. Identified constituents of bovine and rabbit odour which stimulated olfactory receptors located within the proximal-internal region VI of the capsule of Haller's organ in male A. variegatura Peak number Olfactory stimulant a) Identification criteria b) Odour source c) Kovat's index in GC-EL d) Kovat's index in GC-MS e) Kovat's index of standards in GC-MS 0 Response occurrence 3 hexanal MKE steer rabbit I085±0 1084 ±8 1077 1081 1079 1079 2/9 6/10 4 heptanal MKE steer rabbit II84±5 1188 1184 1186 1184 0/9 3/10 6 2-heptenal MKE # steer* rabbit 1336±0 1338±4 1337 1338 1336 1336 4/9 5/10 S nonanal MKE steer rabbit 1400±0 1396 1398 1396 1394 1396 3/9 I/10 11 furfural MKE steer" rabbit 1475±7 1469 ±7 1469 1470 1469 1469 2/9 3/10 13 benzaldehyde MKE steer" rabbit 1535 ±5 1528 ±5 1531 1532 1532 1532 V9 6/10 15 2-methylbenzaldehyde MKE steer rabbit 1600 1622 1622 1621 1621 0/10 1/10 16 3-melhylbenzaldehyde MKE steer rabbit 1623 ±3 1611±5 1627 1628 1626 . 1626 2/9 3/10 17 4-methyIbenzaldehyde MKE steer rabbit 1657 ±3 1643 ±3 1656 1655 1653 1653 2/9 3/10 18 2-hydroxybenzaldehyde MKE steer' rabbit 1677±3 1675±5 1686 1689 1682 1682 5/9 10/10 This table is based on gas chromatography-coupled electrophysiol- ogy (GC-EL) and gas chromatography-coupled mass spectrometry (GC-MS) analyses of bovine and rabbit odour collected on Porapak Q, and the skin wash of steer (both types of analyses were made on the same GC phase DBWAX). a) Different criteria on which identi- fication of a particular vertebrate volatile was based: M -matching mass spectra, K - matching Kovat's index, and E - matching elec- trophysiological activity with that of the synthetic analogue (# the trans isomer of 2-heptenal was employed), b) Analyses were made of bovine or rabbit odour as collected on Porapak, and * indicates that the active compound was also detected by GC-MS in a bovine skin wash, c) Mean Kovat's index (± standard deviation) of active peaks in GC-EL analyses, ä) Kovat's index of the active peak located in GC-MS. e) Kovat's index of the synthetic product corresponding to that of the biologically active peak in GC-MS. I) Number of GC-EL analyses in which a response was observed/out of the total number of analyses with receptors from within region VI of the capsule. Other components of bovine and/or rabbit odours (GC peaks 1, 2, 5, 7,9,10,12,14, and 19 listed in the text and figures) occasionally activated receptors in GC-EL analyses, but could not be identified by GC-MS spike frequency CHO ÇH0 CHO ^> / Ï 1-8 8 10 12 14 16 elullon time [min] Fig. 4. Analysis of a bovine skin wash by gas chromatography-cou- pled elect rophysio logy of olfactory receptors within the proximal region Vl of the capsule of Haller's organ in a male A. variegatum. The lower trace is the flame ionisation detector response (FID) and the upper trace is the summed frequency of all Tiring cells (frequency to voltage converted signal). Hollow vertical bar is 50 impulses/s. Active components are numbered as in Table 1. GC-MS analysis of the active peaks indicated that responses were obtained for (E)-2- heptenal (6), furfural (11), benzaldehyde (13), and 2-hydroxybenz- aldehyde (18). Active constituents 10 and 12 were not characterized. Although the mass spectrum and retention time of peak 10 suggests l-octen-3-ol, response to the synthetic analogue was never recorded from this region of the capsule. Furthermore, the response to com- ponent 12 always accompanied that to furfural (11) in GC-EL ex- periments. The FID trace indicated (E)-2-heptenal (6) at 0.1-0.5 ng in the extract injected RESULTS - CHAPTER 3.3. 48 P. Stcullci. P.M. Guerin: Olfactory receptors of Amblyomma. I 2 4 6 8 10 12 14 16 elution time [min] Fig. 5. Analysis of a rabbit odour, collected on Porapak, by gas chromatography-cou- pled elect rophysiology of olfactory receptors within the proximal region VI of the capsule of Haller's organ in a male A. variegatum. The lower trace is the (lame ionisation detec- tor response (FID); the middle trace is the summed frequency of all firing cells (frequen- cy to voltage converted signal), hollow verti- cal tor is 25 impulses/s; the upper traces are the actual spike trains generated during elu- tion of hexanal (3) and 2-hydroxybenzalde- hyde (18). The aliphatic aldehyde receptors type 1 (all) and type 2 (al2) were both stim- ulated by hexanal (3), but only the 2-hydrox- ybenzaldehyde receptor (s) responded to 2- hydroxybenzaldehyde (18). Hollow horizontal bars indicate the elution time of the active peaks. Solid horizontal bar 1 s; solid vertical bar I mV. Active components are numbered as in Table 1. Receptors sensitive to 2-hepte- nal (6), benzaldehyde (13), and the unidenti- fied peak 19, known to occur in this region of the capsule, were not captured in this recording Table 2. Olfactory receptor types in the capsule of Haller's organ of A. variegatum which responded to specific classes of ver- tebrate volatiles Receptor type Location in Best stimulant Number of the capsule observations* Benzaldehyde receptor region VI benzaldehyde 25 2-hydroxybenzaldehyde region VI 2-hyd roxybenzaldehyde 23 receptor Aliphatic aldehyde receptor 1 region VI hexanal 12 Aliphatic aldehyde receptor 2 region VI heptanal U Aliphatic aldehyde receptor 3 region VI (E)-2-heptenal 13 Lactone receptor region I y-valerolactone 15 * Indicates the number of times the receptor was recorded from in either gas chromalogra- phy-coupled electrophysiology analyses of vertebrate volatiles or in classic single-unit recordings aldehyde were also discovered in steer skin wash. Al- though hexanal, nonanal, furfural, and benzaldehyde were also detected in blanks, they were more abundant in areas permeated with vertebrate odours, i.e. 2 to 4 times more benzaldehyde and furfural, >4 times more nonanat, and > 20 times more hexanal than in blanks. Characterization of receptors responding to the identified vertebrate volatiles The responses recorded to the host-odour components by GC-EL recordings from region VI of the capsule were sometimes very variable. Olfactory receptors) respond- ing to 2-heptenal (6) and the unidentified components 2, 5, 7, 14 of bovine odour in recording B of Fig. 3 were absent in recording A, whereas activity of the receptor sensitive to 2-hydroxybenzaldehyde (18) was only cap- tured in recording A. ïn another GC-EL experiment (Fig. 5), no responses were recorded from receptors for 2-heptenal (6), benzaldehyde (13), and the unidentified components 2 and 19, although these constituents of this rabbit extract proved active on olfactory receptors from approximately the same location within the capsule in other GC-EL recordings. This was due to the fact that the different types of olfactory receptors from within re- gion VI of the capsule from which recordings were ob- tained were probably distributed in two adjacent sensilla (Fig. 1), and the chance of picking up activity of any given receptor was dependent on the exact location of the elec- trode. In this study, we were unable to determine to which sensilla the different receptors captured within re- gion VI of the capsule belong. Careful examination of spike shape and amplitude of receptors responding to RESULTS - CHAPTER 3.3. 49 P. Stcullci. P.M. Gucrin: Olfactory receptors of Amblyomma. I B 150 - S 100 3 a. E - 50 T- *> < ¦ • i -10 -9 -S -7 log [moles] -10 -9 -8 -7 log [moles] bbbbbb bbbbbbbb Fig. 6. A-C Response of a.benzaldehyde-sensitive receptor in the proximal region VI of the capsule of Haller's organ in male A. cariegatum to benzaldehyde and furfural A Dose (moles in stimulus cartridge) of benzaldehyde (n = 6 different ticks) and B dose of furfural (n = 2 different ticks) plotted against spike frequency of the responding receptor calculated from the first 200 ms of the response. Trend lines connect mean values, In A and B the response of each receptor increased with increasing doses throughout the range test- ed. C Representative response of the receptor to benzaldehyde at 10'" moles in the stimulus cartridge. D-F Double successive stimu- lation of the bcnzaldehyde-sensitivc receptor (b) with 10"* moles benzaldehyde in the stimulus cartridge {solid horizontal bar) inter- rupted with 10 7 moles furfural in a second stimulus cartridge (hol- low horizontal bar). Absence of a second excited cell at onset of stimulation with furfural suggests that furfural activated the same receptor as benzaldehyde. D Spike train illustrating response to last 200 ms of stimulation with benzaldehyde and corresponding period from onset of stimulation with furfural. F Spike train resulting from reverse of situation in D, Le. last 200 ms of stimulation with furfural and restart of stimulation with benzaldehyde. Solid horizontal bar in C and hollow bar in E represent I s; vertical bars I mV B -10 -9-8-7 log [moles] 150 , Ot n 100 - n CL F 50 V • 0 ï -** -10 4 -8 •7 log [moles] Fig. 7. A-C Response of receptors sensitive to 2-hydroxybenzalde- hyde and benzaldehyde in the proximal region VI of the capsule of Haller's organ in A. variegatum. A Dose (moles in stimulus car- tridge) of 2-hydroxybenzaldehyde (n = 5 different ticks) and B dose of benzaldehyde (n = 4 different licks) plotted against spike fre- quency of the responding receptor calculated from the first 200 ms of the response. Trend lines connect mean values. In A and B the response of each receptor increased with increasing doses through- out the range tested. C Representative response of the receptor to 2-hydroxybenzaldehyde at 1(T* moles in the stimulus cartridge. D- F Double successive stimulation while recording from a receptor responding to benzaldehyde (solid horizontal bar) interrupted for 1 s (hollow horizontal bar in E) by stimulation with 2-hydroxybenz- aldehyde (both products at 10"* moles in separate stimulus car- tridges). A phasic burst in spike frequency of a second excited cell at onset of stimulation with 2-hydroxybenzaldehyde and at restart with benzaldehyde indicated presence of separate receptors (b and s respectively, in D and F) for these two products in region VI of the capsule of Haller's organ. D Spike train illustrating response to last 200 ms of stimulation with benzaldehyde and corresponding onset period with 2-hydroxybenzaldehyde. F Spike train resulting from reverse of situation in D, Le. last 200 ms of stimulation with 2-hy- droxybenzaldehyde and restart of stimulation with benzaldehyde. Solid horizontal bar in C and hollow bar in E represent 1 s; vertical bars I mV single and/or double stimulations with synthetic ana- logues of the identified stimulants (benzaldehyde, 2-hy- droxybenzaldehyde, furfural, and aliphatic aldehydes) permitted us to clearly discriminate at least five types of receptors in the region V[ of the capsule (Table 2). Proper characterization of receptors responding to methylbenz- aldehyde isomers was not undertaken because of the great difficulty in obtaining reproducible recordings. GC- EL analyses and double successive stimulations permit- ted characterization of separate receptors for the aromat- RESULTS - CHAPTER 3.3. 50 P. Stcullcl, P.M. Gucrin: Olfactory receptors of Amblyomma. I I? ^22, 22?22222222222 o .11 J-. ..... B 333333333333 Rg. 8A-C Response of olfactory receptors located within the prox- imal region VI of the capsule of Hallcr's organ in a male A. variega- tum to the short-chain aliphatic aldehydes (E)-2-hexenal and hex- anal (each at 10~* moles in the stimulus cartridge). B Double succes- sive stimulation with (E)-2-hexenal (solid bar), which stimulated both receptor 1 and receptor 2 weakly, was interrupted for 1 s by stimulation with hexanal {hollow bar) which activated receptor 2 more than (E)-2-hexenal did. Sensitivity of receptor 1 to both alde- hydes was approximately the same. A Spike train illustrating re- sponse to last 300 ms of stimulation with (E)-2-hexenal and corre- sponding period from onset of stimulation with hexanal. C Spike train resulting from reverse of the situation in A, i.e. last 300 ms of stimulation with hexanal and restart of stimulation with (E)-2-hexe- nal. Spikes with underlined numbering are overlapping events; per- ticai bars I mV ic aldehydes (benzaldehyde and 2-hydroxybenzaldehyde) and for different aliphatic aldehydes. Benzaldehyde receptor. One type of receptor in region VI of the capsule was sensitive to benzaldehyde (Fig. 6A and C) and to furfural as suggested by double successive stim- ulations (Fig. 6D-F). The dose-response curve for fur- fural was however much flatter than that for benzalde- hyde (Fig. 6A and B). Since in GC-EL an estimated con- centration of ca. 6 x IO9 molecules of benzaldehyde/cm3 air arriving at the tick preparation increased the base Tiring of this receptor from 5 to ca. 50 impulses/s (calcu- lated on a spike train of 1 s during the peak of the re- sponse), the threshold of this receptor for benzaldehyde is still much lower than this level. Concentration of benz- aldehyde at the preparation was estimated on the amount injected onto the GC column, the width of the benzaldehyde eluting peak, the fraction of the column effluent directed to the preparation, and the dilution fac- tor in the air stream carrying the product to the prepara- tion. Furthermore, responses to furfural were always ac- companied by responses to a later eluting product in GC-EL experiments (peak 12 in Fig. 4) which excited spikes of the same shape and amplitude as furfural and benzaldehyde. This suggested that the unidentified com- ponent 12 may stimulate the same receptor as benzalde- hyde and furfural, and thus share common chemical properties with these two volatiles. B 33 * ,,3333333^s33 3 Fig. 9A-C Response of an olfactory receptor (aldehyde receptor type 3) located within the proximal region VI of the capsule of Haller's organ in a male A. variegation to the unsaturated aldehydes (E)-2-hexenal and (E>-2-heptenal (each at IO"4 moles in the stimulus cartridge). B Double successive stimulation with (E)-2-heptenal (solid bar) interrupted for 1 s by stimulation with (E)-2-hexenal (hollow bar). Absence of a second responding unit at onset of stimu- lation with (E}-2-hexenal or at restart of stimulation with (E)-2-hep- tenal indicated that both products activated the same receptor. This receptor type did not respond to short-chain saturated aliphatic aldehydes. A Spike train illustrating response to the last 300 ms of stimulation with (E)-2-heptenal and corresponding period from on- set of stimulation with (E>-2-hexenaL C Spike train resulting from reverse of the stimulation in A, i.e. last 300 ms of stimulation with (E)-2-hexenal and restart of stimulation with (E)-2-heptenal. At least one other receptor with a spike of smaller amplitude (star) occurs in these traces. Vertical bars 1 mV 2-Hydroxybenzaldehyde receptor. Another receptor in re- gion VI of the capsule was strongly stimulated by 2-hy- droxybenzaldehyde (Fig. 7A and C). Double successive stimulation with benzaldehyde and 2-hydroxybenzalde- hyde permitted a clear distinction between the 2-hydrox- ybenzaldehyde receptor and the benzaldehyde receptor which were sometimes both captured in the same record- ings (Fig. 7D-F). However, the 2-hydroxybenzaldehyde receptor also had the capacity to respond to very high doses of benzaldehyde (at least 10"8 moles in the stimulus cartridge) (Fig. 7B). Three types of aliphatic aldehyde receptors. Three types of aliphatic aldehyde receptors with different specificity spectra were distinguished in region VI of the capsule (Figs. 8 and 9). One receptor (type I) was particularly sensitive to saturated and unsaturated C6 aliphatic alde- hydes, but was only slightly sensitive to heptanal and hardly to nonanal (Fig. 10A and B). Another receptor (type 2) responded mainly to saturated aliphatic alde- hydes such as hexanal and heptanal (Fig. 10C and D), whereas a third receptor (type 3) was excited by unsatu- rated aliphatic aldehydes such as (E)-2-hexenal and (E)-2- heptenal {Fig. 10E and F). These aliphatic aldehyde re- RESULTS - CHAPTER 3.3. 51 P. Steullei. P.M. Guerin: Olfactory receptors of Amhtyomma. I saturated 150 - Q. £ - 50 • • • ¦ - I/ e/ ,0 a ? 'Î ^O 9 ¦ ¦10 -9 -8 -7 log [moles] -9 -8 -9 log [moles] -10-9 -8 -7 log [moles]' unsaturated B 150 i OT T T ? V) S 100 - .T 3 T T E - 50 - T V n . « I T -10 -9 -8 -7 log [moles] •10 -9 -8 -7 log [moles] -10 -9 -8 -7 log [moles] receptor type 1 N'A receptor type 2 W-3 receptor type 3 N=4 Fig. 10A-F. Representative dose response- curves of olfactory receptors located within the proximal region V[ of the capsule of Hatler's organ in male A. variegatum to saturated and unsaturated aliphatic alde- hydes. Solid circle hexanal; hollow circle heptanal; square nonanal; solid triangle (E)-2-hexenal; hollow triangle (E)-2-hepte- naL Ordinales in A-F are spike frequencies of the responding receptors calculated from the first 200 ms of the response, and abscissas dose of products tested (moles in stimulus cartridge). Trend lines connect mean values. In each case, the response of the receptors increased with increasing doses throughout the range tested. A and B Aldehyde receptor type 1 responded equally well to saturated and unsaturated C6 aldehydes (A and B were established with the same receptors, n = 4); C and D aldehyde receptor type 2 was more sensi* live to the saturated C6 and C7 aldehydes than to the corresponding unsaturated products {C and D were established with the same receptors, n = 3); E and F alde- hyde receptor type 3 which was selectively stimulated by unsaturated aldehydes (E and F were established with the same re- ceptors, n = 4) (see also Figs. 9 and 10) dose of aliphatic aldehydes ceptors seemed less sensitive than the benzaldehyde re- ceptor since in GC-EL analyses an estimated concentra- tion of ca. 3 x 10" molecules of hexanal/cmJ air arriving at the preparation was required to evoke responses of ca. 25 impulses/s from a base frequency of ca, 5 impulses/s in receptor type 1, and ca. 15 impulses/s from a base fre- quency of ca. 5 impulses/s in receptor type 2. In GC-EL analyses, responses to hexanal were due to either recep- tor type I or 2, or occasionally both (Fig. 5), heptanal and nonanal mostly excited receptor type 2, and (E)-2-hepte- nal stimulated receptor type 3 (Fig. 3). (E)-2-hexenal was not detected in any of the host-odour extracts analysed in this study. Responses to two unidentified components of bovine odour collected on Porapak (peaks 5 and 7 in Fig. 3B) always accompanied responses to (E)-2-hepte- nal. and spike shapes and amplitudes of the excited re- ceptors were very similar. This suggests that components 5 and 7 may share common chemical properties with 2-heptenal and may likewise excite the aliphatic aldehyde receptor type 3. Nevertheless, we were unable to identify both peaks 5 and 7 by mass spectrometry. Lactone receptor. Finally, when the glass electrode was introduced within the exterio-anterior part of the capsule {region I in Fig. 1), a receptor responsive to y-valerolac- tone was systematically found among other sensory cells (Fig. 11). This receptor also responded weakly to y-buty- rolactonc and 6-caprolactone, hardly at all to bovine odour extracts, and not at all to human breath and hu- man axillary secretion. Nevertheless in GC-MS, a com- pound with three fragment ions (with mass to charge ratios of 56, 85, and 100) characteristic for y-valerolac- tone and eluting at the same retention time as the syn- thetic analogue was Jetected in bovine and rabbit odour collected on Porapak. This suggested that these extracts may contain traces of y-valerolactone but in insufficient quantity to clearly stimulate the lactone receptor in GC- EL. RESULTS - CHAPTER 3.3.____________52________ P. Stcullet, P-M. Gucrin: Olfactory receptors of Amblyomma. I 100 80 60 W 3 (X 40 b 20 0 log [moles] Fig. HA, B. Response of an olfactory receptor within the exterio- anterior region I (Fig. 1) of the capsule of Haller's organ in male A. variegatum to y-valerolactonc A Dose (moles in stimulus cartridge) of Y-valerolactone plotted against spike frequency of the responding receptor calculated from the first 200 ms of the response (n = 5 different ticks). Trend line connects mean values. B Representative response of the receptor to *r-vaieroiactone at 10 T moles in the stimulus cartridge. Horizontal bar 1 s stimulation; vertical bar 1 mV Discussion The following components of rabbit and/or bovine odour stimulate olfactory receptors within chcmosensHla in re- gions I and VI of the capsule of Haller's organ in A. variegatum: short-chain saturated and unsaturated aliphatic aldehydes, furfural, benzaldehyde, methylbenz- aldehyde isomers, 2-hydroxybenzaldehyde, and y-valero- lactone. Except for saturated aliphatic aldehydes, these volatiles were remarkably minor components of the col- lected vertebrate odours. Aliphatic and aromatic aldehydes as well as lactones are not known as kairomones for other haemal op h ago us arthropods. Only propanal-sensitive neurons have been described in tsetse flies (Bogner 1992). These volatiles are furthermore not specific to vertebrate odours. Many phy- tophagous but also some scavenger arthropods are known to bear olfactory receptors sensitive to aliphatic aldehydes (i.e. constituents of the green leaf odour) or to benzaldehyde (Sass 1976; Seelinger 1977; Visser 1986). Benzaldehyde and 2-hydroxybenzaldehyde produced by different plants can act as repellents for several insects (Wallace and Mansell 1976). Finally, aliphatic aldehydes, benzaldehyde. 2-hydroxybenzaldehyde, and lactones also occur in the defensive secretions of various arthropods (Tschinkel 1975; Blum 1981). In A. variegatum. benzaldehyde stimulates a receptor wuhm region VI of the capsule. High amounts of this compound also excite another receptor most sensitive to 2-hydroxybenzaldehyde. Benzaldehyde has already been identified as a component of the aggregation-attachment pheromone of the related species A. hebraeum (Apps et al. 19*Sì. but not in A. variegatum (Diehl et al. 1991 ; Diehl, unpublished). This aromatic aldehyde has been identified from a number of vertebrate sources: preorbitat glands of the muskox (Flood et al. 1989), chin glands of the rabbit (Goodrich 1983), human vaginal secretion (Preti et al. 1977), and mouse urine (Ändreolini et al. 1987). Benz- aldehyde is also reported as a common volatile in air (Welsch and Watts 1990) and in this study was detected in the air of empty stalls (blanks), but at lower amounts than in rooms with steer or rabbits. The benzaldehyde receptor is also stimulated by furfural, a component of both bovine and rabbit odours collected on Porapak, bovine skin wash, and rabbit excrement, but also in hu- man effluvia (Ellin et al. 1974), in human vaginal secre- tion (Preti et al. 1977) and in human urine (Zlatkis and Liebich 1971). 2-Hydroxybenzaldehyde strongly stimulates, another receptor in the region VI of the capsule of Haller's organ. This product, present as a minor constituent of bovine and rabbit odours collected on Porapak and of the bovine skin wash, but absent in our blank odour, was once identified in extracts of replete female A. variegatum (Wood et al. 1975), but a physiological role was never proposed. Presence of 2-hydroxybenzaldehyde in verte- brate odours is nevertheless not well-documented, al- though it has been reported in the anal glands of the beaver (Lederer 1946). Aliphatic aldehydes, found in our concentrates of ver- tebrate odour, are detected by three different types of receptors in region VI of the capsule of Haller's organ of A. variegatum. The type 1 responds best to both saturated and unsaturated C6 aliphatic aldehydes, the type 2 to saturated C6 and C7 aliphatic aldehydes, and the type 3 to unsaturated C6 and C7 aliphatic aldehydes. The aliphatic aldehydes were generally present in our odour extracts at levels well above those of the aromatics. Satu- rated and unsaturated aliphatic aldehydes, as well as the corresponding ketones, alcohols, and fatty acids are well- documented commonly occurring volatiles in vertebrate odours. Some are also common air-pollutants (Welsh and Watts 1990), so it was not surprising to find traces of hexanal and nonanal in our blanks. Unsaturated aliphat- ic aldehydes are reported from goats (Smith et aL 1984), human breath (Krotoszynski et al. 1977), and the rabbit (Goodrich 1983), and saturated aliphatic aldehydes occur in the dog anal sac (Natynczuk et al. 1989), coyote urine (Schultz et al. 1988), skin glands of various bovidae (Burger et al. 1981), anal and chin glands of the rabbit (Goodrich 1983), human effluvia (Ellin et al. 1974; Goetz et al. 1988; Krotoszynski et al. 1977), and human axillary secretion (Labows et al. 1979b). The electrophysiological responses reported here for the human axillary secretion may be due to its aliphatic aldehyde content Equipped with three aliphatic aldehyde receptor types, A. variega- tum would be able to discriminate between volatile mix- tures characteristic of different vertebrates. The ratio be- tween responses of the different aldehyde receptors could thus serve as an odour-specific coding parameter as pro- posed by Tichy and Loftus (1983) for perception of alco- hols by the stick insect, Carausius morosus. y-Valerolactone, present here in traces in bovine and rabbit odour, excites a receptor located in region I of the capsule of A. variegatum. Lactones have been reported from a variety of species within the Bovidae, Camelidae, RESULTS - CHAPTER 3.3. 53 P. Steullct, P.M. Gucrin: Olfactory receptors of Amblyomma. I and Primates. Yeasts of the genus Pityrosporum are re- sponsible for the production of different y-lactones in areas rich in sebaceous glands of humans {Labows et al. 1979a). Several lactones are also reported in human scalp andurinefGoetzetal. 1988; Zlatkis and Liebich 1971), in occipital glands of Camelus bactrianus (Ayorinde et al. 1982), in preorbitai glands of the muskox (Flood et al. 1989), blue and grey duikers (Burger and Pretorius 1987; Burger et al. 1990), and in pedal glands of bontebock (Burger et al. 1977). As adult A. variegatum mostly feed on Bovidae, lactones present in many of these vertebrates could possibly function as cues for host-finding and/or feeding-site selection. In this study, receptor activity was recorded from six distinct regions within the capsule of Haller's organ of A. variegatum, corresponding to sensillum locations ob- served in microscopy. Recordings from five of these loca- tions were highly reproducible in terms of the number of cells captured, spike shapes and amplitudes, spontaneous activity, and stimulus response, i.e. a situation typical for single sensillum recordings (Steullet and Guerin 1992a). By contrast, the recordings were not very reproducible in the posterio-interior region VI of the capsule. Here, the number of cells captured, and spike shapes and ampli- tudes varied from one preparation to another. We imag- ine that depending on the exact location of the tip of the glass recording electrode within region VI, different pop- ulations of receptors from one or both of the two closely associated sensilla in this region of the capsule were being sampled. The seven wall-pore single-walled sensilla within the capsule carry between 21 and 35 receptor cells according to Hess and Vlimant ( 1982). About half of these cells have now been properly characterized: a C02-excited and a C02-inhibited receptor (Steullet and Guerin 1992a), two sulfide receptors (Steullet and Guerin 1992b), a methyl- salicylate receptor (component of the aggregation-at- tachment pheromone, Hess and Vlimant 1986), and as described here benzaldehyde and 2-hydroxybenzalde- hyde receptors, three aliphatic aldehyde receptors, and a lactone receptor. The capsule of the Haller's organ of A. variegatum is thus a very elaborate sense organ contain- ing many olfactory receptors, each with a distinct sensi- tivity spectrum and capable of responding to various ver- tebrate odours such as breath, human axillary secretion, skin wash of steer, and to bovine and rabbit odour ex- tracts. Acknowledgements. We thank Professor Peter Alan Dich! of this Institute for helpful discussions, his interest in this study, and for valuable comments on the manuscript. We are grateful to Dr. JJ.B. Smith, University of Toronto, and to Dr. B.K. Mitchell, University of Alberta (Edmonton), for kindly providing us with their spike analysis programme (SAPID). We also thank Misters Jonczy and Rohrer of Ciba-Geigy Ltd.. St. Aubin (Switzerland) for supplying us with adult ticks, and for access to the animal stalls. We are indebted to the Hasselblad. Roche and Sandoz Foundations, Swiss National Science Foundation (Grant Nos. 3.609-087 and 3!-28684.9O)1 the Ciba-Geigy-Jubilaeums-Stiftung, and the Swiss Office for Educa- tion and Science for funding studies on tick sensory physiology at the University of Neuchätel. This article is part of the thesis being presented by P. Steullet for the Ph.D. degree at the University of Neuchätel. References Andreolini F, Jemiolo B, Novotny M (1987) Dynamics of excretion of urinary chcmosignals in the house mouse (Mus muscuius) during the natural estrous cycle. 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Clin Chem 17:592-594 RESULTS - CHAPTER 3.4. 55 J Comp Physio! A (1993) Il ||| Hl Journal of Comparative £P Physiology A © Springer -Vertag 1993 Identification of vertebrate volatiles stimulating olfactory receptors on tarsus I of the tick Amblyomma variegatum Fabricius (Ixodidae) IL Receptors outside the Haller's organ capsule P. Steullet, P.M. Guerin Institute of Zoology, University of Ncuchâtcl, Chantemerle 22, CH-2000 Neuchâtel, Switzerland Accepted: 30JuIy 1993 Abstract. Bovine odour excites olfactory receptorfs) in a wall-pore olfactory sensillum on the anterior pit of Haller's organ in Amblyomma variegatum. Gas chro- matography-coupled electrophysiology recordings from this sensillum reveal the presence of 4 active compounds in bovine odour. The two strongest stimulants were iden- tified as 2-nitrophenoI and 4-methyl-2-nitrophenol by gas chromatography-coupled mass spectrometry, and by matching electrophysiological activity of synthetic ana- logues. Synthetic analogues of known vertebrate-associ- ated volatiles also stimulate other olfactory receptors in sensilla on the surface of tarsus I: a lactone receptor re- sponding to y -valerolactone and 6-caprolactone; differ- ent fatty acid receptor types responding best to either pen ta noie acid, 2-methylpropanoic acid or to bulanoic acid; three receptors responding to NH3; and one recep- tor responding to 3-pentanone. Gas chromatography- coupled mass spectrometry analysis of vertebrate volatiles revealed presence of a number of these olfactory stimulants in concentrates of rabbit and steer odour, i.e. 2-methylpropanoic acid, butanoic acid, 3-methylbu- tanoic acid, pent ano ic acid, and y-valerolactone. Key words: Tick - Haller's organ - Olfactory receptors - Fatty acids - Ammonia - y-Valerolactone - Nitrophe- nols Introduction While the previous paper (Steullet and Guerin 1993, com- panion paper) deals with the identification of vertebrate volatiles that stimulate olfactory receptors in the capsule of Haller's organ on leg pair I of Amblyomma variegatum (Acari, Ixodidae), this present study aims to identify host- Abbreviations: GC-EL, gas chromatography-coupled electrophysi- ological recording; GC-MS, gas chromatography-coupled mass spectrometry Correspondence to: P.M. Guerin odour volatiles that excite receptors housed in sensilla located outside the capsule. The 7 olfactory sensilla with- in the capsule of Haller's organ carry various host-odour receptors, i.e. CO2-, sulfide-, aliphatic aldehyde-, ben- zaldehyde-, 2-hydroxybenzaldehyde-, and lactonc-recep- tors (Steullet and Guerin 1992a, b, 1993): Adult A. varie- gatum also possess 12 other wall-pore sensilla on the sur- face of the tarsus of the first pair of legs (Hess and VIi- mant 1982, 1983) (Fig. 1). The latter authors observed that the position of sensilla on the tarsus is conserved between tick species, and distinguished between different morphological types of wall-pore single-walled and wall- pore double-walled sensilla (according to Altner's classi- fication, Altner et aL 1977). Based on detailed ultrastruc- tural studies, Hess and Vfimant estimated that the 12 wall-pore sensilla on the surface of tarsus I in A. variega- tum carry between 50 and 65 olfactory receptor cells. On- ly a few of them have been physiologically characterized in A. variegatum, or indeed in any other tick species. Re- ceptors sensitive to 2,6-dichlorophenoI (a tick pheromone component) were described in the DI. I and DILI sensilla (Fig. 1) of A. variegatum (Waladde and Rice 1982; Schoeni 1987), and in a wall-pore sensillum distal to the Haller's organ in Amblyomma americanum and Rhipicepkalus appendicular (Haggart and Davis 1981), and Ixodes ricinus (Thonney 1987). 2-Nitrophenol, a component of the aggregation-attachment pheromone of A. variegatum, excited receptors) in a wall-pore sensillum on the anterior pit of Haller's organ of this species (Schoeni 1987). Finally, NH3-sensitive receptors were dis- covered in sensilla of the anterior pit, and also in more proximally placed sensilla on the tarsus of Rhipicepkalus sanguineus (Haggart and Davis 1980). Materials and methods Animals. Male A. variegatum were reared and maintained in the conditions described previously (Steullet and Guerin 1993). Electrophysiology. Tick was immobilized on a piece of perspex with doublc-sidcd sticky tape. The tip of the sensillum was cut with the RESULTS - CHAPTER 3.4. 56 broken lip of a heat-pulled glass rod (1.5 mm diameter) mounted on a micromanipulator and oscillating at ca. 120 MHz induced by a piezo electric transducer disk (n° 4322.020.177721, Philips) (Gödde 1989). A glass electrode (tip diameter: 10-20 urn) mounted on a micromanipulator and filled with 0.2 M KCl was brought into contact with the cut tip of the sensillum, whereas the reference glass electrode filled with 0.2 M NaCl was introduced into the coxa of the anterior leg. The coxa was previously pinched with blunt forceps to prevent muscle discharge during recordings. Experiments were achieved either under an inverted microscope (Nikon Diaphot TMD) for recording receptor activity in the DLl, DILI, DII.5, DII.6, DHLl, and DIV.1-4 sensilla, or under a stereomicroscope (Olympus SZH) for recordings from the LAILl, VILI, and VTI.4 sensilla (see Fig. 1). Sensilla are named after Hess and YIimant (1982). Amplifica lion, data acquisition and spike analysis were ac- complished as described in Steullet and Guerin (1992a, b). Stimulation. Stimulus delivery system as well as the synthetic chem- icals used to study specificity of the olfactory receptors were as in Steullet and Gùerin (1993). Human breath, human axillary secre- tion, and bovine and rabbit odours collected on Porapak Q were used as sources of natural stimuli (details in Steullet and Guerin 1992b and 1993). Gas chromatography-coupled electropkysiohgy recordings (GC-EL) and gas chromatograph}H:oupted mass spectrometry (GC-MS). GC- EL, with column effluent split, was used to locate active compo- nents of bovine and/or rabbit odour extracts. Two thirds of the effluent was sent to a flame ionisation (FID) or an electron capture detector (ECD), and one third to an electrophysiological prepara- tion of an olfactory sensillum as biological detector. Compounds capable of stimulating olfactory receptors in sensilla on the surface of the tarsus were located due to a strong increase in spike frequen- cy of the responding receptors. GC-MS was subsequently employed to identify these active chromatographic peaks. For further details of the GC-EL methodology and GC-MS, see Steullet and Guerin (1993). Identification of an electrophysiologically active peak: in an extract was first based on a match between its mass spectrum and that of a known product stored in a computer-based library of the GC-MS. The mass spectrum and Kovat's index of the stimulant to be identified were then compared with those of the proposed syn- thetic analogue injected under the same conditions and on the same chromatographic phase (DBWA)C). The biological activity of the synthetic product was subsequently confirmed by electrophysioiogy experiments on the olfactory receptor concerned. Thus, synthetic 2-nitrophenol and 4-methyl-2-nitrophenol (>98% purity) were tested on receptors to confirm their identification as olfactory stim- ulants in GC-EL and GC-MS analysis of odour extracts. Concentrates of air from rooms without rabbits or steer (blanks) were also analysed by GC-MS to check for the occurrence of iden- tified olfactory stimulants in ambient air, and the amount was com- pared with that found in bovine and rabbit odour extracts collected under the same conditions. For this purpose, 2-bromophenol (1.64 fig) was added as internal standard into 1.5 ml of rabbit, bovine, and blank extracts before concentration and analysis by GC-MS. Search for nitrophenois in each extract at the retention time of the corresponding synthetic analogues was made with (heir molecular ions whose mass to charge ratio (M/Z) was respectively 139 for 2-nitrophenol and 153 for 4-methyl-2-nitrophenol. Quantification was achieved by integrating the area under the peak of these charac- teristic ions. Abundance of each product in an extract was normal- ized with reference to 2-bromophenol using the peak area of one of its characteristic fragment ions (M/Z = 172). Results Human breath, human axillary secretion, and rabbit odour collected on Porapak failed to clearly stimulate olfactory receptors in any of the wall-pore sensilla locat- P. Steullet, P.M. Guerin: Olfactory receptors of Amblyomma. Il VÌI.1 Fig. 1. ATarsus of leg I of an adult A. variegatum indicating location and the name ascribed to each olfactory sensillum (stippled). Arrow indicates the slit-like opening to the capsule of Halter's organ which contains 7 additional olfactory sensilla. B Detailed view of the ante- rior pit sensilla of Halkr's organ ed on the surface of the tarsus. Bovine odour collected on Porapak evoked a response of receptorfs) in the wall-pore DILI sensillum on the anterior pit of Haller' organ (Fig. 1). Identification of bovine volatiles stimulating receptoris) in the DIIA sensillum Gas chromatography-coupled electrophysioiogy analy- ses (GC-EL) with the DILI sensillum revealed 4 active compounds in bovine, but none in rabbit odour collected on Porapak (Table I). Although two only occasional but weak stimulants were not identified, the other two were identified as 2-nitrophenol and 4-methyl-2-nitrophenol. Identification of peak 2 as 2-nitrophenol was based on 1 ) matching Kovat's index of the GC-EL active peak with the synthetic analogue (Table I), 2) the presence of the molecular ion (M/Z = 139) of 2-nitrophenol in the un- known in GC-MS analyses at the same retention time as the synthetic analogue, and 3) matching electrophysio- logical activity of the synthetic analogue (Fig. 2). A full mass spectrum was not obtained because of the small amount of peak 2 in the extract and the presence of coeluting products. Peak 3 was identified as 4-methyl-2- nitrophenol on the basis of I) matching mass-spectrum with the synthetic analogue, 2) correspondence of the Ko- vat's index of the unknown in GC-EL with the synthetic analogue (Table 1), 3) matching electrophysiological ac- tivity of the synthetic analogue (Fig. 2). Some 10-20 ng of 2-nitrophenol and 200-300 ng of 4-methyl-2-nitrophenoI were found in 1.5 ml bovine odour extract after collecting 3001 of bovine odour-laden air on Porapak. Rabbit RESULTS - CHAPTER 3.4. 57 P. Steullet. P.M. Gucrin: Olfactory receptors of Amblyomma. If Table 1. Identified constituents of bovine odour which stimulated olfactory receptors in the DII.1 sensillum of male A. variegatum Olfactory stimulant a) Idenli- b) Odour c) Kovat's index d) Kovat's index e) Kovat's index 0 Response fication source in GC-EL in GC-MS of standards occurrence criteria in GC-MS 1 unidentified steer 1803 - - 1/8 rabbit - - - 0/5 2 2-nitrophenol MKE steer 1812±11 1818 1818 7/8 rabbit - 1814 1818 0/5 3 4-methyl-2-nitrophenol MKE steer* I914±10 1922 1919 7/8 rabbit - 1919 1919 0/5 4 unidentified steer 1992±15 - - 1/8 rabbit 0/5 This table is based on gas chromatography-coupled electrophysiol- ogy (GC-EL) and gas chromatography-mass spectrometry (GC- MS) analysis of bovine and rabbit odour collected on Porapak Q. Both types of analyses were made on the same gas chromatographic phasc-DBWAX. a) Different criteria on which identification of a particular odour constituent was based, M - matching mass spec- tra, (in the case of 2-DÌtrophenol identification was based on pres- ence of the molecular ion (M/Z*= 139) in peak 2 at the same reten- tion time as the synthetic analogue), K - matching Kovat's index, and E - electrophysiological activity matching with that of the synthetic analogue, b) Analyses were made of bovine and rabbit odour extract also contained small quantities of both compounds but in insufficient amount to evoke a re- sponse in GC-EL, i.e. 1-5 ng of both nitrophenols in 1.5 ml rabbit odour extract after collection of a similar volume of rabbit odour-laden air on Porapak. By con- trast, extracts of room air without steer or rabbits con- tained no or hardly detectable amounts of either of these substances. odour as collected on Porapak and * indicates that the active com- pound was also detected by GC-MS in a bovine skin wash, c) Mean Kovat's index (± standard deviation) of the active peaks in GC-EL analyses. 6) Kovat's index of the active peak located by GC-MS. e) Kovat's index of the synthetic product corresponding to that of the biologically active peak in GC-MS. 0 Number of GC-EL analy- ses in which a response was observed/out of.the total number of analyses with the DIM sensillum. 2-NitrophenoI and 4-methyl-2- nitrophenol were present at low amounts in rabbit odour extracts, but in insufficient quantity to elicit a response in GC-EL Receptors responding to vertebrate volatiles Apart from receptors) in the D[Ll sensiilum which re- sponded to nitrophenols, receptors housed in this and other sensilla were excited by volatiles normally associat- ed with vertebrate odours. The following types of recep- tors were found: MM "-^ A IJ^ mMM'mm^ OH O2 ECO 16 18 20 elution time [min] Fig. 2A, B, Section of a bovine odour extract analysis by gas chromatography-coupled electrophysiology of the DILI sensillum of a male A. variegatum (for details of technique, see text). The tower trace details part of the chromatogram of a bovine extract obtained with an ECD (electron capture detector); the upper traces (A and B) represent the summed frequencies of all cells recorded from the DILI sensillum (frequency to voltage con- verted signal) during elution (A) of compo- nents of the bovine odour extract and (B) during elution of 10 ng each 2-nitrophenol and 4-melhyl-2-nitrophenol, respectively, at the same retention times as peak 2 and 3 of the extract. Peaks are numbered as in Table 1. Spike trains generated in A during elution of 2-nitrophenol (peak 2) and 4-methyl-2-ni- trophertol (peak 3) are provided. Hollow horizontal bars: approximate elution time of the active compounds; hollow vertical bar 50 impulscs/s; solid horizontal bar I s; solid ver- tical bar 0.3 mV, The great complexity of spike trains recorded from the DILI sensil- lum, which contain 14 receptor cells, prohib- ited us from properly analysing which and how many receptors responded to these ni- trophenols RESULTS - CHAPTER 3.4. 58 P. Steullci. P.M. Guerini Olfactory receptors of Amblyomma. Il g-butyrolactone ^j'.f hH „>|,»fH^| itt 10*8 moles 6-caprolactone 10"7 moles 10"6 moles !4ftt|4)#*^^ 18 1s g-valerotactone 10'1 moles CX •oo B H3C 0 Fig. 3. Responses of an olfac- tory receptor of the DI. I sen- sillum in male A. variegatum to Y-butyrolactone (A), v-valerolactone (B), and 6-caprolactone (Q. Stimulus intensity (number of moles in the stimulus cartridge) is in- dicated above each trace. Due to the multicellular re- sponses, it was often not pos- sible to clearly distinguish the spikes involved in re- sponses to stimulation with low doses of these lactones (1O-* moles in the stimulus cartridge). Horizontal bars 1 s stimulation 1S Nitrophenoï receptor(s). Both 2-nitrophenol and 4- methyl-2-nitrophcnol stimulated receptors) in the DILI sensillum (Fig. 2). Both nitrophenols were equally active. Since an estimation of 10* molecules of 2-nitrophenol/ cm3 air in GC-EL experiments still strongly excited re- ceptor(s) in the DILI sensillum the threshold can be con- sidered at much below this level. However, recordings from the DILI sensillum, which contain 14 receptor cells, were extremely complex (Fig. 2). The great number of chemosensitive units firing-did not permit either clear determination of the exact number of cells responding or if 2-nitrophenol and 4>methyl-2-nitrophenol stimulated the same receptors). Lactone receptor. One receptor in the DI.l sensillum was sensitive to y-valerolactone, found only in traces in bovine and rabbit odour (Fig. 3). Double successive stim- ulations indicated that this receptor also responded to 6-caprolactone. y-Butyrolactone was only a weak stimu- lant at a high concentration (10"* moles in the stimulus cartridge). The difficulty of distinguishing the lactone re- ceptor from the four other receptors housed in the same sensillum during weak stimulation prohibited the estab- lishment of clear dose-response curves (Fig. 3), Acid receptors. Short-chain fatty acid receptors in the DILI sensillum responded best to pentanoic acid (Fig. 4). 3-Methylbutanoic acid and butanoic acid were also ac- tive but only at relatively high concentration (Fig. 4). Nevertheless, the complexity of the multicellular record- ings from the DILI sensillum (with 14 receptor cells) in- hibited us from properly studying the responding recep- tors) and from establishing a dose response-curve. Two receptor cells of another sensillum (DII.5) on the anterior pit also responded to C4 and Cs fatty acids; hexanoic, pentsnotc »ctd 10"' moles 1s butanoic add 10'a motM 10'7 mol« 4ÄII 18 Fig. 4. Responses of olfactory receptors of the DILI sensillum of male A. variegatum to butanoic acid and pentanoic acid. Stimulus intensity (number of moles in the stimului cartridge) is indicated above each trace. Horizontal bars 1 a stimulation. The great com- plexity of spike trains recorded from the DIM iensillum, which contain 14 receptor cells, prohibited us from properly analysing the receptor(s) responding to these short-chain fatty acids heptanoic and nonanoic acids only weakly excited these receptors. By contrast with the DILI sensillum, the DII.5 sensillum only contains three receptor cells. It was thus possible to differentiate the two fatty receptors of the latter sensillum according to spike shape and amplitude as well as by double successive stimulations (Fig. 5). One receptor in this sensillum (type I in Figs. 5 and 6) was most strongly stimulated by butanoic acid but also clear- RESULTS - CHAPTER 3.4. 59 P. Sieullet. P.M. Guerin: Olfactory receptors of Àmblyomma. II WH 2222222222 222222222 2222^22 2 2 2 222 B ! 11 ] 111 1I1 j 1 1 11 IU 2 2 Fig. SA, B. Double successive stimulations of fatty acid receptors in the DII.5 sensillum with 10"* moles of 2-methylpropanoic acid (solid horizontal bar) and IO"8 moles of butanoic acid (hollow hori- zontal bar). Noie that firing of receptor type 1 in- creased at the beginning of stimulation with bu- tanoic acid (in A), whereas firing of receptor type 2 increased at the beginning of stimulation with 2-methylpropanoic acid (in B). Each trace (A and 2 '22 222222'0^229222'2 B) corresponds to a 700 ms spike train. Spikes with 2 2 2z2^2 2 underlined numbering are near or overlapping ^HNB^MHH^^^i^ events. Vertical bar 0.5 mV 2-methylpropanolc acid butanoic acid -10 -9 -8 -7 log [moles] 3-methylbutanolc acid 10 -9-8-7 log [moles] pentanoic acid -10 -9 -8 -7 log [moles] 10 -9 -8 -7 log [moles] Rg. 6. Responses of two types of fatty acid receptors in the DII.5 sensillum located on the anterior pit of the Haller's organ in male A. variegatum to doses (moles in the stimulus cartridge) of 2-methyl- propanoic, 3-methylbutanoic, butanoic, and pentanoic acid plotted against spike frequency calculated for the first 400 ms of the re- sponses (n = 8 different ticks). Trend lines connect mean values; bars are standard deviations. Solid circle: acid receptor type 1 most sensitive to butanoic acid; hollow circle: acid receptor type 2 most sensitive to 2-methylpropanoic acid Iy by 2-methylpropanoic acid, 3-methylbutanoic acid and, to a lesser extent, by pentanoic acid. The second receptor was most sensitive to 2-raethylpropanoic acid (type 2 in Figs. 5 and 6). To establish if the two fatty acid receptors of the DII.5 sensillum are capable of specifically coding for the different short-chain fatty acids, a firing ratio (Q) between the intensity of the response of receptor type 1 and type 2 to the different acids at various concen- trations was calculated (Table 2). Q values for the dose series of 2-methylpropanoic acid were significantly differ- ent from those obtained with a similar dose series of 3- methylbutanoic acid (P £ 0.05, ANOVA with the Gener- al Linear Model procedure of SAS). Both series of Q values were concentration-dependent (P ^ 0.05, Table 2). The butanoic acid and pentanoic dose-series did not elic- it divergent Q values (P > 0.05), and these Q values were concentration-independent (P > 0.05, Table 2). However, Q values for these unbranched fatty acids differed signifi- cantly from those due to the branched fatty acids (P <, 0.05, Table 2). Butanoic and 2-methylpropanoic acids were the most prominent short-chain fatty acids in bovine and rabbit odour, respectively, as collected on Porapak (accounting for > 75% of the total amount of the C4 and C5 fatty acids present). However, no increase in spike frequency of the acid receptors was observed when fatty acids con- tained in bovine and rabbit odour eluted during GC-EL analysis of extracts, most probably because the receptors were not sensitive enough to detect the amounts present. Finally, despite its strong acidic odour, human axillary secretion did also not excite the acid receptors of either the DILI or DII.5 sensilla. Table 2. Firing ratio Q (mean ± SD) of fatty acid receptor type 1 to receptor type 2 in the DII.5 sensillum of 8 different adult A. variegatum in response to stimulation wiih 4 short-chain fatty acids at 4 concen- trations Number of moles in the stimulus cartridge io- 10 -9 10" io- 2-methylpropanoic acid O.7±0.2 3-methylbutanoic acid 1.4+0.5 Butanoic acid 2.3 ±0.3 Pentanoic acid 2.8 ±1.1 0.8 ±0.3 1.3 ±0.6 3.1 ±1.0 0.9 ±0.5 1.8±0.2 2.9 ±0.7 0.9 ±0.4 2.0 ±0.7 2.0 ±0.5 3.2 ±1.2 3.4 ±0.9 2.5 ±0.8 Sb c § Signifies that Q is concentration-dependent for a defined stimulus, and Q for each stimulus is assigned a different letter when significantly different (p£0.05 in both cases, ANOVA with the General Linear Model procedure on SAS) RESULTS - CHAPTER 3.4. 60 P. Steullel. P.M. Guerin: Olfactory receptors of Ambtyomma. II Pig. 7À, B. Responses of two NH3 receptors of the DII.6 sensillum located on the anterior pit of Haller's organ in male A. variegatum. A Dose (moles in the stimulus cartridge) of NH4OH plotted against spike frequency calculated for the first 400 ms of the responses (fi = 6 different ticks). Trend lines connect mean values; bars are standard deviations. Hollow circle: NH1 receptor type 1; solid cir- cle: NH3 receptor type 2. B Representative response of the NH1 receptor types I and 2 to stimulation with 1.4 x 10-9 moles NH4OH in the stimulus cartridge. The upper trace provides detail of 250 ms of the response. A spike of low amplitude from a third receptor also figures on this trace. Horizontal bar 1 s stimulation; vertical bar 0.5 mV 3-pefrfanonff 10 moles 1s Fig. 8. Representative response of a receptor located in one of the DIV sensilla on the tarsus of adult A. variegatum to I0"T moles 3-pentanone in the stimulus cartridge NH3 receptors and others. Two receptors responded strongly to the same range of NH3 concentrations in the DII.6 sensillum such that between 10 l0 and 10"9 moles of NH4OH in the stimulus cartridge was sufficient to evoke a response (Fig. 7). However, the dose-response curve of the two receptors differed significantly (P ^ 0.05, ANOVA with the General Linear Model procedure of SAS). Another receptor in one of the DIV sensilla was also weakly stimulated by high, but not physiologically relevant concentrations of NH3. This receptor was about 100 times less sensitive to NH, than either of the two receptors in the DII.6 sensillum. Except for a receptor in another of the DIV sensilla which responded to 3-pen- tanone (Fig. 8), no further receptors in the wall-pore sen- silla located outside the capsule of Haller's organ on tar- sus I of A. variegatum were characterized. Discussion Olfactory receptors in wall-pore sensilla on the surface of the tarsus of leg I in A. variegatum respond to con- stituents of vertebrate odours. GC-EL analyses of bovine odour presented here demonstrate the response of recep- tor(s) to nitrophenols in the large DILI olfactory sensil- lum on the anterior pit of Haller's organ. Other receptors, which were found in this and other sensilla, were sensitive to either lactones, short-chain fatty acids, NH3, or 3-pen- tanone. However, no responses were obtained from re- ceptors in any of these sensilla to vertebrate odours test- ed under the headings of human breath, human axillary secretion, or rabbit odour collected on Po rapale. This contrasts with the responses of a range of olfactory recep- tors within the capsule of Haller's organ to different con- stituents of the same extracts (Table 3, SteuIIet and Guer- in 1992a, b, 1993). The largest wall-pore sensillum (DII.1) on the anterior pit of Haller's organ presumably bears several receptors for the aggregation-attachment pheromone component 2-nitrophenol as evidenced by the multi unit responses of Schoeni (1987). In the present study, GC-EL experiments employing this sensillum reveals the presence of four ac- tive components in odour of tick-naive steer collected on Porapak, i.e. 2-nitrophenol, 4-methyI-2-nitrophenoL and two unidentified volatiles. 4-Methyl-2-nitrophenol is the most abundant of the 4 stimulants in the odour and is also present in skin wash of steer. GC-MS analysis of air from an unoccupied stall (controls) indicates none, or at most traces of these nitrophenols, suggesting that these products are true vertebrate volatiles although neither of them has previously been reported to our knowledge from vertebrates. However, these compounds are known as air pollutants (Welsh and Watts 1990), but as such are likely to be less prevalent in the ambient air of the African habitat of A. variegatum than in the suburban environ- ment in which controls were made for this study. There can be little doubt about the biogenic origin of nitrophe- nols, as 2-nitrophenol is also a major component of the aggregation-attachmen* pheromone of both A. variega- tum and A. hebraeum (Schoeni et al. 1984; Apps et al. 1988). It is produced in high amounts in dermal glands (type 2) of males after successful attachment and feeding on a host (Diehl et al. 1991), thereby assuring attraction, aggregation, and attachment of conspecifics at the same feeding site (Schoeni et al. 1984; Norval et al. 1989; Delot 1990). The presence of nitrophenols in odours of even tick-naive steer could favour their infestation by pioneer males. A. variegatum does show a preference for parasitiz- ing bulls over goats (Barré et al. 1991), and attach better on cattle than on sheep or rabbits (Norval et al. 1992). The present study shows that quantities of nitrophenols found in bovine odour were higher than in rabbit odour extracts, the latter containing 100 times less 4-methyl-2- nitrophenol. Could it be that Amblyomma has developed RESULTS - CHAPTER 3.4. 61 P. Steullet. P.M. Guerin: Olfactory receptors of Amblyomma. II Tabic 3. Responses and locations of characterized olfactory receptors on tarsus I of male A. variegatum to different vertebrate odours (human breath, human axillary secretion, and bovine and rabbit odour concentrates as collected on Porapak) a) Receptor b) Human Human Bovine Rabbit location Sensillum breath axillary odour odour type secretion Characterized receptors References CI wp-sw - CII wp-sw - cm wp-sw + + + cm wp-sw + + + civ wp-sw + + + CV wp-sw + + + CVI* wp-sw - CVI* wp-sw - evi* wp-sw — DII wp-sw — DIl wp-sw — DILI wp-sw — DIM wp-sw - DII.5 wp-dw - DH.5 wp-dw - DII.6 wp-dw - DIV.group wp-dw - DIVgroup wp-dw (+) ( + ) + + + + + + + + + + + + + + + + + + + + + (+) (+) + + (+) (+) (+) (+) (+) (+) (+) lactone receptor methylsalicylate receptor CO1-excited receptor sulfide receptor type 2 COz-inhibtted receptor sulfide receptor type I benzaldehyde receptor 2-hydroxybenzaldehyde receptor aliphatic aldehyde receptors lactone receptor 2,6-dichlorophenol receptor nitrophenol receptorfs) pentanoic acid receptor(s) 2-methylpropanoic acid receptor butanoic acid receptor NHj receptors § receptor sensitive to high doses of NH3 § receptor sensitive to high doses of 3-pentanone Steullet and Guerin (ibid.) Hess and Vl imant (1984) Steullet and Guerin (1992a) Steullet and Guerin (1992b) Steullet and Guerin (1992a) Steullet and Guerin (1992b) Steullet and Guerin (ibid.) Steullet and Guerin (ibid.) Steullet and Guerin (ibid.) this paper Waladde 1982, Schoeni 1987, this paper Schoeni (1987), this paper this paper this paper this paper this paper this paper this paper + + + Strong response, ++ medium response, (+) stimulant present in the extract but in insufficient quantity to elicit more than a slight response, — no response, a) Name assigned to the sensillum according to its location on the surface of the tarsus (DI-DIV of Fig. 1) or region within the capsule (CI-CVI of Fig. 1 in Steullet and Guerin 1993); * signifies presence of two sensilla in the region from which recordings were made within the capsule; b) morphological type of sensillum as described by Hess and Vlimant (1982): wall-. pore single-walled sensillum (wp-sw), wall-pore double-walled sen- sillum (wp-dw). § NH3 is a common vertebrate-associated volatile, eg. in urine. No receptors from the DIII.2, LAII-I, VTLl1 and VTM sensilla (Fig. I) were characterized a pheromone system which enhances attractivity of what proves to be a suitable host for pioneer males by secreting high amounts of a host-associated volatile? This would not constitute the first report on use of vertebrate-associ- ated volatiles as components of an aggregation-attach- ment pheromone in the genus Amblyomma. 2-Methyl- propanoic acid, nonanoic acid, 2-nitrophenol, and ben- zaldehyde are components of the aggregation-attach- ment pheromone of A. variegatum and/or A. hebraeum, but also vertebrate-associated volatiles to which some tick olfactory receptors are sensitive (Steullet and Guerin 1993). A receptor in the DI. I sensillum on the knoll distal to Haller's organ responds strongly to both y-valerolactone and 6-caprolactone, as confirmed by double successive stimulations. The specificity of this receptor differs from that of another lactone-sensitive receptor found in the capsule of Haller's organ in A. variegatum which is more selectively sensitive to y-valerolactone (Steullet and Guerin 1993). y-Valerolactone is present in traces in bovine and rabbit odour collected on Porapak. But the quantity of y -valerolactone in bovine odour extract was not sufficient to evoke a clear response of this receptor in GC-EL analyses employing the DI. 1 sensillum, as was the case for the lactone receptor in the capsule of Haller's organ (Steullet and Guerin 1993). Lactones are reported from many vertebrate odours (Goetz et al. 1988; Burger et al. 1990), but are unknown as stimulants for other haematophagous arthropods. Two wall-pore sensilla on the anterior pit of Haller's organ of A. variegatum possess fatty acid receptors: the wall-pore single-walled DILI sensillum with receptors) most sensitive to pentanoic acid, and the wall-pore dou- ble-walled DII.5 sensillum with one receptor most sensi- tive to butanoic acid and a second most responsive to 2-methylpropanoic acid. Comparison of the spike fre- quencies of these receptors in response to stimulation with the different acids indicates that the two acid recep- tors of the DII.5 sensillum can specifically code for 2- methylpropanoic acid and 3-methylbutanoic acid, but cannot discriminate between butanoic and pentanoic acid. However, discrimination between these straight- chain fatty acids may be possible with the acid receptors) of the DILI sensillum which are most sensitive to pen- tanoic acid. Equipped with these acid receptors, A. varie- gatum may be able to discriminate between mixtures of short-chain fatty acids which are widespread in verte- brate-associated volatiles (Müller-Schwarze et al. 1974; Ayorinde et al. 1982; Fox 1982; Albone 1984; Goetz et al. 1988; Kanda et al. 1990). GC-MS analysis of the odour extracts collected on Porapak indicated that butanoic was the most abundant fatty acid in bovine odour, whereas 2-methylpropanoic acid predominated in that of rabbit. However, the acid receptors of both the DILI and the DII.5 sensilla were not sensitive enough to respond to the fatty acid amounts present in extracts injected for GC-EL experiments. Furthermore, despite of its acidic odour, human axillary secretion did not clearly excite the RESULTS - CHAPTER 3.4. 62 fatty acid receptors of A. variegatum. This may be due to the fact that human axillary secretion contains an abun- dance of different branched and unbranched, saturated and unsaturated C6 to Cn fatty acids with (E)-3-methyl- 2-hexenoic acid as the major component (Zeng et al. 1991, 1992), but lacks significant amounts of the shorter acids for which the tick possesses receptors. Short-chain fatty acids also act as olfactory stimulants for several blood-sucking insects, i.e. mosquitoes (Lacher 1967) and Triatoma infestans (Bernard 1974). Furthermore, these volatiles elicit probing behaviour in Stomoxys calcitrans (Hopkins 1964) and attraction in the sheep head fly Hy- drotaea irritans (Thomas et al. 1985). Two receptors sensitive to NH3 are present in the DII.6 sensillum, each showing its own response profile. Another receptor in the DIV wall-pore sensilla group also responds to NH3, but only at much higher doses. Similar NH3 receptors exist in another tick species, Rhipi- cephalus sanguineus (Haggart and Davis 1980). NH3 is known as a kairomone for some haematophagous arthropods, i.e. as a probing stimulus for Stomoxys calci- trans (Gatehouse 1970), and attractant for Tabanidae (Hribar et al. 1992). Although NH3 is widely represented in vertebrate odours, none of the tick NH3 receptors re- sponded to the extracts tested here. One reason may be that the porous polymer (Porapak), which we used to collect vertebrate odours, is known to have a low affinity for polar low molecular weight volatiles such as NH3 and amines (Sugisawa 1981). Finally, as already described by Waladde (1982) and Schoeni(1987), receptors in the DLl and the DILI sensil- la are highly sensitive to the common tick sex pheromone product, 2,6-dichIorophenol (Table 3). No clear responses were obtained from these receptors in single-unit record- ings with whole odour extracts of vertebrates, or to indi- vidual components of these extracts in GC-EL analyses. These findings confirmed our GC-MS analyses which in- dicated that the halogenated phenol was not present in detectable amount in the vertebrate odours collected for this study. Although Lees (1948) suggested that the olfactory re- ceptors responsible for host-odour perception in ticks are mainly located within the capsule of Haller's organ, this study clearly demonstrates that sensilla located elsewhere on the tarsus also respond to some vertebrate-associated volatiles. However, these sensilla do not only contain ol- factory receptors. Although human breath delivered di- rectly to the tarsus activated receptors in both the DII.5 and DII.6 sensilla (Steullet, unpublished), the same stimu- lus delivered in a temperature- and humidity-controlled airflow to the preparation did not excite any receptor. This suggests that thermoreceptors might be involved in the response to breath. In some insects, grooved double- walled sensilla contain olfactory receptors together with a cold unit (Altneret al. 1981; Steinbrecht 1984). Breath components, essential for the arousal of resting A. varie- gatum to initiate host-finding, seem to be exclusively de- tected by olfactory receptors within the capsule of Haller's organ (Steullet and Guerin 1992a, b), while re- ceptors sensitive to other vertebrate-associated volatiles and/or pheromone compounds are distributed among P. Stcuilet, P.M. Guerin: Olfactory receptors of Amblyomma. II both the capsule sensilla and those situated on the sur- face of the tarsus I. Table 3 summarizes responses of all characterized olfactory receptors on tarsus I of A. varie- gatum to the different vertebrate odours tested in our studies (Steullet and Guerin 1992a and b, 1993). This array of characterized olfactory receptors certainly equips A. variegatum for a finely tuned image of its odor- ous environment, and opens a new avenue of research on the behavioural responses of this tick species to the iden- tified stimulants and their role in host-finding. Acknowledgements. We thank Professor Peter Alan Diehl of this Institute for helpful discussions, his interest in this study, and for valuable comments on the manuscript We are grateful to Dr JJ.B. Smith, University of Toronto, and to Dr B.K. Mitchell, University of Alberta (Edmonton), for kindly providing us with their spike analysis programme SAPID. We thank Misters Jonczy and Rohrcr of Ciba-Geigy Ltd., St-Aubin (Switzerland) for supplying us with adult ticks, and for access to the animal stalls. We are indebted to the Hasselblad, Roche and Sandoz Foundations, and the Swiss National Science Foundation (Grant Nos. 3.609-087 and 31- 28684.90», the Ciba-Geigy-Jubilaeums-Stiftung, and the Swiss Office for Education and Science for funding studies on tick sensory phys- iology at the University of Neuchätel. This article is part of the thesis being presented by P. Steullet for the PhD. degree at the University of Neuchätel. References Albone ES (1984) Mammalian semiochemistry. John Wiley & Sons, Chichester New York Altner H, Sass H, Altner I (1977) Relationship between structure and function of antenna! chemo-, hygro-, and thermoreceptive sensilla in Periplaneta americana. Cell Tissue Res 176:389-405 - Altner H, Routil Ch, Loftus R (1981) The structure of bimodal chemo-, thermo-, and hygroreceptive sensilla on the antenna of Locusta migratoria. 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J Neurosci Methods 29:77-83 Goetz N, Kaba G1 Good D, Hussler G, Bore P ( 1988) Detection and identification of volatile compounds evolved from human hair and scalp using headspace gas chromatography. J Soc Cosmet Chem 39:1-13 Haggart DA, Davis EE (1980) Ammonia-sensitive neurons on the first tarsi of the tick, Rhipicephalus sanguineus. J Insect Physiol 26:517-523 Haggart DA, Davis EE (1981) Neurons sensitive to 2,6- dichlorophenol on the tarsi of the tick Ambiyomma americamm (Acari, Ixodidae). J Med Entomol 18:187-193 Hess E, Vlimant M (1982) The tarsal sensory system of Ambiyomma variegatum Fabricius (Ixodidae, Metastriala). I. Wall pore and terminal pore scnsilla. Rev Suisse Zool 89:713-729 Hess E, Vlimant M (1983) The tarsal sensory system of Ambiyomma variegatum Fabricius (Ixodidae, Metastriata). III. Mapping of sensory hairs and evolution of the relative importance of sensory modalities during post-embryonic development. Rev Suisse Zo- ol 90:887-897 Hopkins BA (1964) The probing response of Stomoxys calcitrata (L.) (the stable fly) to, vapours. Anim Behav 12:513-524 Hribar U, Leprince DJ, Foil LD (1992) Ammonia as an at tractant for adult Hybomitra lasiophthalma (Diptera: Tabanidae). J Med Entomol 29:346-348 Kanda F, Yagi E. Fukuda M, Nakajima IC Onta T. Nakata O (1990) Elucidation of chemical compounds responsible for foot mal- odour. Br J Dermatol 122:771-776 Lacher V (1967) Elektrophysiologtsche Untersuchungen an einzel- nen Geruchsrezeptoren auf den Antennen weiblicher Moskitos (Aedes aegypti L). J Insect Physiol 13:1461-1470. Lees AD (1948) The sensory physiology of the sheep tick, Ixodes ricinus. S Exp Biol 25:145-207 Müller-Schwarze D, Müller-Schwarze C, Singer AG, Silverstein RM (1974) Mammalian pheromone: identification of active component in the subauricular scent of the male pronghom. Science 183:860-862 Norval RAl, Andrew HR, Yunker CE (1989) Pheromone-media- tion of host-selection in bont ticks [Ambiyomma hebraeum Koch). Science 243:364-365 * Norval RAI, Peter T, Meltzer MI (1992) Comparison of the attach- ment rates of males of the ticks Ambiyomma hebraeum and A. variegatum to cattle, sheep and rabbits in the absence of aggre- gation-attachment pheromone. Exp Appi Acarol 15:145-152 Schoeni R (1987) Das wirtsgebundene Aggregationspheromon der tropischen Buntzecke Ambiyomma variegatum (Acari: Ixodidae). Thèse Université Neuchâtel, Switzerland Schoeni R, Hess E, Blum W, Ramstcin K (1984) The aggregation-at- tachment pheromone of the tropical bont tick Ambiyomma var- iegatum Fabricius (Acari: Ixodidae): isolation, identification and action of its components. J Insect Physiol 30:613-618 Steinbrecht RA (1984) Arthropoda: chemo-, hygro-, and thermore- ceptors. In: Bereiter-Hahn J, Matolsky AG, Richards KS (eds) Biology of the integument. Springer, Berlin, pp 523-553 Steullet P, Guerin PM (1992a) Perception of breath components by the tropical bont tick, Ambiyomma variegatum Fabricius (Ixodi- dae). I. C02-excited and CXVinhibited receptors. J Comp Phys- iol A 170:665-676 Steullet P, Guerin PM (1992b) Perception of breath components by the tropical bont tick Ambiyomma variegatum Fabricius (Ixodi- dae)TI. Sulfide receptors. J Compi Physiol A 170:677-685 Steullet P, Guerin PM (1993) Identification of vertebrate volatiles stimulating olfactory receptors on tanus I of the tick Ambiy- omma variegatum Fabricius (Ixodidae) L Receptors within the Haller's organ capsule. J Comp Phyiiol A 173: ¦-¦ Sugisawa H (1981) Sample preparation: isolation and concentra- tion. In: Tcranishi R, Flath RA, Sugisawa H (eds) Flavor re- search recent advances. Marcel Delcker Ine, New York Basel, pp 15-51. Thomas G, Schomaker CH, Been TH, van den Berg MJ, Prisjs HJ (1985) Host finding and feeding in Hydrotaea irritant (Diptera, Muscidae): the role of chemical senses. Vet Parasitol 18:209-221 Thonney F (1987) Etude morphologique et itructurale des récepteurs sensoriels du tarse I de la tique Ixodet ricinus L, Thèse Université Neuchâtel, Switzerland Waladde SM (1982) Tip-recording from ixodid tick olfactory sensil- Ia: responses to tick related odours. J Comp Physiol 148:411- 418 Waladde. SM, Rice MJ (1982) The sensory basis of tick feeding behaviour. In: Obenchain FD, Galun R (eds) Physiology of ticks. Pergamon, Oxford, pp 71-118 Welsh DI, Watts CD (1990) Collection and identification of trace organic compounds in atmospheric deposition from a semi- rural site in the U.K. Int J Environ Anal Chem 38:185-198 Zeng X-N, Leyden JJ, Lawlcy HJ1 Sawano K, Nohara I, Preti G (1991) Analysis of characteristic odors from human male axillae. JChemEcol 17:1469-1492 Zeng X-N, Leyden JJ, Brand JG, Spielman AI, McGinky KJ, Preti G (1992) An investigation of human apocrine gland secretion for axillary odor precursors. J Chem Ecol 18:1039-1055 GENERAL DISCUSSION 64 IV. GENERAL DISCUSSION 4.1. Location and number of the tick olfactory sense organs Several behavioural studies have indicated that the site of odour perception in ticks is essentially located on the tarsus of the first leg pair (Hindle and Meni man 1912; Lees 1948) where typical olfactory sensilla are exclusively found in ticks (Hess and Vlimant 1986; Vlimant personal communication). Consequently, this study has only investigated the functionality of the tarsal wall-pore sensilla in Amblyomma variegatum, although some terminal pore sensilla on the tarsus or elsewhere on the tick (Hess and Vlimant 1982, 1983, 1986) might detect vapours at very close distance as Stadler and Hanson (1975) demonstrated in tobacco hornworm larvae. Compared to many adult insects, ticks possess relatively few olfactory sensilla and hence a low number of receptors (less than 100 per tarsus according to Hess and Vlimant 1986). In blood-sucking insects, Mclver (1987) found a direct correlation between the approximate number of chemosensitive neurones per antenna and the distance travelled by the insect to the host. Thus, Pediculus humanus which lives on the host has only ca. 50 olfactory receptors, compared to the 12000 of Stomoxys calcitrans. On the other hand, Cimex lectularius which lives a short distance from its host, i.e. in bedsteads or under the wallpaper, possesses only some 200 chemosensitive neurones. Ticks, who do hardly respond over distances of 10-20 meters to a host, possess about the same number of olfactory receptors as Cimex. In ticks, the number of olfactory sensilla also tends to decrease in more advanced genera. Rather primitive ticks, such as Ixodes ricinus, carry 20 olfactory setae per tarsus whereas the highly specialized one-host tick, Boophilus microplus, has only 14 (Hess and Vlimant 1986; Thonney 1987). However, this reduction does not lead to a decrease in the olfactory capability (number of receptors), but to a concentration of the olfactory receptors within fused sensilla (Hess and Vlimant 1982). Most tarsal sensilla are grouped into two clusters, accounting for about half of all tick olfactory receptors, to form the Haller's organ on the dorsal side of the tarsus with an anterior pit and the posterior capsule. GENERAL DISCUSSION 65 4.2. The capsule of Haller's organ, a complex olfactory organ Whereas the anterior pit of Haller's organ carries a group of structurally different sensilla (wall-pore single-walled, wall-pore double-walled, and no pore sensilla), the capsule consists of a cavity which encloses only wall-pore single- walled sensilla, i.e. primitively a total of 9 according to Leonovitch and Belozerov (1992), 7 in A vahegatum, A americanum and I. ricinus (Foelix and Axteil 1972; Hess and Vlimant 1982; Thonney 1987), and only 4 in Hyalomma asiaticum and B. microplus (Leonovitch 1978; Waladde and Rice 1982). Furthermore, the degree of enclosure within a capsule varies from one tick species to another. In some Ixodes species, the capsule is completely open and forms a kind of pit (Homsher and Sonenshine 1979), whereas in most other Pro- and Metastriate ticks the capsule is a deep cavity with a small opening of variable shape, i.e. round in I. ricinus (Thonney 1987) and slit-like in A variegatura (Hess and Vlimant 1982). The biological significance of this encapsulation is thought to protect some olfactory sensilla against mechanical damage (Foelix and Axteil 1972) and/or desiccation. Ticks do indeed lead a tough life with long non-parasitic phases surviving in sometimes quite hostile conditions and are exposed to violent grooming by the host during feeding-site selection, biting, and feeding phases. Interestingly, the wall-pore sensilla inside the capsule are more delicate than the wall-pore setae outside the capsule. Thus in A. variegatum, the capsule sensilla have ten times thinner walls (ca. 0.1 urn) and bigger pores than the wall-pore single-walled sensilla outside the capsule (Hess and Vlimant 1982). In I. ricinus, the most exposed capsule sensiIlum also possesses thicker walls than less exposed ones (Thonney 1987). Larger and numerous pores as well as remarkably thin walls favouring diffusion of odour molecules into the capsule sensilla essentially compensates for enclosure within a capsule. The present study has shown that the molecular diffusion and air turbulence around the tarsus permits access of vapours to the confined sensilla (chapter 3.1.). Therefore, encapsulation does not limit the spatial field of perception of the capsule sensilla, such that olfactory stimulants carried by wind from any direction are perceived (chapter 3.1.). Consequently, the capsule of Haller's organ is a sensory organ with a specialized architecture designed to protect olfactory sensilla without restricting vapour perception or sensitivity. The capsule contains olfactory receptors essential for successful completion of the life cycle in most tick species. Lees (1948) proposed that the capsule of I. ricinus comprises most of the olfactory functions whereas the anterior pit is essential for hygroreception. Such a functional distinction between the anterior pit and the capsule is however too simplistic, as we now GENERAL DISCUSSION 66 know from electrophysiological experiments in A variegatum that many olfactory receptors are also located outside the capsule on the tarsus. Thus, nitrophenol, lactone, fatty acid, NH3, and 2,6-dichlorophenoI receptors are found in the anterior pit of Haller's organ (chapter 3.4.). However in A variegatum, volatiles in vertebrate breath which are primordial tick arousers only stimulated receptors within the capsule , i.e. C02-receptors and sulfide- receptors (chapters 3.1. and 3.2.). This might also be the case for other tick species, although Waladde and Rice (1982) reported responses to human breath in receptors of the anterior pit of B. microplus. However, the latter finding might have been due to temperature changes associated with the stimulation method employed as was the case for receptors responding to human breath in the DILS and 6 sensilla of A variegatum in this study (chapter 3.4.). Moreover, the capsule of Haller's organ of A variegatum is a complex olfactory organ housing many receptors (21-35) of different specificities which respond to various host-associated volatiles (CO2, H2St mercaptans, saturated and unsaturated aliphatic aldehydes, lactones, furfural, and benzaldehyde-related compounds), to products which can be considered as both host-odour volatiles and as tick pheromone components (benzaldehyde, 2-hydroxybenzaldëhyde), and to the tick pheromone component, methylsalicylate (chapters 3.1., 3.2., and 3.3.). The peculiar life style of ticks and the low, albeit highly diversified, number of olfactory receptors per tick species concentrated in just a few wall- pore sensilla on the first legs (used both as "antennae" and for running) might in the end justify some special protection. 4.3. Specificity and sensitivity of the olfactory receptors Ticks possess an outstanding variety of tarsal olfactory receptors inside but also outside the capsule. The present study reveals that A variegatum can detect a vast range of volatiles such as CO2, H2S, mercaptans, NH3, branched and unbranched short-chain fatty acids, saturated and unsaturated aliphatic aldehydes, lactones, furfural, benzaldehyde, 2-hydroxybenzaldehyde, other benzaldehyde-related compounds, methylsalicylate, and both nitro- and chlorophenols (Table 4.1. and chapters 3.1., 3.2., 3.3., and 3.4.). The diversity of olfactory receptors is even greater as several receptors were not successfully characterized. Some of them responded to unidentified components of human breath (chapters 3.1. and 3.2.), bovine and rabbit odours (chapters 3.3. and 3.4.), and others were not stimulated by any of the tested synthetic volatiles or host odours. Each characterized receptor of A. variegatum appears to have an unique response spectrum. Each has shown a response to GENERAL DISCUSSION 67 a limited range of compounds from the same chemical class, but a particular class of olfactory stimulants was in most cases monitored by at least two receptors with overlapping specificity spectra, i.e. acid receptors in the DII.1 and DII.5 sensilla (chapter 3.4.), aldehyde receptors 1, 2, 3 in the capsule (chapter 3.3.), sulfide receptors 1, 2 (chapter 3.2.), lactone receptors in both the capsule and Dl.1 sensillum (chapter 3.3. and 3.4.), benzaldehyde and 2- hydroxybenzaldehyde receptors in the capsule (chapter 3.3.). Most of the olfactory receptors investigated in A. variegatum could not be considered as either "specialists" or "generalists", but rather of an intermediate form, the "specialized generalist" as proposed by Kaissling (1971). A. variegatum is thus equipped with paired or grouped receptors which allow odour quality coding as proposed by Tichy and Loftus (1983) and Visser (1986). This sensory organisation is widespread in insect olfaction, i.e. stick insects (Tichy and Loftus 1983) and cockroaches (Selzer 1984). The olfactory system of A. variegatum appears to be able to provide a maximum of information about quality of volatiles with a minimum number of receptors and seems to be, by and large, as sophisticated as that of most insects. With the different types of olfactory receptors, ticks seem to be capable to differentiate the complex of host odours from others in its environment. Furthermore, some special features also allow refined evaluation of volatile concentrations. Thus, CO2 concentration is monitored by two antagonistic types of receptors which work in a different range. This consequently permits detection of a precise concentration over a wide range. The C02-inhibited receptor is sensitive to very small concentration changes between 0 and 0.2%, whereas the C02-excited receptor best monitors concentrations above 0.1% (chapter 3.1.). Systems with excited and inhibited receptors have already been described in insects, and have been ascribed different functions. Thus in mosquitoes, lactic acid respectively excites and inhibits two types of receptors (Davis and Sokolove 1976). The sensitivity of the "LA-excited" receptors, but not that of the "LA-inhibited" receptors, depends on the physiological state of mosquitoes. The inputs of both types of receptors are thought to interact via CNS integration to finally dictate the behavioural sensitivity to lactic acid (Davis et al. 1987). Some of the volatiles detected by A. variegatum are commonly known as olfactory stimulants for other blood-sucking arthropods, whereas others are unusual. Tsetse flies possess receptors sensitive to CO2 and propana! but also to acetone, 2-butanone, 1-octen-3-ol, and methylphenol isomers (Bogner 1992, Den Otter et al. 1992). Furthermore, indole-related compounds and carotenoid metabolites caused strong EAG responses in Glossina (Hall 1990). On the GENERAL DISCUSSION 68 other hand, mosquitoes bear chemosensitive neurones for CO2, various fatty acids, and amines, but also for lactic acid, terpineol, and citral (Lacher 1967; Davis and Sokolove 1974; Geier, personal communication). Terpene receptors in mosquitoes are actually associated with plant-feeding behaviour (Bowen 1992). Finally in Stomoxys calcitrans, Lewis (1972) mentioned olfactory receptors responding to CO2, fatty acids, NH3, and amines, but also to esters and alcohols. Although haematophagous diptera possess a greater number of olfactory receptors than ticks (Mclver 1987), the diversity of the chemosensitive neurones in both taxonomic groups seems to be about the same, but in contrast to ticks, several receptors of each type occur on the antennae of these insects. Stomoxys, Glossina and mosquitoes may then not necessarily detect more subtle differences in host-odour composition than ticks, but may have a higher sensitivity thus responding to host odours from longer distance. Such a high sensitivity might be obtained via a high spatial convergence of the numerous chemosensitive neurones in the brain. Amplification along the integration process is already well-established in several insects (cf. reviews by Boeckh and Boeckh 1979; Light 1986; Boeckh and Ernst 1987; Visser and de Jong 1988) Table 4.1. provides a list of the best characterized olfactory receptors in male A. variegatum along with an approximate estimation of the sensitivity threshold based on GC-EL experiments. The 2,6-dichlorophenol receptor in the DI.1 sensilium, the 2-nitrophenol receptor in the DII. 1 sensillum, and the methylsalicylate receptor, benzaldehyde receptor and 2-hydroxybenzaldehyde receptor within the capsule were the most sensitive with less than 108 molecules/cm3 air of the stimulant already exciting these receptors (chapters 3.3. and 3.4.). The aliphatic aldehyde receptor (type 3) responding to (E)-2- heptenal and both sulfide receptors excited by H2S were also highly sensitive with thresholds estimated at between 108 and 109 molecules/cm3 air (chapters 3.2. and 3.3.). By contrast, CO2 receptors responded only to much higher changes in stimulus intensity (chapter 3.1.) making the C02-excited receptor ca. 106 to 107 times less sensitive than for instance the 2-nitrophenol receptor. The relative lack of sensitivity of CO2 receptors compared with that of other olfactory receptors has already been noted in insects (Boeckh et al. 1965). Presence of high ambient levels of CO2 (0.04% or even more en the litter zone) might go some way to explain the low sensitivity of the CO2 receptors. As defined by Weber's law, the resolving power of receptors should be better when the background level of the stimulant is low rather than high. On the whole, the estimated thresholds of the olfactory receptors of A. variegatum lie in GENERAL DISCUSSION 69 approximately the same range as most insect olfactory receptors, with the possible exception of some receptors for pheromone components, i.e. the bombycoi receptor in Bombyx (Boeckh et al. 1965). 4.4. Correlation between receptor specificity and sensillum ultrastructure Interestingly, most of the stimulants identified in this study were detected by receptors located in wall-pore single-walled sensilla (chapters 3.1., 3.2., and 3.3.; Table 4.1.). By contrast, only a few stimulants for receptors in wall-pore double-walled sensilla have been clearly identified, i.e. branched, unbranched short-chain fatty acids, and NH3 excited separate receptors enclosed in wall- pore double-walled C sensilla (DII.1 and DII.5 sensilla) (chapter 3.4.); NH3 and 3-pentanone weakly stimulated receptors within wall-pore double-walled A (DIV sensilla); but stimulants for the 4 to 7 receptors of wall-pore double-walled B sensilla (DIM.2, LAII. 1, VM. 1, and VII.4 sensilla) remain uncharacterized. However, the presence of a tubular body at the base of these wall-pore double- walled sensilla indicates that they have a mechanosensory modality. The ventral or lateral location of three of these sensilla may suggest a possible tactile function despite the fact that they appear to be typical wall-pore olfactory sensilla. But, further investigations on this type of sensillum are evidently needed. Correlations between receptor type and the sensillum ultrastructure have also been observed in insects: different classes of stimulants have been identified for receptors in wall-pore single-walled sensilla, i.e. fatty acids, aldehydes, alcohols, terpenes, esters, whereas wall-pore double-walled sensilla principally house fatty acid and amine receptors (Altner et al. 1977). In A. variegatum, pheromone and host-odour receptors are not enclosed in separate sensilla, i.e. 2,6-dichlorophenol and lactone receptors in the Dl.1 sensillum (chapter 3.4.). Neither is the perception of pheromone products is confined to special hairs restricted to a particular site of the tarsus, i.e. the methylsalicylate receptor was located within the capsule, whereas nitro- and chlorophenol receptors are found on the anterior pit. This suggests that by contrast with many insects A variegatum has not developed a highly specialized and distinct pheromone perception system separated from the host-odour receptors (for insects, see Visser 1986). GENERAL DISCUSSION 70 sonsHIum senslllum receptor typ« best stimulant a) threshold b) estimated name morphology [molecules/cartridge) threshold (molecules/cm3 airi DLl wp-sw A 2,6-dichloTophenol 2,6-dichlorophenol 10l2.i0l3 IO? -IO» DI.l wp-sw A lactone f-valerci acton e 1015 ? DIM wp-sw A nitrophenol 2-nitrophenol 1012.1013 107 .108 DIM wp-sw A 2,6-dichlorophenol 2,6-dichlorophenol 10l2-l0l3 10' -10» DIM wp-sw A fatty acid pentanoic acid 1014 ? DII.5 wp-dwC fatty acid (type 2) butanoic acid IO« ? DII.6 wp-dwC Tatty add (type 1) 2-methylpropanoic acid 10l2.10l3 ? DILB wp-dw C NH3 NH3 (NH4OH) IO".«" 1 DIV wp-dw A NHa NH3 (NH4OH) 1016 Ì DIV wp-dw A 3-pentanone 3-pentanone lOlß-1016 Ì capsule wp-sw B methylsaticylate methylsalicylate 1012.1013 10' IO« Capsule wp-sw B lactone f-valerc4actone IOI3.I0W 1 capsule wp-sw B aldehyde (type 1) hexanal 10»2,i0l3 10». ioio capsule wp-sw B aldehyde (type 2) heptanal 1013.10» 109- ioio capsule wp-sw B aldehyde (type 3) (E>2-heptenal 10l2.10l3 IO» 109 capsule wp-sw B bentaldehyde benzaldehyde 10l2.J013 10? IO» capsule wp-sw B 2-hydroxybenMldehyde 2-hydroxybenzaldehyde 10l2-10l3 10? 108 capsule wp-sw B C02-inhibited CO2 10W-iois e IO" IO" capsule wp-sw B C02*excited CO2 iole e 10« capsule wp-sw B sulfide (type 1) H2S ìoS-io10 8 IO« 109 capsule WD-SW B sulfide (type 2) H9S 109-lQlO 8 10» 109 Table 4.1. Characterized olfactory receptors of male A. variegatura. The estimated sensitivity for the best stimulant for each receptor is provided, a) minimum amount (in molecules) of stimulant in the stimulus cartridge which evoked a response; all the amount applied to the filter paper within the cartridge did not evaporate at once; g indicates that the stimulus within the cartridge was a gas; b) approximate concentration of the stimulant in air (molecules/cm^ air) sufficient to elicit a response as estimated by gas chromatography-coupled electrophysiology. This estimation was based on the minimum amount of stimulant injected onto the GC column necessary to elicit a response, the width of the eluting peak, the fraction of the column effluent directed to the preparation, and the dilution factor in the air stream carrying the product to the preparation. ? indicates that the threshold was not estimated with gas chromatography-coupled electrophysiology. Name as well as morphological type of sensillum housing the characterized receptor are also provided (nomenclature based on Hess and Vlimant 1982, 1986). No receptors within the wp-dw B sensilla (DIII.2, LAII. 1, VILI, and VII.4 sensilla) were characterized. GENERAL DISCUSSION 71 4.5. Conservatism in the evolution of the tick olfactory system The results of the present work and of previous electrophysiological studies on tick olfactory receptors suggest a high ultrastructural and functional homology among Ixodidae (Table 4.2.). Thus, receptors sensitive to 2,6- dichlorophenol have been described in the anterior pit of A. variegatum (Waladde 1982; Schoeni 1987, chapter 3.4.), A. americanum (Haggart and Davis 1981), Dermacentor variabilis (Sonenshine 1991), Rhipicephalus appendiculatus (Waladde 1982), I. ricinus (Thonney 1987), and B. microplus (de Bruyne and Guerin 1993). The aggregation-attachment pheromone components of A. variegatum, 2-nitrophenol and methylsalicylate, stimulated, respectively, receptors in the DIM sensillum and in the capsule of A variegatum (Hess and Vlimant 1986; Schoeni 1987; chapter 3.4.), but also of B. microplus (de Bruyne and Guerin 1993). Methylsalicylate also excited a receptor in the capsule of I. ricinus (Guerin, unpublished). NH3 receptors have been found in the anterior pit of A. variegatum (Guerin et al. 1992 and chapter 3.4.), and in Rhipicephalus sangineus (Haggart and Davis 1980). The Dl. 1 sensillum bears a lactone receptor in A. variegatum (chapter 3.4.), A. hebraeum (Steuilet, unpublished) and also in B. microplus (de Bruyne, unpublished). Finally in the capsule, sulfide receptors were discovered in both A. variegatum and Hyalomma dromedarii (chapter 3.2.). Nevertheless, whereas fatty acids stimulated only receptors outside the capsule in A. variegatum (Hess and Vlimant 1980 and chapter 3.4.), pentanoic acid was reported by Sinitsina (1974) to excite receptors in the capsule of H. asiaticum. From the above, the specificity of olfactory receptors in ticks would appear very homogeneous. Although a systematic comparative electrophysiological study on chemosensitive neurones of different tick species would be imperative for any firm conclusions, the tick olfactory system seems to have only weakly evolved. Based on very fragmentary knowledge, mites also seem to possess, at least partially, a closely-related olfactory system. A methylsalicylate receptor has been found on the dorsal side of the tarsus of the first leg pair of the mite, Phytoseiulus persimilis (de Bruyne et al. 1991). Moreover, many pheromone components of mites are furfural-related compounds or hydroxybenzaldehyde-related compounds (Kuwahara 1991). These are all chemically close to some stimulants for receptors of A. variegatum (chapter 3.3.). Most of the known tick stimulants also constitute commonly occurring semiochemicals for many phytophagous insects, i.e. CO2 (Bogner et al. 1986), fatty acids (Visser 1986), aldehydes (Visser 1986), benzaldehyde (Visser 1986; Hansson et al. 1989), 2-hydroxybenzaldehyde GENERAL DISCUSSION 72 (Wallace and Mansell 1976), or for scavenger insects, i.e. aldehydes, NH3, fatty acids, HgS, mercaptans (Waldow 1973). This and the fact that tick ancestors were presumably scavenger or phytophagous Acari suggests that the tick olfactory system has not significantly evolved. Tick ancestors were more likely generaiist and opportunistic species with morphological and physiological potential to explore new environments and occupy new ecological niches. Parasitism is indeed thought to be an innovation of such "chance feeder" species (Kim 1985). The olfactory system of tick ancestors was probably generalist enough to efficiently detect and recognize novel environments and novel diets without significant modifications. The lack of physiologically relevant differences in the peripheral olfactory system among ticks suggests that the different life cycles, host-finding strategies and host specificities has mostly evolved at the level of the integration of the sensory information rather than at the level of the sensory inputs. Such an hypothesis was already proposed by Hess and Vlimant (1986). Consequently, further studies on behaviour and on the CNS integration process seem to be highly essential to really understand host specificity in ticks. Furthermore, commonly occurring constituents in the pheromone communication system of ticks also suggests that intraspecific communication systems in ticks have only partially evolved without significant evolutionary specialization of the peripheral sensory system in contrast to many insects (Hansson et al. 1989). Vogt et ai. (1991) proposed that the pheromone sensory system of insects had evolved from a non-specialized olfactory system via modifications of odour-binding-proteins. However, such an evolution has not apparently occurred systematically in ticks. According to our current knowledge, only the tick pheromone component 2,6- dichlorophenol presumably involves a significant evolutionary specialization of the peripheral olfactory system in these organisms. By contrast, the aggregation-attachment pheromone components emitted by fed male Amblyomma to mark the infested host and make it more attractive for conspecifics do not constitute of highly specific or autonomous compounds. Except for methylsalicylate, the other components of the aggregation- attachment pheromone of A. variegatura (2-nitrophenol, nonanoic acid) and A. hebraeum (2-nitrophenol, nonanoic acid, 2-methylpropanoic acid, and benzaldehyde) have also been found in bovine and/or rabbit odour collected on Porapak (chapters 3.3., 3.4., and appendix). These compounds have also been reported in the literature as vertebrate-associated volatiles, i.e. benzaldehyde from muskox, rabbit, human, and mouse (Flood et al. 1989; Goodrich 1983; Preti et al. 1977; Andreolini et al. 1987), and nonanoic and 2-methylpropanoic GENERAL DISCUSSION 73 acid from chimpanzee, grysbok, rabbit and human (Fox 1982; Burger et al. 1981 ; Goodrich 1983; Kanda et al. 1990). This suggests that, to favour feeding and meeting of the sexes on an adequate host, A. variegatum and A. hebraeum have developed a pheromone communication system using volatiles associated with uninfested hosts and perceived by what were presumably pre- existing host-odour receptors. A methylsalicylate receptor has been also found in phylogenetically different tick species and even in mites, suggesting that this receptor was already present before A. variegatum incorporated the volatile in its aggregation-attachment pheromone. Though absent in the aggregation- attachment pheromone of A. variegatum, benzaldehyde and 2-methylpropanoic acid (present in that of A. hebraeum) were also detected by receptors of A. variegatum. From a fitness viewpoint, it would appear more astute to emit an aggregation-attachment pheromone composed of substances autonomous of the odour of uninfested hosts. However from an evolutionary viewpoint, the chance of developing an aggregation-attachment pheromone composed of host-associated volatiles which are perceived by the olfactory receptors necessary for the survival of the parasite was certainly higher. Indeed, it only involves an adjustment of the host-finding behaviour of male and female A. variegatum to respond to the increased amounts of these compounds released by mature males (see also chapter 4.6.) and not a significant modification or specialization of the peripheral olfactory system, confined as it is to a relatively few sensilla on the first leg pair. 4.6. Perception of vertebrate volatiles and host-finding Hidden in the litter zone, unfed resting adult A. variegatum await for a host (mostly large ruminants) passing nearby to evoke a succession of behavioural responses, i.e. arousal, initiation of locomotion, orientation and attraction, contact with the host, feeding-site selection, and finally attachment to the host. The establishment of a host-parasite relationship in adult A variegatum involves two phases: some pioneer males first parasitize a large ruminant (primary host infestation) and if these males succeed in establishing a feeding site they become sexually mature and emit an aggregation-attachment pheromone to attract conspecific females and males (secondary host infestation). Whereas males can colonize and select an adequate uninfested host, females only parasitize an already infested one, where a blood-meal can be guaranteed and meeting of the sexes is assured. Various olfactory cues intervene along the successive behavioural events of host-finding in male and female A. variegatum, although host-location is likely to depend also on other GENERAL DISCUSSION 74 senses as suggested by studies on vision (Kaltenrieder et al. 1989; Kaltenrieder 1990) and on IR detection (Poffet 1988). However in this chapter, only the role in host-finding of the olfactory stimulants identified in the present study will be discussed here. Receptor characterized Location (aensfllum) Tick species Reference 2,6-dichlorophenol DLl/DILI Amblyomma variegatum Waladde 1982; Schoeni 1987 anterior pit * Amblyomma americanum Haggart and Davis 1981 DLl/DILI Rhipicephcdus appendiculatus Waladde 1982 anterior pit * Dermacentor variabilis Sonenshine 1991 DLl/DH. 1 Boophilus microplus de Bruyne and Guerin 1993 DLl Ixodes ricinus Thonnev 1987 2-nitrophenol DILI Amblyomma variegatum Schoeni 1987; Steullet and Guerin 1993b DILI Boophilus microplus de Bruyne and Guerin 1993 7- valero lactone DLl/capsule Amblyomma variegatum Steullet and Guerin 1993a, b DLl Amblyomma hebraeum Steullet, unpublished DLl Boophilus microplus de Bruyne, unpublished ammonia DII.6 / DIV group Amblyomma variegatum Steullet and Guerin 1993b anterior pit */ DIV croup Rhipicephalus sanguineus Hasgart and Davis 1981 fatty acids DII.1/DIL5 Amblyomma variegatum Steullet and Guerin 1993b capsule Hyalomma asiaticum Sinitsina 1974 methylsalicylate capsule Amblyomma variegatum Hess and Vhmant 1986 capsule Ixodes ricinus Guerin, unpublished capsule Boophilus microplus de Bruvne, unpublished sulfide capsule Amblyomma variegatum Steullet and Guerin 1992b capsule Hyalomma dromedarii Steullet and Guerin 1992b Table 4.2. Characterized olfactory receptors of A variegatum also reported in other tick species. * Indicates that the receptor could not be surely ascribed to a definite wall-pore sensillum of the anterior pit (DII. 1, or DII.5, or DII.6) because of use of tungsten electrodes. GENERAL DISCUSSION 75 Most of the volatiles detected by A. variegaium are widespread in nature and rather unspecific to vertebrates. Resting adult A. variegaium waiting in the litter zone are confronted by very large amounts of gases exhaled or released by eructation at the level of the ground by large ungulates. Therefore, breath components constitute the most abundant vertebrate-associated cues for the unfed resting ticks. The breath component CO2 is an essential cue during host- finding for many blood-sucking insects as well as for ticks (i.e. Garcia 1962; Nevill 1964; Guglielmone et al. 1985; Beelitz and Gothe 1991; Gillies and Wilkes 1968, Turner 1971, Warnes and Finlayson 1985, French and Kline 1989). This product arouses adult A. variegatum and induces them to move as demonstrated in activation bioassays (chapter 3.2.), in the wind tunnel (chapter 3.1.) and in the field (Yunker and Norval 1991). CO2 also evoked upwind orientation in the wind tunnel (chapter 3.1.), but did not efficiently attract adult A. variegatum from more than 1-2 meters in the field (Barré 1989; Barré et al. 1991 ; Norval et al. 1992a). Despite its intrinsic unspecificity, CO2 might provide specific information to A. variegatum. Since the amount of vertebrate-emitted COg is directly correlated with animal size, a common host such as a buffalo represents a stronger source of CO2 than a small mammal. Furthermore, CO2 exhaled at ground level by a grazing herbivore is more easily detectable by a waiting adult A variegatum in the litter zone than CO2 emitted from an upright human some 1.5 meters above the ground. Thus, the host-finding strategy of the parasite and the behaviour of its usual host can serve to determine the effective specificity of a kairomone such CO2. The C02-excited receptor of A. variegatum resembles C02-excited receptors described for tsetse flies (Bogner 1992) and Lucilia cuprina (Stange 1974). On the other hand, the C02-inhibited receptor of A. variegatum, which at least equals the high sensitivity of the CO2- excited receptor of Aedes aegypti (Kellogg 1970), is unknown to-date for other blood-sucking arthropods. Such a C02-inhibited receptor nevertheless exists in the temporal organ of the Japanese house-centipede (Yamana et al. 1986, Yamana and Toh 1987) and in wall-pore single-walled sensilla of the termite Schedorhinothermes lamanianus (Ziesmann et al. 1992). Interestingly, A. variegatum, the Japanese house-centipede, and Schedorhinothermes lamanianus spend a part of their life time in microhabitats (litter, soil, and nest, respectively) where CO2 concentration is higher than normal ambient values (0.03-0.04%). Such CC>2-inhibited receptors might monitor CO2 changes around relatively high ambient levels better than classical C02-excited receptors. The ambient CO2 level to which each of these organisms is confronted in its microhabitat falls into the working range of the C02*inhibited GENERAL DISCUSSION 76 receptor (0-0.2% for Ambiyomma, 0-1% for the centipede, and 0-5 % for the termite). Considering the working range of the C02-inhibited receptors, A. variegatum appears to be equipped better for detecting minute CO2 changes than either the centipede or the termite. The C02-inhibited receptor of A variegatum is extremely sensitive to CO2 shifts, i.e. 0.001-0.002% (chapter 3.1.) and could serve to first alert resting ticks of the presence of a vertebrate. In the wind tunnel, concentrations of 0.1% to 0.2% were the most effective levels to strongly arouse resting ticks and initiate locomotion (chapter 3.1.). Adult A. variegatum may consequently detect shifts in CO2 levels long before eliciting any active search. Thus, active search is only initiated when the potential host is not too far away and when CO2 levels reach a certain threshold, i.e. 0.1%. The C02-excited receptor best monitors concentrations above 0.1% (chapter 3.1.), and should therefore intervene during initiation and maintenance of the active search. Perritt et al. (1993) recently found that even 9 ppm (0.0009%), corresponding to the sensitivity threshold of the CO2- inhibited in A. variegatum, elicited questing in D. variabilis and movement in A. americanum. The breath product, H2S, which stimulated two types of sulfide receptors within the capsule of Haller's organ of A. variegatum, also alerts resting individuals but without strongly inducing them to walk (chapter 3.2.). This compound is unknown to-date as a kairomone for other haematophagous arthropods, but another sulfur-containing product, dimethyldisulfide, is an attractant for the New World screwworm Cochliomyia hominivorax in the field. Omitting dimethyldisulfide from an attractant odour mixture (swormlure-4) significantly reduced catches of Cochliomyia hominivorax in wind-orientated traps (Green et al. 1993). H2S is generally present at low concentrations in vertebrate breath (i.e. 0.007 to ca. 0.7 ppm in human, Tonzetich 1977) but is very abundant in gases released by ruminants during eructation (ca. 100 ppm in the rumen, Hungate 1966). Considering the sensitivity thresholds of the CO2- inhibited receptor (0.001-0.002%) and the sulfide receptors (0.0001 ppm) in A variegatum (chapters 3.1. and 3.2.) as well as the concentrations of CO2 (25%) and H2S (100 ppm) in the rumen (Hungate 1966), resting A variegatum may detect an eructation from a host from a longer distance with the sulfide receptors than with the C02-receptors alone. In this context, H2S might be, long before C02. the first alerting signal for the presence of a suitable host such as a ruminant in the vicinity of the waiting tick. Whereas H2S alone activated resting adult ticks but without often inducing them to move, CO2 and H2S do not act synergistically as an arousal signal (chapter 3.2.). However, in GENERAL DISCUSSION 77 preliminary bioassays on the locomotion compensator, mixtures of CO2 and H2S significantly altered the behaviour of excited walking male A variegatum by frequently eliciting reorientating behaviour (stopping, changing direction, and moving for a short distance upwind). This behaviour was totally different from that induced by CO2 or HgS alone (Table 4.3. and Rg. 4.1.). Further investigations on host-finding behaviour of A. variegatum in relation with the eructation cycles of the host might be worthwhile to better understand the exact role of HgS and CO2 in host selection. prestimulation stimulation a) Proportion time spent in reorientating (mean) H9.3 COa COa/HgS 0.05 0.07 0.03 0.06 0.15* 0.18* b) number of reorientating behaviours / SO s (mean) HgS_______COg COg/HgS 0.81 1 0.38 0.94 0.44 2.06' c) reorientating angle y [0J (mean) H2S COg COVHaS 61.3 65.9 615 84.6 8L8 110.3' Table 4.3. Reorientating behaviour of excited male A, variegatum walking on a locomotion compensator when stimulated with either H2S, CO2, or mixtures of both (for method, see chapter 2.6.). Ticks were considered to evoke reorientating behaviours if they stopped or moved slowly (< 0.5 cm/s). Mean walking speed of A. variegatum on the locomotion compensator was about 2.7 cm/s. During reorientating behaviour, ticks often raised the first pair of legs in the air, initiated large turns, and even moved backward briefly. Such behaviour could not be satisfactorily described by the locomotion compensator. Consequently, reorientating was analysed from the video tapes, a) The proportion time spent by each tick in reorientating during the prestimulation and stimulation period was compared pairewise with a Wilcoxon signed rank test b) The frequency of reorientating behaviour recorded in the prestimulation and stimulation periods was compared pairewise with a sign test for each tick, c) The reorientating angles y, expressing the amplitude of the change in orientation between the beginning and end of the reorientating behaviour elicited during the prestimulation and stimulation periods were compared with a Wilcoxon-Mann-Whitney test Tracks are more tortuous when ticks are submitted to stimulation with a mixture of CO2 and H2S than tracks elicited during stimulation with either H2S or CO2 alone (see Fig. 4.1.). * Indicates that behaviour during stimulation was significantly different from that elicited during prestimulation periods. For each stimulus, 16 ticks were observed. GENERAL DISCUSSION 78 *< v. B \... 0 \ \ •*-*„__y y «s /" V^ X / "^-.. r Fig. 4.1. Representative tracks of three different males of A uariegatum submitted to A 0.15% CO2, and B 0.15% CO2 and H2S (aqueous solution of 1 mg Na2S/ml at the source). White arrow indicates wind direction (0.1 m/s); star represents the beginning of track; dashed lines are tracks made during prestimulation and poststimulation (30 s each), whereas continuous lines are tracks during stimulation (30 s); small bold arrow indicates a reorientating behaviour (walking speed < 0.5 cm/s); horizontal bar 50 cm. A host infested by attached male A. variegatum arouses and attracts many more resting conspecific ticks than an uninfested host. Thus, 20% of unfed males released within a few meters of uninfested bulls had fixed on these animals after 2 days, against hardly 2% on uninfested goats (Barré et al. 1991), indicating a preference for bovidae. However, 30% of released unfed males had attached after 2 days on bulls previously infested by pioneer male A. variegatum, and about 10% on infested goats (Barré et al. 1991). By contrast, GENERAL DISCUSSION 79 no unfed females were observed on uninfested bulls or goats 2 days after release, whereas about 10% of females had attached after the same period of time on infested bulls and goats (Barré et al. 1991). This behaviour was mediated by three volatiles emitted in large amounts by fed attached males: 2- nitrophenol, nonanoic acid and methylsalicylate (Schoeni et al. 1984; Diehl et al. 1991). 2-Nitrophenoi which stimulated very sensitive receptor(s) in the anterior pit of Haller's organ of A variegatum has also been found in bovine odour (chapter 3.4.). This product greatly improved the stimulant effect of CO2 on locomotion, but only weakly excited resting ticks when presented in absence of CO2 in a wind tunnel (Steullet 1987). However, 2-nitrophenol on its own clearly attracted excited adults in the field (Hess and De Castro 1986; Norval et al. 1991a), and in the wind tunnel (Steullet 1987). 2-Nitrophenol is evidently for long-range attraction the most important component of the aggregation- attachment pheromone. Held experiments demonstrated that low doses of the components of the aggregation-attachment pheromone added together to a CC>2-baited trap improved capture of unfed males but not of females. Much higher doses were needed to attract females (Barré et al. 1991). Thus, it may be considered that males are behaviourally sensitive to even low amounts of 2- nitrophenol, levels encountered in uninfested hosts such as steers (chapter 3.4.) or in weakly infested ones. By contrast, females are more likely to respond only to higher levels which can be found in vertebrates well infested with male ticks. Indeed, the production rate of the aggregation-attachment pheromone by feeding males, frequently found in aggregations of tens or even hundreds of individuals on the same host, is high (ca. 1.8 ug of 2-nitrophenol/hour male, Diehl et al. 1991). This shows how finely host-finding in A variegatum may be tuned to amounts of compounds such as 2-nitrophenol parsimoniously serving as a host-odour kairomone and component of the aggregation-attachment pheromone in this tick species. Furthermore, females only attach on hosts infested by fed males releasing the aggregation-attachment pheromone (Schoeni et al. 1984; Schoeni 1987; Barré 1989). Two components of the pheromone were found to evoke attachment in females, i.e. 2-nitrophenol and methylsalicylate (Schoeni et al. 1984; Schoeni 1987; Norval et al. 1991b). By contrast, males can also attach on hosts in the absence of the aggregation- attachment pheromone, but the rate of attachment greatly depends on host type, i.e. 98% on cattle against 37% on rabbits and only 19% on sheep (Norval et al. 1992b). Feeding-site selection and attachment in males might be mediated by olfaction in conjunction with contact and heat perception. Nevertheless, the role in host-finding of vertebrate volatiles found to stimulate GENERAL DISCUSSION 80 receptors of male A. variegatum in this study (chapters 3.3. and 3.4.) remains largely unknown. NH3, lactones, aliphatic and aromatic aldehydes, furfural, and short- chain fatty acids have not yet been properly investigated in behaviour tests. NH3, which is found in vertebrate urine but also in sweat (Spector 1956), has nevertheless been reported as an olfactory stimulant for haematophagous insects and tick species other than A. variegatum. NH3 is an arousal signal for R. sanguineus (Haggart and Davis 1980). The soft tick Omithodoros erraticus exhibited a marked preference for ammonia vapours than for acetic acid (El- Ziady 1958). Furthermore, NH3 attracts Hydrotaea irritans (Thomas et al. 1985) and Tabanidae (Hribar et al. 1992). NH3 also evoked probing in Stomoxys calcitrans according to Hopkins (1964) but not to Gatehouse (1970). Lactones, reported from many bovidae and primates (i.e. Burger et al. 1987; Goetz et al. 1988; Flood et al. 1989), and in urinary volatiles of the pine vole (Boyer et al. 1989), remain unknown as stimuli for haematophagous arthropods. Benzaldehyde and 2-hydroxybenzaldehyde which are reported to be emitted by some tick species (Wood et al. 1975; Apps et al. 1988) but also to be associated with vertebrate odours (chapter 3.3.; appendix; Lederer 1946; Preti et al. 1977; Andreolini et al. 1987; Goodrich 1983; Flood et al. 1989), stimulated some very sensitive capsule receptors of A. variegatum (chapter 3.3.). These aromatic aldehydes are nevertheless unknown to-date in the literature as olfactory stimulants for other blood-sucking arthropods, but some electrophysiological experiments undertaken in this laboratory have revealed that sandflies also carry receptors sensitive to benzaldehyde (Dougherty and Guerin, personal communication). Saturated and unsaturated aliphatic aldehydes, which stimulated 3 receptors of differing but overlapping specificity in the capsule of Haller's organ of A. variegatum (chapter 3.3.), are common vertebrate-associated volatiles (i.e. Ellin et al. 1974; Burger et al. 1981; Schultz et al. 1988; Goetz et al. 1988; Natynczuk et al. 1989). These products are also widespread in many other natural sources (i.e. green leaf odour). Except for tsetse flies which carry propanal-sensitive neurones (Bogner 1992), no other blood-sucking arthropod is known to hold aliphatic aldehyde receptors or to behaviourally respond to such products. Short-chain fatty acids excited several different olfactory receptors with overlapping specificity in A. variegatum (chapter 3.4.). These volatiles, like aliphatic aldehydes, are often associated with vertebrate odours (i.e. Ayorinde et al. 1982: Fox 1982: Goetz et al. 1988; Goodrich 1983). Fatty adds also GENERAL DISCUSSION 81 constitute well-known olfactory stimulants for some other blood-sucking invertebrates. Mosquitoes and the bug Tn'atoma infestans possess acid receptors (Lacher 1967; Bernard 1974), and various short-chain fatty acids also evoke probing in Stomoxys calcitrans (Hopkins 1964) or attraction in Hydrotaea irritans and Cochlyomyia hominivorax (Thomas et al. 1985; Green et al. 1993). The relative abundance of short-chain fatty acids varies significantly between animal type. In the present study, extracts of bovine odour collected on Porapak contained butanoic acid as the major fatty acid but 2-methylpropanoic acid predominated in rabbit odour (chapter 3.4., appendix). A. variegatura could presumably differentiate with its olfactory receptors between various acid and aldehyde mixtures, and thus between potential hosts. Subtle variations in amounts of these fatty acids and in the relative abundance of each acid in a complex odour might evoke different behaviours in ticks and be partially responsible for host specificity. At such it is not surprising that most results reported in the literature are often contradictory. Butanoic acid, for example, is reported as both an attractant and repellent for I. ricinus (Totze 1933, Lees 1946). Future behavioural studies on attraction, induction of attachment and host specificity as related to short-chain fatty acids and the other olfactory stimulants identified in the present study are clearly necessary and will have to carefully take into account concentration effects as well as mixtures. Our current knowledge would nevertheless suggest that host-finding in adult A. variegatura is mediated by fine tuning of its olfactory system to host-related volatiles. SUMMARY - RESUME 82 V. SUMMARY 1. Several olfactory receptors within wall-pore single-walled sensilla (type A and B) and wall-pore double-walled sensilla (type C) on tarsus I of the tick Amblyomma variegatum respond to various host-associated volatiles. By contrast, receptors in wail-pore double-walled sensilla (type A and B) located outside the Haller's organ are stimulated by neither host-odours (bovine and rabbit odours, human breath, human axillary secretion), nor by synthetic compounds associated with vertebrate odours or tick pheromones. The function of the latter sensilla thus remains unknown. 2. Neither the anterior pit, nor the capsule of Haller's organ is exclusively dedicated to the perception of host odours or pheromones. Pheromone receptors are also not restricted within specialized sensilla, but are generally confined to the same setae as host-odour receptors. However, receptors to breath components are only present in the capsule. The capsule of Haller's organ is consequently an essential sense organ for host-finding since breath is the main cue in initiating this behaviour. CO2 which is perceived by capsule receptors is indeed a strong locomotor stimulant, whereas HgS, a breath constituent stimulating receptors in the capsule, also arouses resting adult A. variegatum. On the other hand, the anterior pit of Haller's organ may also play a role in host-finding as it bears for instance receptors for 2-nitrophenol, a volatile used by A. variegatum in selecting adequate hosts and a suitable feeding-site. 3. Enclosure of wall-pore singie-walled sensilla (type B) within a capsule which protects them against mechanical damage and desiccation does not restrict vapour perception. Molecular diffusion in air and air turbulence around the tarsus are likely to permit access of odours to the confined sensilla and permit perception of olfactory stimulants independent of wind direction. 4. About half of the receptors within the capsule have been characterized: - a C02-excited receptor best monitoring changes in CO2 concentration above 0.1% (1000 ppm) - a COg-inhibited receptor highly sensitive to minute changes in COg levels (0.001 to 0.002% or 10 to 20 ppm) around ambient SUMMARY-RESUME 83 - two sulfide receptors highly sensitive to H2S (estimated threshold at ca. 0.1 ppb) but showing a dissimilar overall response to small sulfides and mercaptans - three aliphatic aldehyde receptors (bovine and rabbit odour): one highly sensitive to C6 saturated and unsaturated aldehydes; another best responding to Cq and C7 saturated aldehydes; and a third stimulated by Cq and C7 unsaturated aldehydes - a benzaldehyde receptor (bovine and rabbit odour) also responding but to a lesser extent to furfural (bovine and rabbit odour) - a 2-hydroxybenzaldehyde receptor (bovine and rabbit odour) also weakly responding to benzaldehyde - a receptor sensitive to y-valerolactone (bovine and rabbit odour) - a methylsalicylate receptor (component of the aggregation-attachment pheromone of A. variegatum) - several other receptors stimulated by either breath, bovine and/or rabbit odour but whose adequate stimuli have not been identified. 5. About half of receptors in the two wait-pore single-walled sensilla (type A) and in the two wall-pore double-walled sensilla (type C) have also been characterized as responding to host-associated volatiles and/or pheromones: - short-chain fatty add receptors (bovine and rabbit odour) responding best either to pentanoic acid, butanoic acid, or 2-methylpropanoic acid - two NH3 receptors (vertebrate odours) which differ from each other by their response intensity - a receptor sensitive to 7-valerolactone (bovine and rabbit odour) and to 6- caprolactone - receptor(s) to 2-nitrophenol (a component of the A, variegatum aggregation-attachment pheromone and of bovine and rabbit odour) also sensitive to 4-methyl-2-nitrophenol (bovine and rabbit odour) -a 2,6-dichlorophenol receptor (tick sex pheromone component) also weakly responding to 2-nitrophenol. SUMMARY-RESUME 84 6. Receptors of A. variegatum, which have been characterized, can not be considered "generalist" but show responses to a limited range of components from the same chemical class. Sulfides and mercaptans, short-chain fatty acids, aliphatic aldehydes, aromatic aldehydes, and lactones are all detected by at least two receptors with overlapping specificity spectra. All receptors on the tarsus are unique in terms of response characteristics. Thus, A variegatum possesses an olfactory system which provides a maximum of information about host-odour quality with a quite few number of olfactory receptors. 7. This work also suggests that development of the aggregation-attachment pheromone of the genus Amblyomma did not require a high specialization of the olfactory system. Two of the three components of the pheromone of A. variegatum (2-nitrophenol and nonanoic add) and all components of the pheromone of Amblyomma hebraeum (2-nitrophenol, benzaldehyde, 2- methylpropanoic acid, and nonanoic acid) have also been identified in bovine and rabbit odour. Though both benzaldehyde and 2-methylpropanoic acid do not seem to be constituents of the aggregation-attachment pheromone of A. variegatum, the latter species does possess receptors for these volatiles. It seems that male A. variegatum and A. hebraeum have developed a pheromone system by producing higher amounts of compounds associated with odour of vertebrates in order to reinforce attractivity of colonized hosts for conspecifics. Mimicry of a part of the host bouquet in the aggregation- attachment pheromone to favour meeting of the sexes on adequate hosts would thus not have involved a profound modification of the olfactory system used for host finding as would have been the case with the use of a more autonomous pheromone system. SUMMARY - RESUME 85 RESUME 1. Ce travail a permis de mettre en évidence différents récepteurs olfactifs des sensilles à pores (type A et B) ou à fentes (type C) du tarse de la patte I de la tique Amblyomma variegatura répondant à diverses classes de volatiles associés à l'odeur des vertébrés. Par contre, aucun récepteur des sensilles à fentes (type B) situées en dehors de l'organe de Haller n'a montré de réponses évidentes aux odeurs de vertébrés (odeur de lapin, de bovin, haleine et sueur humaine) et à différents analogues synthétiques de composés de l'odeur des vertébrés ou des phéromones de tiques. La fonction de ces dernières sensilles reste donc énigmatique. 2. Aucun des deux groupes de sensilles formant l'organe de Haller (capsule et "anterior pit") est dédié spécifiquement à la perception des odeurs d'hôte ou des phéromones. Seule la détection des volatiles de l'haleine semble être l'apanage exclusif de récepteurs olfactifs de la capsule. La capsule représente donc un organe sensoriel essentiel pour la recherche de l'hôte chez A variegatum étant donné que l'haleine constitue le déclencheur principal de ce comportement. Le CO2, perçu par des récepteurs de la capsule, est en effet un puissant déclencheur de la recherche active alors que l'HgS, autre composé de l'haleine détecté par des récepteurs de la capsule, éveille les tiques en repos. Cependant, "!'anterior pit" joue aussi un rôle important dans la recherche d'un hôte puisqu'il est notamment le siège de la perception du 2-nitrophénol, volatile essentiel dans la sélection d'un hôte adéquat. 3. L'entrée des volatiles dans la capsule renfermant sept sensilles à pores (type B) n'est pas entravée. La diffusion des molécules dans l'air et les turbulences crées autour du tarse suffisent en effet pour que les volatiles pénètrent dans la capsule indépendamment de la direction du vent. 4. La moitié environ des récepteurs contenus dans la capsule ont pu être caractérisés. Il s'agit de: - un récepteur excité par le CO2 répondant particulièrement à des changements de concentration supérieurs ou égaux à 0.1% (1000 ppm) SUMMARY-RESUME 86 - un récepteur inhibé par le CO2 extrêmement sensible pour des très petits changements de concentration (0.001 à 0.002% ou 10 à 20 ppm) autour des valeurs ambiantes de CO2 - deux récepteurs à H2S au seuil de sensibilité estimé à environ 0.1 ppb, mais dont un seul récepteur répond bien à des stimulations de petits mercaptans (éthyl mercaptan) et de dimethyl sulfide - trois récepteurs aux aldéhydes aliphatiques (odeur de bovin et de lapin): l'un particulièrement sensible aux aldéhydes saturés et insaturés à 6 carbones, un deuxième aux aldéhydes saturés à 6 et 7 carbones, et un troisième aux aldéhydes insaturés à 6 et 7 carbones - un récepteur au benzaldéhyde (odeur de bovin et de lapin) répondant également mais moins intensément au furfural (odeur de bovin et de lapin) - un récepteur au 2-hydroxybenzaldéhyde (odeur de bovin et de lapin) répondant aussi faiblement au benzaldéhyde - un récepteur sensible au 7-valérolactone (odeur de bovin et de lapin) - un récepteur au méthylsalicylate (composant de la phéromone d'agrégation et d'attachement de A. variegatum) - plusieurs autres récepteurs répondant soit à l'haleine, soit aux odeurs de bovin et/ou de lapin mais dont les stimuli n'ont pas été identifiés. 5. On a pu également mettre en évidence plusieurs récepteurs olfactifs stimulés par des volatiles associés aux vertébrés et/ou aux phéromones de tiques dans deux sensilies à pores (type A) et deux sensilles à fentes (type C) de "!'anterior pit" de l'organe de Haller: - des récepteurs aux petits acides gras (odeur de bovin et de lapin) répondant particulièrement bien soit à l'acide pentanoique, soit à l'acide butanoique, ou à l'acide 2-méthylpropanoique. - deux récepteurs au NH3 (odeur de vertébrés) différant par l'intensité à laquelle ils répondent - un récepteur sensible au 7-valérolactone (odeur de bovin et de lapin) et au 6-caprolactone SUMMARY - RESUME 87 - au moins un récepteur au 2-nitrophénol (composé de ta phéromone d'agrégation et d'attachement de A. variegatum mais aussi composé mineur de l'odeur de bovin et de lapin) et sensible au 4-méthyl-2- nitrophénol (odeur de lapin et de bovin) -un récepteur au 2,6-dichlorophénol (phéromone sexuelle des tiques) répondant faiblement au 2-nitrophénol. 6. Parmi tous les récepteurs caractérisés, aucun n'est un véritable "généraliste" répondant à un large spectre de volatiles. Ils sont plutôt sensibles à un groupe de substances de parenté chimique étroite. Une catégorie de stimuli (ex.: sulfides et mercaptans, acides gras, aldéhydes aliphatiques, aldéhydes aromatiques, lactones) est généralement détectée par au moins deux récepteurs au spectre sensiblement différent. Parmi tous les récepteurs caractérisés, il n'existe pas deux récepteurs olfactifs exactement identiques sur le même tarse. Armée d'un nombre limité de récepteurs olfactifs (moins de 100) comparativement à la majorité des insectes adultes, A variegatum possède un système sensoriel lui permettant de détecter un maximum de volatiles différents. 7. Ce travail suggère également que la phéromone d'agrégation et d'attachement des tiques du genre Amblyomma ne résulte pas d'une forte spécialisation du système olfactif. Deux des trois composés de la phéromone de A. variegatum (2-nitrophénol et acide nonanoique) et tous les composés de la phéromone de Amblyomma hebraeum (2-nitrophénol, benzaldéhyde, acide 2-méthylpropanoique et acide nonanoique) ont en effet été clairement identifiés dans l'odeur de bovin et de lapin. Bien que n'étant pas des composés de la phéromone de A. variegatum, le benzaldéhyde et l'acide 2-méthylpropanoique possèdent leur propre récepteur chez cette dernière espèce. Il semble donc que les mâles de A. variegatum et A. hebraeum ont développé un système phéromonal d'agrégation et d'attachement en produisant en grande quantité des substances déjà associées à l'odeur des vertébrés qui renforcent ainsi l'attractivité des hôtes déjà infestés pour leurs conspécifiques. REFERENCES 88 Vl. REFERENCES Aeschlimann A (1967) Biologie et écologie des tiques (Ixodoidea) de Côte d'Ivoire. Acta Trop 24:281-405 Aeschlimann A (1976) Les tiques, leur biologie et les maladies qu'elles transmettent. 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J Insect Physiol 38:759-764 REFERENCES 90 Boyer ML, Jemiolo B, Andreolini F, Wiesler D, Novotny M (1989) Urinary volatile profiles of pine vole, Microtus pinetorum, and their endocrine dependency. J Chem Ecol 15:649-662 Burger BV, le Roux M, Garbers CF1 Spies HSC, Bigalke RC, Pachler KGR, Vessels PL, Christ V, Maurer KH (1977) Further compounds from the pedal gland of the bontebok (Damaliscus dorcas dorcas). Z Naturforsh 32c:49-56 Burger BV, le Roux M, Spies HSC, Truter V (1981) Mammalian pheromone studies, V. Compounds from the preorbitai gland of the grysbok, Raphicerus melanotis. Z Naturforsh 36c:344-346 Burger BV, Pretorius PJ (1987) Mammalian pheromone studies, Vl. Compounds from the preorbitai gland of the blue duiker, Cephalophus monticola. Z Naturforsh 42c:1355-1357 Burger BV, Pretorius PJ, Spies HSC, Bigalke RC, Grierson GR (1990) Mammalian pheromones VIII. Chemical characterization of preorbitai gland secretion of grey duiker, Syfvicapra grimmia (Artiodactyla: Bovidae). J Chem Ecol 16:397-416 Davis EE1 Haggart DA, Bowen MF (1987) Receptors mediating host-seeking behaviour in mosquitoes and their regulation by endogenous hormones. Insect Sci Appi 8:637-641 Davis EE, Sokolove PG (1976) Lactic acid-sensitive receptors on the antennae of the mosquito, Aedes aegypti. J Comp Physiol 105:43-54 de Bruyne M, Dicke M, Tjallingii WF (1991) Receptor cell responses in the anterior tarsi of Phytoseiulus persimilis A-H. to volatile kairomone components. Exp Appi Acarol 13:53-58 de Kramer JJ (1985) The electrical circuitry of an olfactory sensillum in Antheraea polyphemus. J Neurosci 5:2484-2493 de Kramer JJ, Hemberger J (1987) The neurobiology of pheromone perception. In: Prestwich GD, Blomquist GJ (eds) Pheromone biochemistry. Academic Press, Orlando, pp 433-472 Delot J (1990) Les phéromones d'agrégation-fixation chez la tique Amblyomma variegatura (Fabricius) (Ixodida): une étude en Guadeloupe.Thèse, Ecole Nationale Vétérinaire d'Alfort (France) Den Otter CJ, Van der Goes van Naters WM (1992) Single cell recordings from tsetse (Glossina m.morsitans) antennae reveal olfactory, mechano- and cold receptors. Physiol Entomol 17:33-42 REFERENCES 91 Diehl PA, Guerin PM, Vlimant M1 Steullet P (1991) Biosynthesis, production site, and emission rates of aggregation-attachment pheromone in males of two Amblyomma ticks. J Chem Ecol 17:833-847 Ellin Rl, Farrand RL, Oberst FW, Crouse CL, Billups NB, Koon WS, Musselman NP, Sidell FR (1974) An apparatus for the detection and quantitation of volatile human effluents. J Chromatogr 100:137-152 El Ziady S (1958) The behaviour of Ornithodoros erraticus (Lucas, 1849), small form (Ixodoidae, Argasidae, towards certain environmental factors. Ann EntSoc Am 51:317-336 Fallis AM, Raybould JN (1975) Response of two African simuliids to silhouettes and carbon dioxide. J Med Entomol 12:349-351 Flood PF, Abrams SR, Muir GD, Rowell JE (1989) Odor of the muskox. A preliminary investigation. J Chem Ecol 15:2207-2217 Foelix RF, Axtell RC (1971) Fine structure of tarsal sensilla in the tick Amblyomma americanum. Z Zellforsch 114:22-37 Foelix RF, Axtell RC (1972) Ultrastructure of Haller's organ in the tick Amblyomma americanum L. Z Zellforsch 124:275-292 Fox GJ (1982) Potentials for pheromones in chimpanzee vaginal fatty acids. Folia primatol 37:255-266 French FE1 Kline DL (1989) 1-octen-3-ol, an effective attractant for Tabanidae (Diptera). J Med Entomol 26:459-461 Garcia R (1962) Carbon dioxide as an attractant for certain ticks (Acarina, Argasidae and Ixodidae). Ann Ent Soc Am 55:605-606 Garcia R (1965) Collection of Dermacentor andersoni (Stiles) with carbon dioxide and its application in studies of Colorado tick virus. Am J Trop Med Hyg 14:1090-1093 Gatehouse AG (1970) The probing response of Stomoxys calcitranslo certain physical and olfactory stimuli. J Insect Physiol 16:61-74 Gillies MT, Wilkes TJ (1968) A comparaison of the range of attraction of animal baits and of carbon dioxide for some West African mosquitoes. Bull Entomol Res 59:441-456 GIadney WJ1 Grabbe RR, Ernst SE, Oehier DD (1974) The gulf coast tick: Evidence of a pheromone produced by males. J Med Entomol 11:303-306 Gödde J (1985) Low cost storing of two electrical biosignals from DC to 20 kHz at more than 80 dB dynamic range. Pflügers Arch 403:324-327 REFERENCES 92 Gödde J (1989) Vibrating glass stylets: tools for precise microsurgery on cuticular structures. J Neurosci Methods 29:77-83 Goetz N, Kaba G, Good D, HusslerG, Bore P (1988) Detection and identification of volatile compounds evolved from human hair and scalp using headspace gas chromatography. J Soc Cosmet Chem 39:1-13 Goodrich BS (1983) Studies of the chemical composition of secretions from skin glands of the rabbit Oryctolagus cuniculus. In: Müller-Schwarze D, Silverstein RM (eds) Chemical signais in vertebrates 3. Plenum Press, New York London, pp 275-290 Gray JS (1985) A carbon dioxide trap for prolonged sampling of Ixodes ricinus L populations. Exp Appi Acaro! 1:35-44 Green CH, Hall MJR, Fergiani M, Chirico J (1993) Attracting adult New World screwworm, Cochliomyia hominivorax, to odour-baited targets in the field. Med Vet Entomol 7:59-65 Guerin PM, Stadler E, Buser HR (1983) Identification of host plant attractants for the carrot fly, Psilae rosae. J Chem Eco! 9:843-861 Guerin PM, Steullet P, Kröber T, Diehl PA, Vlimant M, de Bruyne M, Cordas T, Falk-Vairant J, Kuhnert, Lösel PM (1992) The chemical ecology of ticks at the host vector interface. In: Munderloh, UG Kurtii TJ (eds) Proceedings of the 1st international conference on tick-borne pathogens at the host- vector interface: an agenda for research. Minnesota University Saint Paul, Minnesota, pp 314-323 Guglielmone AA, Moorhouse DE, Wolf G (1985) Attraction to carbon dioxide of unfed stages of Ambiyomma triguttatum triguttatum Koch, under field conditions. Acarologia 26:123-129 Hafez M1 Bishara SI (1982) Reactions ot three species of Rhipicephalus ticks to host odour and light (lxodidea:lxodidae). J Egypt Soc Parasitol 12:309- 318 Haggart DA, Davis EE (1980) Ammonia-sensitive neurons on the first tarsi of the tick, Rhipicephalus sanguineus. J Insect Physiol 26:517-523 Haggart DA, Davis EE (1981) Neurons sensitive to 2,6-dichlorophenol on the tarsi of the tick Ambiyomma amehcanum 2 times lower., and + that the stimulus was not detected in the blank. APPENDIX 108 Table A acetoni tri le p,w dimethyl sulfoxide P 4-methyl-2-pentanone W 2-(2-butoxyethoxy)ethanol p, w 2-methyl-3-buten-2-ol W dimethyl sulfone P 2-ethoxyethanol P benzothiazole P styrene W phenol P.w 2-propanol p, w -y-nonanolactone p, w 2-butoxybutanol p, w 4-methylphenol P, w nonenal W 2-ethylpbenol P 2-ethvlhexanol P, W 3-ethylphenol P Table B cc-pinene 2-ethylhexanol camphene pentadecane und e cane camphor ß-pinene jupmene 2-heptanone indene L-limonene 1-phenylethanone dodecane a-terpineol 3-methylbuten-l-ol 2-(2-butoxyethoxy)ethanol styrene dimethyl sulfone 2-nonanone phenol tetradecane 4-methylphenol 4-ethylphenol Table 8.2. Tentative gas chromatography-coupled mass spectrometry identification of some components of bovine (A) and rabbit odour (B) but not found to stimulate olfactory receptors of A. variegatum during gas chromatography-coupled electrophysiology analyses. Identification was based on comparison of mass spectra of unknowns with those of standards in a computer-based library (percent match > 90% in each case), p presence in bovine odour as collected on Porapak Q; w presence in the bovine skin wash extract. APPENDIX 109 bovine odour peak average of 4.45 to 4.48 min 1I ¦. Ji" "A Jt'"*V"i*V"iJ*- ii* i«. "ii* "uà ri* it« il« »li">M ri* Ut hexanal retention time: 4.39 min (. [I1J IH _____________ t/t •» w "jl»bi * * il* iii "a« "il» i«ó ri« ri» '»«i"»»*'"«i* ii* Ü« »m bovine skin extract peak average of 4.37 to 4.39 min £==*^ II >**»!* M4 LJi Ta w »o iè* il» \i* li* m li* li* ït* li* hexanal „ retention time: 4.39 min t/t ¦ » » «* M ¦w tè* " Ìm ih ili 'ü* ti« Ïm' li* i«. ih il» rabbit odour peak average of 5.87 to 5.90 min yr •¦ » <* «1 ' il m m il* »J* 'ili ili iti »ti ih i** *t*""*i« hexanal retention time: 5.91 min H HUC* ¦a il '»V%'ìm''iìì "tU'liw''.i.fa j»j" i& "»*¦'"»!»"»»*, Fig. 8.1. Gas chromatography-coupled mass spectrometry analyses. Comparison of the mass spectrum and retention time of an unknown stimulant found in bovine odour (A)1 rabbit odour (B) and in the bovine skin extract (C) with that of synthetic hexanal injected under the same conditions as for the natural odour in each case. APPENDIX 110 A Fig. 8.2. Gas chromatography-coupled mass spectrometry analyses. Comparison of the mass spectrum and retention time of an unknown stimulant found in bovine odour (A) and rabbit odour (B) with that of synthetic heptanal injected under the same conditions as for the natural odour in each case. t - bovine odour peak average of 6.94 to 6.96 min yt ¦. i* é» u h ili di il. tl. it« it. ij. ii* ]i« im heptanal retention time: 6.89 min J JU VC -. 1» ' *V'A m i«V ^li~ i.a"i.i' lii'Hi tit li« il» "Hi APPENDIX 111 »OSO • i ~i r bovine skin extract peak average of 10.20 to 10.22 min •noe Tora «ora «> (Ora 4000 1000 1000 i» »T 1000 Jl ti* , , II. ill i I ut -» 0 40 «0 OO 100 130 140 1(0 XÉO 1OO fio 140 1(0 1*0 !burnus« ¦ora « (E)-2-heptenal retention time: 10.08 min ¦eoo r> TOOO «era tara 4 ora 10 1000 I* lobo IO OO J. Jl ti T........ V* - io «e (o m ìóo lio i4o ite i«o lóo »io i4o ico joo Fig. 8.3. Gas chromatography-coupled mass spectrometry analyses. Comparison of the mass spectrum and retention time of an unknown stimulant found in the bovine skin extract with that of synthetic (E)-2-heptenal injected under the same conditions as for the extract. Full identification of (E)-2-heptenal in bovine and rabbit odour as collected on Porapak, based on a full mass spectrum, was not feasible because of coeluting products which obscured the spectrum. Presence of the molecular ion of heptenal (Af+=112) was however detected in a peak in bovine and rabbit odour at the same retention as the synthetic analogue. APPENDIX 112 bovine odour peak average of 11.80 to 11.83 min £== JULb ff"*mm üi"%W Um lit" i»«" ti« ti*' tl> ria t nonanal retention time: 11.73 min V» ¦ > Até " ' «V M__l** )!• 140 la '. ti* m " m " a» " «J« " ita ,. rabbit odour peak average of 10.35 to 10.37 min v i »a» AA»M< J.( ^-4X.M^.............¦ , ¦ •a »*•_ li« 14« i*» ih ri« m m m i*7 nonanal retention time: 10.40 min ko. «j.LB.J,,J,jl l\,H» tt j LO«« il* l ¦*i ^ -. ». ' * i A A" i« Fig. 8.4. Gas chromatography-coupled mass spectrometry analyses. Comparison of the mass spectrum and retention time of an unknown stimulant found in bovine odour (A), rabbit odour (B) and in the bovine skin extract (C) with that of synthetic nonanal injected under the same conditions as for the natural odour in each case. APPENDIX 113 bovine odour peak average of 13.13 to 13.15min M-A- /t -. JQ «. w A X iw ih w''iti''Ji A* it* »in Ma furfural retention time: 13.06 min a '»a it« a* ii» if» it» it» »j» »t> *t« I« rabbit odour peak average of 112S to 1127 min E-S «>.t. rfe,>. .^,. I» 1» !.. iti tt*li«>•¦ lis furfural retention time: 11.28 min -. M «• « H IH li* it. ,» li. in. Fig. 8.5. Gas chromatography-coupled mass spectrometry analyses. Comparison of the mass spectrum and retention time of an unknown stimulant found in bovine odour (A) and rabbit odour (B) with that of synthetic furfural injected under the same conditions as for the natural odour in each case. Presence of furfural was indicated in the bovine skin extract by its molecular ion (Af+=96) in a peak eluting at the same retention time as the synthetic analogue. APPENDIX 114 A bovine odour peak average of 14.26 to 14.29 min Ii M+ LJIf1L'/ m il« ili ti* ti* li* ti* ili it* ti* it« -ft^w benzaldehyde retention time: 14.21 min JLd 1/1 -, Jo 'J*" "**" " " "**" " "ti* lì* ti* til it« ti* ti* li* lì* ti* Br= rabbit odour peak average of 12.07 to 12.08 min "UJuL it^x- ahi benzaldehyde retention time: 12.10 min IC------& Fig. 8.6. Gas chromatography-coupled mass spectrometry analyses. Comparison of the mass spectrum and retention time of an unknown stimulant found in bovine odour (A) and rabbit odour (B) with that of synthetic benzaldehyde injected under the same conditions as for the natural odour in each case. Presence of benzaldehyde was indicated in the bovine skin extract by its molecular ion (Af+=106) in a peak eluting at the same retention time as the synthetic analogue. APPENDIX 115 bovine odour ion 60.00 " 4 1 U. ti 7 6 ";" "i" p 9 10 „ ö »« "in 11 : I I ».*» i I I1TMt 5 H.It ll.M I*.M KM 1».M M.— ti.«* 1*.M ton 73.00 2 14.1« L3 U-M li.** Ii IT.Il I »¦*! il.l* I It,, I "i'I. ,1 , .I.J., I- rabbit odour •B ll.M H.M TI.** «-« MBBMl ton 60.00 11MMB IBMMB MMM 1 7 BMOM U.ti MBBM 4 ti. ii 5 ...t MMM I K 6 8 9 10 11.IB I 1 \ 1T.„ i .1 T V 1— ., U M M-M K .M 11.M 1*.** ti'.** ii.» UHM ton 73.00" 3 I* UWIII IMOMt MMM 10.» MMM —M 2 ».«:» .a. I i U.M l I. , I T ".-V11 -r v M.M 1 la. -• 11 — 14,** 14.** IS.H ».M u!m r*« — bovine skin extract 1 acetic acid 2 propanoic acid 3 2-m el h y I propanoic acid 4 buta noie acid 5 3-m ethyl buia noie acid 6 penta noie acid 7 hexanoic acid 8 heptanoic acid 9 octanoic acid 10 nonanoicacid 11 decanoic acid Fig. 8.7. Gas chromatography-coupled mass spectrometry analyses. Full identification of short-chain fatty acids (C2 to C^q) in bovine and rabbit odour, based on full mass spectra, was often not feasible because of coeluting products which obscured the spectra. Presence of these short-chain fatty acids in bovine odour (A), rabbit odour (B), and in the bovine skin extract (C) was however indicated using 2 characteristic ions (M/Z=60; and M/Z=73) for single ion monitoring. Peaks eluting at the same retention time as the synthetic analogues contained these ions. APPENDIX 116 »IM» 1 bovine odour peak average of 15.83 to 15 87 min •COO looo (ODO •» SOOO «0«0 4 )000 JOCO 1000 0 a/E -> 3 1* LU 10 Jul 100 o «o co «a im li o ilo i«o ilo îèo »o ito »*o i*c »ou ¦ « 7-valeroIactone retention time: 15.74 min •ooo ÏOOO «ooo' «1 * ! WJ »0 «000 1000 ta oo 10OO 100 I C I . 'o 4o «'o oo iio iio ïio i«o i«o im iiô lie aio lie Fig. 8.8. Gas chromatography-coupled mass spectrometry analyses. Comparison of the mass spectrum and retention time of an unknown stimulant found in bovine odour with that of synthetic -y-valerolactone injected under the same conditions as for the natural odour. if-Valerolactone was detected in an extact of rabbit odour with 3 characteristic ions (Af/Z=56, 85, and 100, respectively) in a peak eluting at the same retention time as the synthetic analogue. APPENDIX 117 bovine odour U,ì* H.*i U.M 1«,» jt.Tl ton 119.00 11UMr rabbft odour ion 119.00 ru> -J.I •• >•¦•¦ ¦ 4» Ht« ¦(.*¦ .<. U .Ta n.i B 1 2-meïhyIbenzatdehyde (15.68 min) 2 3-methylbenzatdehyde {15.96 min) 3 innethylbenzaldehyde (16M min) l/l -,______!•_ UJma.Ui rabbK odour peak average of 16.10 to 16.11 min sr "^—ar ili 3-methylbenzaldehyde retention time: 15 96 mm Fig. 8.9. Gas chromatography-coupled mass spectrometry analyses. Full identification of methylbenzaldehyde isomers, in bovine (A) and rabbit odour (B). based on full mass spectra, was often not feasible because of numerous coeluting products which obscured the spectra. Presence of methylbenzaldehyde isomers in bovine and rabbit odour was indicated using 3 characteristic ions (M/Z=91, 119, and 120, respectively) in peaks eluting at the same retention time as the synthetic analogues. C Comparison of the mass spectrum and retention time of an unknown stimulant found in bovine odour with that of synthetic 3-methylbenzaldehyde injected under the same conditions as for the natural odour. APPENDIX 118 bovine odour -peak average of 16.93 to 1656 min M IAiJ tt-X-ff 3^^^:1^¾.'¾¾*¾. »U.i.UU,- Wl - » ** . ** 2-hvdroxybenzatdehyde retention time: 16.85 min \u a u* m "il* i*. a. ».» ^' ^ rabbit odour peak average of 17.02 to 17.04 min. t» M M li» dr U. ^¦¦ic^*;.: "iu"X"ifr 2-hydroxybenzaIdehyde retention time: 16.85 min I1U M, p, J111L,,,,...... , —,— — /i -. w M «* M I*» li» m i*« m tit ri» m m lit Ut B bovine skin extract peak average of 16.82 to 16.84 min Ht Um' tit 2-hydroxybenzaldehyde retention time: 16.85 min U. ¦¦ » ?¦ J M" 'm 1J. .v ,L ,u ^r-13; Fig. 8.10. Gas chromatography-coupled mass spectrometry analyses. Comparison of the mass spectrum and retention time of an unknown stimulant found in bovine odour (A), rabbit odour (B) and in the bovine skin extract (C) with that of synthetic 2- hydroxybenzaldehyde injected under the same conditions as for the natural odour in each case. APPENDIX 119 bovine odour rabbft odour Ion 139.00 .»•¦»• !•.. ^w ¦ r—/¦V...t-f^/\yTV.^if>AT< »¦" "" "— »¦« M = ' ion 153.00 11.» !».*• 11.« nlT^i-i L *-*• K.H u.*a 1 2-nltrophenol (1549 min) 2 4-methyl'2-nHrophenol (16.55 min) 1 2-nttrophenol (19.15 min) 2 4-methyl-2-nItrophenol (20.55 min) bovine odour peak average ol 16.53 to 1656 mtn SSSSS* r lM*wLjKA*\ 111. J I A.,..!,,.,,,,.......,. .I7-- 1» H ». H A im w M IM ii« 11« li« >1« lU IU lU ill il» »4« 4-methyi-2-nitrophenol^T retention time:16.55 min .LxX.A ä t,.....y.,i.....,"'.".....i......... i . ¦• f M — H M H^m M W U.Ï1«itilit ti. il.lt. li. IT Fig. 8.11. Gas chromatography-coupled mass spectrometry analyses. Presence of 2- nitrophenol and 4-methyl-2-nitrophenol in bovine (A) and rabbit odour (B) was indicated by their respective molecular ions (AZ+= 139 for 2-nitrophenol and M+= 153 for 4-methyl- 2-nitrophenol) in peaks eluting at the same retention time as the synthetic analogues. Retention times of the synthetic analogues are provided under figures A and B. C Comparison of the mass spectrum and retention time of an unknown stimulant found in bovine odour with that of synthetic 4-methyl-2-nitrophenol injected under the same conditions as for the natural odour. Identification of 2-nitrophenol based on a filili mass spectrum was not feasible.