IMPRIMATUR POUR LATHESE Stimuli chimiques dans le comportement sexuel de la tique du bétail Boophilus microplus de M. Marien de Bruyne UNIVERSITÉ DE NEUCHATEL FACULTÉ DES SCIENCES La Faculté des sciences de l'Université de Neuchâtel sur le rapport des membres du jury, Messieurs P. Guerin, P.-A. Diehl, R. Stocker (Fribourg) et J.-L Connat (Dijon) autorise l'impression de la présente thèse. Neuchâtel, le 2 juillet 1996 Le doyen: 77 R. Dändliker To the cover: To most of us this looks like a rising sun over a cornfield, radiating a bright day ahead of us. To a male tick this looks like a female tick, firmly attached to the skin of a vertebrate host, promising excellent chances for spreading his genes. The latter organism of course, has got it right. "Subjectively, first of all, we are inevitably the centre of perspective of our own observation. " (... Scientists) "are now beginning to realise that even the most objective of their observations are steeped in the conventions they adopted at the outset and by forms or habit of thought developed in the course of research; so that, when they reach the end of their analysis they cannot tell with any certainty whether the structure they have reached is the essence of the matter they are studying, or the reflection of their own thought. " Pierre Teilhard de Chardin in "The phenomenon of man" 1955 In French; English translation of 1959 "We live together, we act on, and react to, one another; but always and in all circumstances wewre by ourselves: :The martyrs-go hand in hand into the arena; they are crucified alone. Embraced, the lovers desperately try to fuse their insulated ecstasies into a single self-transcendence; in vain. By its very nature every embodied spirit is doomed to suffer and enjoy in solitude. Sensations, feelings, insights, fancies—all these are private atid, except through symbols and at secondhand, incommunicable. We can pool information about experiences, but never the experiences themselves. From family to nation, every human group is a society of island universes. Aldous Huxley in "The doors of perception" 1954 l PREFACE The taste of tick sex Put in plain English, the subtitle above is basically all this thesis is about,. In science we have the habit of writing in code rather than in prose and certainly not in poetry. This means that every word used should have a precisely defined meaning. So on the cover title I specify "Boophilus microplu? as the scientifically correct name of the particular species of tick under investigation, I use "chemical stimuli" rather than taste and I write "mating behaviour" to put my approach to sex in an ethological context rather than suggesting a genetic or a reproductive physiology approach. However, scientists live in a world of illusions just like the rest of the human species and holding to the idea that words are precise is one such illusion. As humans we largely communicate via auditory and visual cues. We talk or write a lot and make signs and facial expressions. Auditory and visual communication exists in arthropods too but is overall less important than chemical communication. In the discipline of chemical ecology, natural compounds that carry information, used during interactions between organisms are called semiochemicals or more precisely infochemicals (Dicke and Sabelis, 1988). Much has been written about them. Substances mediating specific behaviours have been defined and redefined, ordered in classes and subclasses. Pheromones were defined as chemicals used for communication between two or more animals of a single species (Karlson and Luscher, 1959). They can be classified in terms of the behaviours they elicit from receiving conspecifics such as aggregation, trail following, dispersal, opposition and sexual behaviours. However, confusion occurs commonly between behaviour and the function or effect of that behaviour. For instance, the function of an alarm pheromone is clearly to transmit a state of alarm in the local population of a certain species. The actual behaviour it elicits can be different between species, ranging from dropping down from a leaf in aphids, to aggressive defense behaviour in ant soldiers, or simple dispersal. Another complication lies in the definition of behaviour. If a group of randomly placed animals aggregate over a certain amount of time in a specific spot in an arena this is,ußen termed aggregation behaviour. However such an experiment does not say anything about the behaviours involved in each individual animal which lead to the overall effect of aggregation. A chemical signal emitted by females or males eliciting behaviour from the opposite sex that leads directly or indirectly to mating is usually refered to as a sex pheromone (Shorey, 1973). Throughout this thesis I have avoided the use of such words where possible, precisely because they do have a more or less defined meaning. So even though the topic we are dealing with definitely suggests we are looking for pheromones, I take a careful approach and will not try to run ahead of my results. In part II of the general introduction I will therefore give a review of chemical stimuli that are tick derived or associated with ticks without consistently hanging such confusing labels like "mounting sex pheromone" on them and not rigorously ordering them into arbitrary classes. The three plain English words used above (tick, sex and taste) do mark the biological stage that we set for ourselves. Ticks are the very peculiar group of animals that we in our department are so familiar with but that need introduction to the reader who is generally more confronted with other arthropods. Most work on pheromones has been done on insects and comparisons are often made but not always valid. 2 PREFACE Sex is one of two important resources for any animal, the other being food. Most chemical signals that scientists have decoded carry information about food sources or mate quality. Arguably, sex is the most important of the two. The role of the whole biological machinery of an organism is ultimately to reproduce. Food is only the fuel for the engine. This does not mean that the chemical signals involved could not be similar or the same. Why animals have sex, i.e., exchange gametes, in order to reproduce is actually still a big question to which the answer is not so well known as suggested in some first-grade text books. With the word taste we enter into the fascinating realm of perception. Ticks live in a world of illusions just as humans do. All animals, by means of their nervous systems, respond to changes in their environment by adapting their behaviour to it. The illusion in the mind of the tick and the corresponding behavioural response can be evoked by the experimental scientist by manipulating the appropriate physical elements of the right environment:-' What ,are those elements that^evoke the response that ultimately allows the individual, and with it the species, to survive in its natural environment and how does he perceive them? By writing taste I already suggest that the dominant sensory modality in tick sex is chemical. I will argue here that this is at least partly the case. Incidentally, the title might just as well have been "the smell of tick sex" were it not for the fact that I have not been able to show a behavioural function in mating of the only smelly compound I have isolated. The difference between smell and taste is anyway hard to define. Thus I have set the stage for a study on sensory input and behavioural output, involving stimuli of a chemical nature that play a role in mating of a particular species of tick. This brings us to the technical aspects of my work. Throughout, I have tried to address three différents aspects of this study.. Hence a multi-disciplinary approach was inevitable, involving electrophysiological techniques for measuring sensory input, behavioural experiments for motor output and analytical chemistry for resolving complex mixtures of compounds. These techniques, each related to different domains of science have been employed here mainly to try to answer some proximate questions, asking ourselves-how things'work^ratherjlhan-iwhy things work that way. The main body of the results arising from this approach therefore only allows me to answer such questions. In the discussion I will, however, also try to address some of the ultimate questions, i.e., the why's of mating behaviour in the cattle tick and draw comparisons to what has been described for other ticks. 3 CONTENTS PREFACE The taste of tick sex 02 CONTENTS 04 GENERAL INTRODUCTION 1: Boophilus microplus: a special kind of tick 1.1. Tides; a group of obligate blood feeding Arthropods 05 1.2. Boophilus microplur. a special case 1.2.1 Taxonomy and distribution 07 1.2.2 Host specificity and life-cycle 09 1.2.3 Economic importance 10 1.2.4 Reproductive biology 11 2: Sensory physiology of ticks 12 3 : Tick associated chemical stimuli in tick biology 3.1. Volatile signals .—.. 16 3.2. Low-volatile cuticle associated signals 18 3.3. Substrate deposited signals '' 18 RESULTS 1 : The on-host sexual behaviour of Boophilus microplus males and their 20 in vitro responses to females and dummies in a host simulating arena unpublished results introduction 20 materials and methods 22 results 24 discussion 31 2: Isolation of 2,6-dichlorophenol from the cattle tick Boophilus 37 microplus: receptor cell responses but no evidence for a behavioural response published in J. Insect Physiol. 40:143-154 introduction . 37 materials and methods 38 results 42 discussion 45 references 47 3 : Cholesteryl esters and other contact chemostimuli in the mating 49 behaviour of the cattle tick Boophilus microplus manuscript for submission to ArchJnsèct BJswénuPhysiot. introduction - ¦- 49 materials and methods 5I results 54 discussion 62 GENERAL DISCUSSION 1: The ghost of Bombyx mori: Concepts in pheromone research 69 2: Evolution of chemical signals and chemosensory systems in ticks 70 3: The tick mating system 71 4. No need for a volatile mating signal 72 5. An alternative role for 2,6-dichlorophenol 73 6. Chemical and mechanical signals in mating behaviour 74 7. Contacting the right female or choosing the right male 75 8. Future prospects 76 REFERENCES 77 ACKNOWLEDGEMENTS 87 SUMMARY 88 RÉSUMÉ 90 Appendices: I Chemical compounds in tick sensory physiology and chemical ecology II Distribution of chemoreceptive sensilla in B. microplus III Bioassay results with other SPE column fractionations IV Bioassay results with cholesterol, cholesteryl oleate and guanine 4 GENERAL INTRODUCTION 1 Boophilus microplus a special kind of tick 1.1. Ticks; a group of obligate blood feeding Arthropods Ticks (Acari: Metastigmata, see TABLE I) are members of the Arthropod subphylum Arachnida and just like spiders, harvestmen and scorpions differ from insects and crustaceans by the lack of antenna and mandibles, but possess the characteristic scissor-like appendages called chelicera. Together with the omnipresent mites, they form one of the most species rich taxa in the Animal kingdom; the Acari. Among the Acari, that have radiated from the predatory lifestyle so common in other classes of Arachnida to almost every lifestyle and habitat imaginable, ticks stand out as a small group of specialised ectoparasites of relatively large body size that feed on blood of vertebrates. Because .of their size. and. detrimental-effect to human economy and health, they have been long since known tomanand were referred to as "disgusting parasitic animals" by Aristotle in the 4th century BC. Unfortunately, man's opinion about these fascinating creatures has not changed much over the last 2300 years. The group, sometimes referred to as superfamily Ixodoidea, sometimes as suborder Ixodida or metastigmata, consists of three families. The Argasidae or soft ticks are considered most primitive. They are ectoparasites of small mammals and birds that feed intermittently, taking short (from a few minutes to several hrs) bloodmeals. The Nutalliellidae, exhibit some characters shared with either Argasidae or Ixodidae, as well as some unique features but are represented by only one species from. Africa, of which only a few female specimens are described. Finally, the Ixodidae or hard ticks are characterised by the presence of a sclerotized shield (scutum), covering the dorsal side of the idiosoma either completely in males, or only partially in females (FIG 1). They are generally parasites of reptiles or mammals often developing close associations with their hosts. Each life stage takes a long (several days) bloodmeal while staying firmly attached to the host's skin: TABLE 1: SysltnratiaposiuoWVfBoophilusynicropIus^HightT order systematics after Krantz (1970) taxon name characters Phylum Arthropoda Body segmented, jointed appendages Subphylum: Chelicera ta No antennae or mandibles Class Arachnida Two pairs of mouthparts (chelicera and pedipalpi) and 8 legs in nymphs and adults, 6 legs in larvae Subclass: Acari Ticks and mites, without any clear body segmentation, possessing a gnathosoma (false head) Order Parasitiformes Suborder Metastigmata Respiratory openings grouped behind coxa IV, Haller's organ. Superfamily Ixodoidea Same as Metastigmata, i.e.. ticks Family Ixodidae Hard ticks, posses a sclerotized dorsal shield (scutum) and mouthparts are visible from above. Palpal tarsus (segment IV) small and placed vemrally on segment III. Subfamily Rhipicephalinae Genus Boophilus small, inornate, no apparent festoons, palpal article I reduced, short mouthparts Species microplus 5 GENERAL INTRODUCTION 1 FIGURE 1. Elements of lick morphology as demonstrated on two species of the Rhipiccphalinae A: Rhipicephalus sanguineus (Latreille) unfed female, dorsal view, after Pomerantsev (1959). B: Boophilus microplus (Canestrini) male, dorsal view C: B. microplus undistended female, ventral (left) and dorsal (right) views of idiosoma (body). Morphological features include: 1. claws, 2. pulvillus, 3. tarsus, 4. tibia, 5. genu, 6. femur, 7. trochanter, 8. capitulum (gnathosoma), 9. chelicera, 10. pedipalp, 11. porose areas, 12. Haller's organ, 13. scutum, 14. festoon, 15. eye, 16. coxa, 17. gonopore (genital aperture), 18. fovea dorsalis, 19. spiracular plate, 20. anus, 21. caudal process. A Melasma» Ixodtdac RhipK^phaliiuc Hyalomminac Prostri« la AfT-asidac Boophilus —Rhipicephalus Rhipiccnlof Dcmuocntor Hyalomma Haemaphysalis Amblyomminac | Amblyomroa "Aponommi Ixodes "Argas HacmaphysaÜnac txodinae ArRasinae Omithodorinae TJrttithodorus Doophilus Rhipicephalus ~Hym!omma Rhipiccntor DermacentOf Amblyotnroa 2 iaenuphysalis Amblyomma 1 AponocTuni Ixodes Ar&zs Omithodoms FIGURE 2. Two proposed phylogenies of important lick genera A: After Hoogstraal and Aeschliman (1982) based on morphological characters and host associations. B: After Black and Piesman (1994) based on mitochondrial DNA analysis. Species of the genus Amblyomma are found in two separate groups, shown by numbering them as 1 and 2. An overview of tick systematics was given by Keirans (1992) with a complete list of valid species; there are some 850 species of ticks in the 3 families and 19 genera. Fossil evidence for the time and place of origin of ticks is virtually absent, so most theories on tick evolution are based on assumptions and extrapolations of present day distribution, host associations and morphologies. The scenario of the development of the various 6 GENERAL INTRODUCTION 1 tick genera that is now most commonly agreed upon is that produced by Hoogstraal and Aeschlimann (1982) (FIG 2A), although it's assumptions have recently been criticised (Klompen et al., 1996). Tick taxonomy and evolutionary biology is extremely rudimentary compared to that of insects. In fact, no formal cladistic analysis had been made until Black and Piesman (1994) published a phylogeny of the important tick genera based on mitochondrial DNA. They managed to confirm largely the phylogeny proposed by Hoogstraal and Aeschlimann but with some important discrepancies (FIG 2B). Both phylogenies, however, place the genus Boophilus in close relation with Margaropus species and the genus Rhipicephalus. The genera Amblyomma, Aponomma and Haemaphysalis branch off before and the members of the genus Ixodes (Prostriata) are considered the most primitive Ixodidae. It is the special place of the Boophilids among the ticks that will be discussed here as it is exemplified by the experimental animal of our choice Boophilus. microplus — a tick species that is special in more than one respect. 1.2. Boophilus microplus: a special case 1.2.1 Taxonomy and distribution The members of the genus Boophilus Curtice 1891 (type species B. annulatus) are all small inornate tick species that are highly specialised parasites of Bovidae. They can be readily distinguished by the apparent absence of festoons, relatively short stout mouthparts, presence of thick ridges on the palps and reduction of article I (Arthur, 1960, FIG 1). Hoogstraal and Kim (1985) postulate that short broad mouthparts and small body size are advanced characters and Boophilus then represents an extreme case. This genus is probably one of the most recently evolved Ixodid genera and speciation might still not be completed (Aeschlimann, personal communication). There are now five species of Boophilus almost universally recognised: TABLE 2: The five commonly recognised species of Boophilus. B. microplus (Canestrini, I877) Southern cattle tick (US), Asian blue tick (RSA), Common cattle tick (ArgJ, Cattle tick (Aus.) B. annulatus '(Say, 1821)............ Cattfe tick of the western hemisphere (US), In Russian literature also as B. calcaratus B. decoloratus (Koch, 1844) African blue tick (RSA) B- zeigyi Aeschlimann & Morel, 1965: no common name known B. kohlsi Hoogstraal & Kaiser, 1960; no common name known A question has arisen as to whether B. annulatus and B. microplus are distinct species since hybridization occurs. Even though Fl females appear normal, the testes of Fl males are absent or vestigial, leading Newton et al (1972) to conclude they are separate biological species. B. annulatus and B. microplus can be easily separated from the members of the B. decoloratus group by the absence of a conspicuous seta on a protuberance of the ventromedial side of the first palpal segment. The fact that the form of the genital opening is also markedly different for the two groups (Feldman- Muhsam and Shechter, 1970) confirms their distinctness. The genital opening of B. decoloratus, B. geigyi and B. kohlsi resembles that of the Rhipicephalus sanguineus group. The present day distribution of the five species is strongly influenced by man's habit of transporting cattle around the world (FIG 3). It is believed that originally B. microplus was indigenous to the oriental region only (Morel, 1969) whereas B. annulatus probably originates from the Near East. The latter species has spread 7 GENERAL INTRODUCTION 1 around the Mediterranean and was probably introduced in the Americas and West-Africa with Iberian cattle. B. microplus has been introduced into Madagascar and East-Africa on Indian cattle breeds and from Madagascar it was transported to South- Africa in 1896. It too has reached South-America from where it has spread to its present distribution. More! (1969) also suggests that the spread of B. decoloraius into West-Africa's savannah was secondary. This means that originally the five species could have been distributed either parapatrically or allopatrically with B. microplus in tropical Asia, B. annulatus in the West and Central Asian steppes, and B. decoloratus and B. geigyi in East- and West-Africa respectively. B. microplus was probably introduced in Australia with Brahman or Balinese cattle from Indonesia in the nineteenth century (Roberts, 1965). Roberts also comments on the large variability between populations of this species in certain morphological characters such as the size and shape of the adanal plates, the caudal process in males and the scutum in females; with most characters varying considerably, even between individuals from the same host. This points to the high morphological variability generally observed in this species, complicating species determination. Incidently, Spicket and Malan (1978) noted that when Australian B. microplus are mated with South African ones females produce very little viable offspring suggesting the existence of sibling species. FIGURE 3. Distribution of the five recognised Boophilus species in the world. A: Worldwide distribution of B. microplus and B. annulatus with arrows indicating presumed secondary spread through cattle importations. B: species of the B. decoloratus group in Africa and the Near East. Modified alter Morel (1969), Feldman-Mühsam and Shechter (1972), Pomerantzev (1950), Roberts ( 1965), Wharton ( 1974) and Rawlings and Mansingh ( 1987). 8 GENERAL INTRODUCTION 1 1.2.2 Host specificity and life cycle Not only is the sole source of nutrients for Ixodid ticks found in vertebrate blood but their whole life history is strongly correlated with that of their host. Unlike most of the Argasidae, hard ticks attach for several days to the skin and thus pass a considerable part of their life in the particular chemical, thermal and mechanical environment of the host's skin. Most of the important processes in their life-cycle take place there, even though the time spent off the host may be much longer. Ecologically, ticks can be divided in endophilic and exophilic species (Sonenshine, 1993). The former, also called nidicolous, are closely associated with caves, nests or burrows of their host while the latter live freely in various biotopes and need to somehow locate their hosts more actively. Exophilic ticks employ one of two strategies to do this. They either actively hunt for the host or sit and wait. B. microplus is a typical example of the latter "ambush" type^It has considerably.limited the.duration of the "wait" part of the strategy by going-through a non^parasitic:phase only once in its lifetime, i.e., as larva. More than 90% of the known Ixodid tick species exhibit the typical three-host life-cycle (Hoogstraal and Aeschlimann, 1982) where each life-stage; larva, nymph and adult, feed on different individual host animals and drop to the ground between bloodmeals to moult. The three hosts may be from different species or from the same species, as is necessarily the case with endophilic tick species. Some species in the genera Hyalomma and Rhipicephalus have developed either an obligatory or facultative two-host life-cycle in which larvae and nymphs feed on the same individual host. The one-host life-cycle, with all stages feeding on the same host and even moulting in situ is almost unique to Boophihts and Margaropus species and may have evolved only once in this group. The three species of the genus Margaropus are closely related to Boophilus and are exclusively African parasites of giraffes or horses. Only a few other species show a one-host pattern, sometimes related to extreme weather conditions notably the winter or moose tick, Dermacentor albipictus. The larvae of B. microplus, being the only free living life stage, are therefore responsible for the host specificity of this species. Most Ixodidt'Species ,exhibit£Some:fonn-..oÊ:host specificity and many species have members of the large mammalian group of Ungulates as hosts, especially in the adult stage (Hoogstraal and Aeschliman, 1982). Together with members of the genus Aponomma, highly specific for certain reptiles, Boophilus species are among the most host specific of ticks with all stages predominantly feeding on members of the Ungulate family Bovidae. B. microplus specialises on cattle and buffaloes, occasionally parasitising horses (Equidae). The latter phenomenon seems to be more prevalent in B. decoloratus which is a bit less specific, while B. kohlsi is almost exclusively found on goats (Feldman-Muhsam and Shechter, 1970). The life-cycle of B. microplus is relatively short (Nunez et ai, 1985). Under favourable conditions (warm and humid, i.e., tropical) engorged females take about 14 days, after dropping from the host, to lay most of their eggs. These in turn hatch within 20 days, again depending on temperature, and larvae climb vegetation to attach to a passing host after 7 days (Davey, 1987). Development is rapid once the larvae attach to a suitable host and, most importantly, independent of climatic conditions. After 6 days on the host, nymphs appear that will moult into adults 7 days later. The first engorged females drop only 20 days after the larvae attached. By comparison, the parasitic phase of D. albipictus is strongly influenced by climate and takes up to 150 days in winter (Drew and Samuel, 1989). Thus, unlike most ticks which complete one life-cycle per year or less and at times include a diapause (Belozerov, 1991), 9 GENERALINTRODUCTION 1 B. microplus can make it through a full cycle in just 60 days and can have up to 6 generations per year. Moreover, egg production is high, averaging 2500 per female and eclosion is usually around 80%. Even though mortality on the host can be remarkably high, this tick has a high potential for rapid population increase in pastures regularly grazed by cattle (Mount et al, 1991). 1.2.3 Economic importance Ticks are second only to mosquitoes as important vectors of disease. In fact the first demonstration of transmission of a disease by an arthropod vector was when Smith and Kilborne implicated B. artnulatus in the causes of Texas fever in 1893 (Uilenberg, 1992). A great variety of diseases are transmitted to domestic animals. Human diseases are less prevalent, though the discovery of Lyme disease in 1977 has led to a renewed interest in tick biology. Members of the genus Boophilus are efficient vectors of two important cattle diseases: Babesiosis (Texas fever or red water) and Anaplasmosis (gall sickness). B. microplus transmits highly pathogenic agents that cause these diseases, Babesia bigemina, B. bovis and Anaplasma marginale world-wide. Apart from transmitting diseases, these ticks also cause anaemia, weight loss, reduced milk production, secondary infections and diminished value of hides. The cattle tick is undoubtedly one of the most important pests of domestic animals (Wharton, 1974). Tt causes considerable damage to the cattle industry, particularly in areas where extensive cattle ranching is an important source of income such as the southern US, Northern Argentina, South Africa and Australia (including New Guinea and some Melanesian islands). Interestingly, there are less problems with B. microplus in India and other South Asian regions since Indian cattle breeds (Bos indicus) develop resistance to this tick and hence they occur in relatively low numbers on Zebu and Brahman cows (Wharton and Norris, 1980). The various European (Bos taurus) breeds used mostly in ranching are, however, highly susceptible and develop heavy tick loads. If no control measures are taken, some animals can die simply by loss of blood or secondary infection of wounds. In the US, where B. microplus occurs together with B. annulatus, heavy losses and expensive control measures led to a majoprcampaign in the early half of this century and the two species have been eradicated from Texas and Florida. Ever since, a quarantine zone has been permanently maintained along the Mexican border to prevent reintroduction. Tn South Africa, B. decoloratus is less of a problem, but there are reports that B. microplus is spreading and replacing this species in certain areas (Norval and Short, 1984). In Australia B. microplus is prevented from being introduced to areas outside its present range by continuous expensive control measures which are helped by climatic conditions that do not favour permanent establishment. However, since most of the methods used are chemical, the biggest problem in control of B. microplus is its capacity to develop resistance to the acaricides employed. This species has developed resistance against all known classes of acaricides even to the relatively recently introduced synthetic pyrethroids (Nolan, 1985). Especially in Southern Queensland, at the fringes of the tick's distribution range, numerous strains are characterised by different simple- and cross-resistance mechanisms (Wharton, 1974, Nolan, 1985). The variety of B. microplus that is used in our experiments is one such organophosphorous resistant strain from Biarra, Southern Queensland, discovered in 1966. Recent developments have led to the emergence of a commercial vaccine against antigens in the tick's gut cells as a new hope in the continuing struggle against B. microplus (Willadsen el al., 1995). It is the first time that such a vaccine has been developed 10 ^1S-SHT1V, :-. ¦ i- GENERAL INTRODUCTION 1 against an arthropod — another illustration of the unique economic importance of this tick 1.2.4 Reproductive biology Autogeny and repeated gonotrophic cycles occur in Argasidae but Ixodid ticks go through one single gonotrophic cycle and feeding is necessary for the females to produce a batch of eggs (Oliver, 1989). In the prostriata (genus Ixodes) mating can take place before or during feeding and spermatids are produced in the late nymphal and early adult stages. All metastriate ticks mate on the host during feeding and a blood-meal is necessary for males before meiosis and spermatid production can occur. Allmost all tick species are bisexual and generally produce a 1:1 sex ratio among progeny. Parthenogenesis has been reported as possible in many species of ticks including B. microplus (Stone, 1963, Thompson et al, 1980), but is probably of little practical significance-since-larvae, produced are barely viable. Some Haemaphysalis species however have parthenogenetic geographical races and it is the sole method of reproduction of at least two species of Ambtyomma (Oliver, 1983, 1989). Copulation consists of transfering incapacitated spermatids via a spermatophore which is deposited onto the female gonopore (Feldman-Muhsam and Borut, 1983). The spermatophore is a complicated structure (Oliver, 1982) which, after being deposited, evaginates into the female tract, leaving part of it outside. It contains proteins, sugars, symbionts and a polypeptide that regulates final sperm maturation (Shepherd et al., 1982) as well as a proteinaceous factor which signals the presence of sperm to the reproductive system of the female and initiates vitellogenesis (Diehl et a!., 1982, Sahli et ai, 1985). Rapid feeding and subsequent engorgement is similarly stimulated by a factor present in the spermatophore (Pappas and Oliver, 1972). In most species, males are known to be able to copulate repeatedly with the same or different females, i.e., several spermatophores are regularly found in female genital tracts. il GENERAL INTRODUCTION 2 Sensory physiology of ticks All animals need to be informed about their environment, in order to escape its dangers (desiccation, freezing, prédation) and to utilise its resources (food, shelter, mates). This information is present in numerous forms (thermal, radiated, mechanical and chemical stimuli) and animals have developed various sensory systems to perceive them, concomitant with such behaviours that enable them to adapt to, or change the environment, so as to maximise their fitness. Ticks possess relatively simple sensory systems that nevertheless are sufficient for their survival. It is therefore of considerable biological interest to study their physiology and we have set about doing so in the context of the mating behaviour of B. microplus. At the peripheral level, arthropod sensory systems usually include a variety of small organs embedded in the^cuticle called sensilla (see Zacharuk, 1985). They contain 1) sensory neurons, 2) some non-sensory associated cells and 3) a modified zone of cuticle very often in the shape of a hair, plate or pore. The typology of such structures has been described for insects (Altner and Prillinger, 1980) but analogous sensilla can be found in ticks (Hess and Vlimant, 1986). For a detailed summary of the distribution of sensilla on the legs and palps of B. microplus see appendix II. We can now examine the potential sensory cues that can play a role in mating by looking briefly at the different senses in ticks in general and B. microplus in particular. Heat A source of warmth is generally accepted to be the major cue in inducing feeding for different species of ticks. It also plays an important role in host location. Ticks readily orient to warm objects (Totze, 1933, Krijgsman, 1937, Lees, 1948) but these reactions have been observed over relatively short distances. This, and some experiments with low emissive material, led Lees (1948) to believe that only air temperature was involved (convected or conducted heat) while radiated heat was not perceived. Experiments with A. variegatum however, indicate heat perception overmuch larger distances where infrared radiation might play a role (Robert, 1985, Poffet, 1988). A cold receptor was first described in a terminal pore (tp) sensillum on the anterior tarsi of R. appendiculatus (Waladde et ai, 1981) and later warm and cold receptors were characterised in two no-pore (np) sensilla more distally on the anterior tarsi of A. variegatum (Hess and Loftus, 1984). The resolving power of these units for abrupt temperature changes was below 1°C. Although the first pair of legs was known to play a prominent role in orientation to heat sources (Lees, 1948), it has also been observed that masking or ablation of the anterior tarsi does not lead to complete insensitivity to heat (Totze, 1933). As mating of B. microplus takes place on the host, a relatively high temperature and possibly a steep gradient are part of the natural environment for this tick. Humidity The importance of humidity for the survival of ticks is well documented. As small animals, exposed for long periods to differing climatic conditions, ticks need to deal with the problem of desiccation. Oriented responses to humidity have been shown to be greatly influenced by physiological state. Lees (1948) recorded a marked avoidance (negative taxis) of sudden increases in humidity. Longer term reactions include kinetic 12 GENERAL INTRODUCTION 2 effects, which, depending on the hydration state, resulted in arrestment of ticks in areas of higher humidity. Hygroreceptors have so far not been demonstrated in ticks but may be present in the anterior pit of Haller's organ. Attached ticks and those moving through the pelage of their bovine hosts are permanently exposed to high humidities (Kuhnert, 1995). Light Ticks do not possess compound eyes like insects, nor do they have big simple eyes with well developed lenses as spiders do. Some ticks are reported as eyeless because no apparent eye-like structures are present on the cuticle (e.g. Ixodes and Haemaphysalis species). However, Binnington (1972) has shown that even such eyeless species have photoreceptors at several points under the cuticle and optical nerves with corresponding centres in the synganglion. Clear responses to direct light have been shown in eyeless ticks such as Ixodes ricinus (Lees, 1948). Light perception is probably monochromatic, with sensitivity peaking around 500 nm (blue/green) and a marked low sensitivity beyond 600 nm (red), as has been demonstrated by electroretinogram (ERG) recordings in eyes of D. variabilis, A. variegatum and H. dromedarii (Camroll and Pickens, 1987, Kaltenrieder el ai, 1989). Many ticks, including B. microplus, have been shown to orient away from light sources and respond to changes in overall light intensity (Totze, 1933). Lees (1948) demonstrated that this negative phototaxis disappears with ageing in unfed I. ricinus but is restored in engorged ticks. A sudden decrease in light intensity (shading) leads to questing activity in host seeking ticks in repose position. In addition, a marked orientation toward areas of lower intensity of reflected light (i.e. darkness, skototaxis) was present in this species. Some hunting ticks such as Hyalomma asiaticum and H. dromedarii apparently use this to focus on a silhouette during the approach to a host (Leonovich, 1986, Kaltenrieder et ai, 1990). The eyes of B. microplus are not well developed but are visible under the microscope as a slightly elevated and modified zone of cuticle at the edge of the scutum. There is no apparent order in the arrangement of photoreceptive.ceHs.vAcertam,sensitLvitydn"Ä::./w/c/'0/?/w5 larvae to shading has been demonstrated (Waladde and Rice, 1982) and can therefore be expected in adults too. However, it is unlikely that visual perception of mates plays a major role in mating behaviour. Mechanical stimuli Waladde and Rice (1982) have observed that sound (i.e., air borne oscillations) in the 80-800 Hz range activates B. microplus larvae. However, chordotonal organs have never been found in ticks and the only convincing report for orientation to sound is from an argasid tick species, Ornithodorus concanensis that is attracted to the chirps (3-8 kHz) produced by its bird host (Webb et ai, 1977) These authors imply that the first pair of legs is involved in the perception. Vibrations (i.e., substrate bome oscillations) are an important means of communication and prey location in spiders and scorpions. These stimuli are perceived by so-called slit sensilla, small intracuticular mechanoreceptive sensilla (Brownell and Farley, 1979). A number of different but similar structures has been found on the legs of ticks (Hess and Vlimant, 1984 & 1986). These structures may be involved in the perception of vibrations and/or sounds, as was previously suggested by Schulze and Schröder (1949). Unfortunately, very little research has been done on the perception of vibrations by ticks. A. variegatum can perceive strong vibrations in much the same range as described by Waladde and Rice 13 GENERAL INTRODUCTION 2 (30-300 Hz; Stämpfli-Pauchard, 1989) but reports that Hyatomma species respond to vibrations in the sand produced by the tread of camels and other large mammals have so far not been confirmed (Leonovich, personal communication). Another possible role of these mechanoreceptor organs is in the perception of gravitational pull (Robert, 1985). Interesting responses have been described by Lees (1948) in I, ricinus when climbing up and down vertical structures. These ticks are capable of evaluating the difference between upward and downward direction and get arrested just below the tip of vertical rods after several up and down walks. However they show no preference for top or bottom end of a tube with closed ends, confirming results obtained by Krijgsman (1937) with B. anmtlatus larvae. Other tactile stimuli such as physical disturbance, substrate contact or texture probably play a major role in tick sensory ecology. Ticks questing for hosts on vegetation respond to mechanical stimuli and attach to passing adequate substrates (Lees, 1948). This is the principle behind the flagging method so often us¥d to collect free-living stages of ticks. Ticks show distinct preferences when offered substrates with different textures (Totze, 1933). Another commonly observed phenomenon is the tendency to push into crevices and assemble at edges and obstacles (thigmotaxis) noted by many authors but never properly quantified. Male B. microplus, when removed from their host, are arrested by and attach to any rough surface or in comers and edges (personal observations and Kuhnert, 1995), whereas larvae form clumps by holding on to one-another. However, some of these effects can be explained without the explicit need for sensory perception. Ticks do bear many mechanoreceptive hairs with uninnervated shafts (np sensilla) on legs and other body parts (Hess and Vlimant, 1986, Vlimant unpublished). Like those described in insects, they posses the enlarged dendritic structures called tubular bodies, associated with the flexible membrane of the sensillar socket at the base (Altner, 1977). They differ from insect mechanoreceptors by having two tubular bodies instead of one which is characteristic for Arachnida (Foeïix, 1985). These bodies are placed on opposite sides of the sensillum and might thus be more sensitive to the direction of deflection of the hair. Ticks generally show negative anemotaxis (Lees, 1948). Although the receptors involved in-tlffiorientation to wind are not known they probably are mechanoreceptive seta. Chemical stimuli The discrimination between olfaction and taste (gustation) is based on definitions that differ widely between various fields of interest (Chapman, 1995). Henceforth we will define olfaction as the sense carried by wall-pored sensilla (wp) with shafts innervated by sensory cells that probably project directly to a defined group of nerve cells in the glomeruli of the central nervous system. In contrast taste sensilla are typically terminal pored (tp) and the first synapses of their receptor cells are in the region of the brain directly linked with the body region on which they lie (palpal lobe, pedal lobes etc.). No assumptions are made about criteria used in definitions elsewhere such as the medium of transport of chemical cues, the nature of the chemicals perceived, the transduction mechanisms involved or the specificity/sensitivity spectrum of receptors. Ticks are characterised, among other things, by Haller's organ, a compound sensory organ consisting of a capsule and an anterior pit on the dorsal aspect of the tarsus of leg I. Originally described by Haller in 1881 as an auditory organ, its structure has since been accurately investigated and found to comprise various sensilla with a relatively stable number of sensory cells and a gland associated with the capsule (Foelix and Axtell, 1972, Balashov and Leonovich, 1976, Hess and Vlimant, 1986). An 14 GENERAL INTRODUCTION 2 olfactory function for this organ was first suggested by Lahille (1905) based on observations on B. annulatus. Hindle and Merriman (1912) then unequivocally established the location of olfaction on the tarsus of the fowl tick Argas persicus (Argasidae). It has since been established that more olfactory sensilla are present on the surface of the anterior tarsus outside Haller's organ but none have been found elsewhere. These olfactory wall-pored (wp) sensilla are of two types: single-walled (wp-sw) and double-walled (wp-dw). The former have branched dendrites innervating the shaft whereas the latter have unbranched dendrites and cuticular canals running between the two walls (spoke wheel structure, Foelix and Axtell, 1971). Binnington (1987) showed that sensory nerves from the anterior legs of B. microplus pass the pedal ganglion and project onto olfactory glomeruli. Thus the olfactory system seems analogous to that of insects with leg pair I functioning, like antennae. This is also clear from behavioural.observations.(Lees, 1948) where leg waving suggests a function of air-sampling similar; to that ; shown by insect antennae. Lees (1948) also demonstrated the important role of olfactory stimuli in host seeking by I. ricinus. In spite of its considerable importance, the entire olfactory system in ticks comprises only cof 90 peripheral nerve cells (Hess and Vlimant, 1986) as compared to ca 40.000 sex pheromone receptors alone in male moths (Boeckh et al, 1965). Roughly half of these are located in Haller's organ. The physiological responses of some of these cells have been characterised (Sinitsina, 1974, Haggart and Davis, 1979, 1980, 1981, Waladde, 1982, Steullet, 1993) and in A. variegatum responses were found to CO2, H2S, NH3, short fatty acids, ketones, lactones, and aldehydes which have all been isolated from host odours (Steullet, 1993). In.-addition, various phenolic compounds as well as benzaldehyde- and salicylaldehyde-based compounds which can be both host- and/or tick-produced are perceived by olfactory hairs on the tarsus. It.seems that the tick olfactory system uses a minimum amount-of cells in an optimal way with very little replication of receptor types. Responses to gustatory stimuli have been poorly investigated. In fact, early authors suggested that a sense of taste was absent in ticks (Totze, 1933). However, several authors have since, .shown ;-.that.:Xermmal,:pore.v(tp)- sensilla are present on the tarsi, especially of leg I, and on the pedipalps. In addition, such sensilla are located on the idiosoma as well (Vlimant, personal communication). A role in the mating behaviour of three ixodid ticks has been suggested for the so-called claw sensilla at the tip of the tarsus I (Phillips and Sonenshine, 1993) but physiological data for these and other tarsal contact chemo receptors are lacking except for reports on responses to salts of one of the paired claw sensilla in two Ixodes and one Hyalomma species (Elizarov, 1963, Guerin et al, 1992). The compact group of tp sensilla on the apical surface of the palpal segment IV has been suggested to play a role in perception of an off-the- host contact pheromone in some Argasid ticks and A. hebraeum (Leahy et ai, 1975a, Rechav et ai, 1977). This "palpal organ" is probably a complex mechano-gustatory organ capable of perceiving many different contact stimuli (Grenacher, personal communication). Additional gustatory receptors are described in pores in the hard cuticle of the cheliceral digits of B. microplus (Walladde and Rice, 1977) and electrophysiological recordings from these sensilla in Dermacentor species revealed responses to 20 hydroxy-ecdysone (Taylor et al, 1991). Summarising we can state that the signals most likely involved in the mating behaviour in B. microplus are of chemical and/or mechanical nature perhaps modified by thermal and humidity cues. 15 GENERAL INTRODUCTION 3 Tick associated chemical stimuli in Tick biology* Many tick species have been investigated for the role of chemical signals in their behaviour but research has mostly concentrated on economically important species most notably Dermacentor variabilis and D. andersoni, two American ticks that infest dogs, various cattle pests such as Amblyomma species and Rhipicephalus appendiculatus, the camel tick Hyalomma dromedari! and the fowl tick Argas persicus. With regard to the use of infochemicals in intraspecific communication no clear picture has emerged of the distribution, variety and common principles within the Ixodidae. There is practically no knowledge available for Boophilus microplus, excepting an early report by Chow et al. (1972). In view of its economical importance, but maybe more because of its special position in tick biology, an investigation into chemically mediated behaviour of this species is clearly needed. Before proceeding with such a study it is appropriate to give a brief introduction to chemical signals mediating behaviour in other species of ticks, restricted to those stimuli associated with ticks rather than their hosts or their environment. 3.1. Volatile signals For most Ixodid tick species the first and major task to accomplish in life is to locate a host. It is on the host that they will find both food and mates. Volatile signals from the host play a crucial role in helping ticks to reach this goal and various host odours have been shown to excite olfactory receptors in the tick Amblyomma variegatum (Steullet, 1993). It is in this same genus of ticks that volatile signals, produced by ticks already feeding on the host, were found to play a role in host seeking when combined with CO2 (Norval et ai, 1989). This signal, produced by male ticks, is now commonly referred to as "aggregation attachment pheromone" or also "attraction-aggregation- attachment pheromone" and was first described as attracting female and male A. maculatum on the host to feeding males. Its presence was necessary for;,females to attach to a host (GIadney 1971, Gladney at ai, 1974). Such a signal is also present in A. hebraeum and attracts nymphs as well (Rechav et al., 1976, 1977). Schöni et al (1984) isolated 2-nitrophenoI, methyl salicylate and nonanoic acid from extracts of fed male A. variegatum and showed that this mixture of compounds induced aggregation as well as pairing of males and females in a bioassay off the host. It is probably produced in dermal glands on the ventrolateral cuticle of male ticks and only after a few days of feeding (Diehl et al., 1991). Apps et al. (1988) isolated benzaldehyde instead of methyl salicylate and 2-methyI propanoic instead of nonanoic acid in the headspace of A. hebraeum which might explain the species specific behavioural responses to this pheromone (Rechav, 1978). Olfactory receptors for components of the pheromone of both species are found in Haller's organ of A. variegatum (Hess and Vlimant, 1986, Diehl et ai, 1991, Steullet and Guerin, 1994a&b) and field experiments confirm earlier findings that 2-nitrophenol attracts females of this species over a considerable distance whereas methyl salicylate and nonanoic acid play a role in pairing and attachment (Hess and de Castro, 1986, Schöni et ai, 1984). Dongus and Gothe (1995) demonstrated the existence of a volatile signal from fed male Hyalomma truncatum that attracts conspecific ticks. Consequently, a signal, combining attraction for information on chemical compounds in this and following chapters refer Io appendix I 16 GENERAL INTRODUCTION 3 to the host with aggregation and sexual pairing on the host, may not be exclusive for Ambfyomma species. In the 1970's Berger identified a compound which apparently induced male ticks on the host to detach and move towards females (Berger et ai, 1971, Berger, 1972). It was promptly declared a female produced sex attractant in agreement with ideas that were then developing in research on moth pheromones. However the compound, 2,6- dichlorophenol, as well as the behaviour it causes were controversial (Oliver, 1974). The rather unusual phenomenon of the biosynthesis of a chlorinated organic compound by an arthropod was confirmed when radiolabeled Cl injected into nymphs of A. americanum was found incorporated in 2,6-dichlorophenol produced by the adults (Berger, 1974) and the compound has since been found in 14 species in the genera Ämblyomma, Haemaphysalis, Hyalomma, Dermacentor and Rhipicephalus (Sonenshine, 1985). However, it was consistently found in males as well as in females whenever it was properly investigated (Kellunr- & Berger, 1971, McDowell & Waladde, 1986, Sonenshine et al.t 1984, Price et al., 1994). Only in A. americanum and H. dromedari is there evidence that males produce considerably less of this compound (Kellum & Berger, 1971, Silverstein et al, 1983). A complex of dermal glands, associated with two small pored plates on the alloscutum of metastriate ticks, the foveae dorsales, was subsequently linked with biosynthesis and emission of this compound (Sonenshine et al., 1981). These foveal glands are present in all life-stages of metastriate ticks but are not present in prostriata (Schulze, 1942, Dinnik and Zumpt, 1949). Behavioural responses evoked by 2,6-dichlorophenol include leg movements of attached males and subsequent detachment (e.g. Berger, 1972, Chow et al., 1975), effects which it has been shown can be evoked by many other, non chemical, stimuli such as an airstream, heat or physical disturbance. In H. dromedarii it attracts males on the host over a distance of 1 cm but appears not to be attractive when offered in an airstream off the host (Khalil et ai, 1981) whereas A. variegatum and A. hebraeum males in the field are attracted over considerable distance to sources of 2,6-dichIorophenol:ywhen.combiaed:withiC02r(Norval et ai, 1991). Orientation to a wide range of concentrations of 2,6-dichlorophenol applied to the skin of a rabbit was observed in fed males of A variabilis and D. andersoni (Sonenshine et al, 1976). Unfed males and unfed or fed females did not show such responses. However some of these tests were done in the presence of "preserved" female ticks and the statement that 2,6-dichlorophenol is sufficient to elicit orientation and copulation is therefore not fully convincing. The role of this compound in mating behaviour of tick species is still not fully resolved. Olfactory receptors for 2,6-dichlorophenol have been found in five species of Ixodid ticks in two sensilla: one identical to the one containing 2-nitrophenol receptors and the second more distal from Haller's organ on the tarsus of leg I (Chow, 1979, Haggart and Davis, 1981, Waladde, 1982, Thonney, 1987). This product has remained the only identified volatile implicated in sexual behaviour in ticks. However, Wood et al. (1975) report the presence of two other phenolic products, 4-methyl phenol and phenol in some Rhipicephalus species and their potential to cause detachment and attraction of male ticks. In other tick species, various distinct chemical signals have been described to influence behaviour of male ticks from a distance but have so far not been identified (Gothe 1987, Leonovich, 1981, Petney and Bull, 1981, Andrews and Bull, 1982a, Rechav, 1983. Andrews et ai, 1986) 17 GENERAL INTRODUCTION 3 3.2. Low-volatile, cuticle associated signals Lipophilic chemical signals of low volatility, present on the cuticle are known to play various roles in arthropod behaviour. Cuticle hydrocarbons have especially been implied in the mating behaviour of members of a wide variety of insect orders. They also mediate host recognition in parasitoids and play a major role in the social organisation of bees (Howard and Blomquist, 1982) as well as in the biology of its acarine parasite Varroa jacobsoni (Rickli, 1994). A variety of long chain hydrocarbons have been reported to be present on the cuticle of ticks (e.g. Estrada-Pena et ai, 1992) including B. microplus (McCamish and Canneti, 1980) but have so far not been implicated in tick behaviour. In D. variabilis and D. andersbni, mating behaviour is mediated by chemicals on the cuticle since hexane extracts applied to previously washed and biologically inactive females induce typical courtship behavioural responses after the male contacts the female (Hamilton and Sonenshine, 1988). Cholesteryl oleate and other cholesteryl esters have been isolated from cKese extracts and their activity in combination with 2,6-dichlorophenol has been demonstrated in this bioassay (Hamilton et al., 1989, Sonenshine et al., 1991). Cholesteryl esters have also been reported to induce mating responses in R. appendiculatus (Hamilton et ai, 1994), However, this signal is not considered to be very specific. In the two Dermacentor species another chemical signal inside the female gonopore induces male ticks to copulate, i.e., produce a spermatophore and insert it (Sonenshine et al., 1982) and prevents interspecific copulations. Since mating behaviour is aborted when the anterior reproductive tract is surgically removed, but restored when the gonopore is treated with extracts of this tissue, this signal must not normally be present on the exterior parts of the cuticle. Allan et al. (1988) suggest that fatty acids, predominantly myristic (14:0), palmitic (16:0) and stearic (18:0) acid are responsible since they are present in an extract of the anterior reproductive tract and, at least in D. variabilis, are as active as the extract. However these compounds are present on other parts of the cuticle as well, and the methyl esters of these free fatty acids, though not present in the extract, can induce the same activity. Taylor et al. (1991) describe male copulatory responses to 20-hydroxy-ecdysone and other steroids departed on the gonopore and account also for electrophysiological responses from gustatory receptors in the chelicera to this hormone. The exact composition of this chemical signal as well as the relevance of its components for species specific behavioural responses remain to be established. In two African camel ticks H. dromedari! and H. anatolicum excavation such a genital signal is present but apparently not species specific (Khalil et ai, 1983). There is some evidence for a similar signal in A. americanum but the regulation of genital probing behaviour appears to be different for A maculatum (Allan et ai, 1991). In Argasidae the coxal organ produces a secretion that induces male mating responses when smeared over the cuticle of nymphs (Schlein and Gunders, 1981). The coxal organ has an osmoregulatory function in argasid ticks and is absent in Ixodidae. However an exocrine gland associated with it is present and is thought to play a role during moulting in argasid as well as ixodid ticks (Binnington, 1975). 3.3. Substrate deposited signals Many arthropod species are known to aggregate and several cases are known in which a chemical signal deposited on the substrate is involved (Ishii and Kuwahara, 1968, Schofield and Patterson, 1977, Mumcuogli et ai, 1986), often in combination with other stimuli such as mechanical contact with objects or conspecifics. An association 18 GENERAL INTRODUCTION 3 with faeces and/or other excretory products is commonly demonstrated e.g. in flour mites (Levinson et al ,1991) and the acarine bee parasite V.jacobsoni (Donzé and Guerin, 1994). In ticks, aggregation of free living life stages on filter papers impregnated with tick derived material was first demonstrated in Argasidae (Leahy et al, 1973, 1975b, Leahy, 1979). This water or saline (0.9% NaCl) soluble non-volatile interspecific signal is commonly, though not consistently, referred to as "assembly pheromone". The arrestment of groups of ticks after 1 hr on such contaminated filter paper discs has been observed in petri-dish bioassays in numerous species of Argasid ticks. Off-host aggregation is induced by products from all life-stages and all stages appear to respond, though strongest responses are often recorded from males responding to products of females. Fed ticks appear to produce more of this material whereas unfed ticks respond better. Similar effects were observed in four Ixodes species (Graf, ,1975, Treverrow et al, 1977, Uspensky and Emelyanova, 1980, Hâjkova and -Leahy, 1982), two Dermacentor species, A. americanum and Heamaphysalis leporispalustris (Leahy et al, 1983) as well as in H. dromedarii (Hajkova et al, 1980). In the latter species, significant responses were only obtained from males responding to female contaminated papers. It would seem therefore that arrestment in response to substrate deposited material from ticks is a widespread phenomenon in argasid and ixodid ticks though the existence of such a pheromone in the Dermacentor species was later denied (Taylor et al, 1987). The immediate locomotory and other behavioural responses have never been recorded, so knowledge on the mechanisms involved is lacking and end results are apparent only after 30 min. In addition, the role of this behaviour in tick ecology is unclear. It has been suggested that ticks aggregate to prevent loss of water and increase the chances of locating hosts or mates. George (1981) observed that even though responses in bioassays of two argasid species were interspecific their clusters in nature were usually separated. In Argas walkerae the active material is associated with tick excreta (Gothe et al, 1984). However, in Ixodes and Aponomma species, papers brought in contact with nymphal exuviae produced similar effects (Treverrov et al, 1977, Uspensky and Emelyanova, 1980) which.- might^diçate-a-: secretory instead of excretory origin of some chemicals involved. Otieno et al (1985) demonstrated the presence of guanine in active fractions and its role in aggregation of two argasid species and also showed responses from R. appendiculatus to this compound. In addition many other purines as well as ammonium salts induce arrestment. Xanthine and hypoxanthine were shown to be present in pellets of five species of Argasidae and xanthine increases arrestment to guanine (Dusbâbek et al, 1991a & b). Guanine has long since been known as the primary end product of the nitrogen excretion cycle of many arachnids including mites and ticks (Kitaoka, 1961, Hamdy, 1972, Hamdy and Sidrak, 1982). It is excreted in the form of white pellets from the anus and distinct from the dark faeces which mainly contain haematin. Perception of this chemical signal is eliminated after ablation or masking of the palps (Leahy et al, 1975a, Gothe et al., 1984) so contact chemoreceptors on the palpal organ are likely to be involved but mechanical factors (Dusbâbek et al, 1991a) and humidity (Hassanali et al, 1989) are reported to modify behavioural responses. 19 _______________________RESULTS 1_____________________ The on-host sexual behaviour of Boophilus microplus males and their in vitro responses to females and dummies in a host-simulating arena MARIEN DE BRUYNE unpublished results INTRODUCTION Mating behaviour in Boophilus microplus follows the general pattern described for other Ixodid tick species. To permit transfer of a spermatophore to the genital aperture of a female the male needs to accomplish four things: 1) he has to detach himself from the host's skin, 2) locate the female, 3) pass to her ventral side and 4) locate the gonopore. How these goals are achieved, and particularly the types of signals which play a role in inducing and guiding the male's behaviour, is still a matter of dispute. Sonenshine (1985) describes the mating behaviour in Dermacentor variabilis as follows: 1) attached, feeding males detach under the influence of the sex pheromone 2,6-dichIorophenoI. 2) initial random searching behaviour leads to detection of pheromone which is followed by short-range orientation to the female, 3) after making contact, males mount and palpate the female dorsum then turn to crawl under the female via the posterior end, and 4) ventral positioning then leads to location of the gonopore, insertion of the chelicera and deposition of the spermatophore after some 10 to 20 min. Females are described as fairly passive in this process. Though' they often raise their bodies to facilitate male passage, they are apparently not capable of hindering males by refusing this move. It is not entirely clear in what way the first two phases are regulated by the pheromone, especially the distinctness of the de novo detection of 2,6-dichIorophenol in phase 2, after mediating detachment in phase 1. Hamilton and Sonenshine (1988) score 6 phases in their behavioural bioassay with D. variabilis and D. andersont orientation, contact, mounting, turning, move to female's venter and location of gonopore. They conclude that 2,6-dichlorophenol regulates the first two phases and a contact sex pheromone the remaining four. In both these descriptions, however, it is not clear which events mark the transition between the different phases or how they were quantified. A third chemical signal associated with the gonopore had already been described for these same two species (Sonenshine etaiy 1982) The mating behaviour of Aponomma hydrosauri and other Australian reptile ticks follows a similar pattern (Andrews and Bull, 1980, 1982a&b). However, these authors discriminate between two volatile signals, on'e responsible for detachment of fed males inducing unoriented movements on the host and another affecting fed and unfed males for attraction at short range. In addition, males of different species show courtship behaviour after contact with females of all three species, presumably mediated by a third signal, but mating is successful only with conspecific females. They argued that this is because the female body lift occurs only in response to conspecific males 20 RESULTS 1 (Andrews, 1982). They also observed that differences in body size and leg orientation during the ventral positioning make it impossible for males of one species to insert their mouthparts into the gonopore of females of other species and suggest that such mechanical factors play an important role in mating. B. microplus is different from other tick species previously investigated for mating behaviour, by being a one-host tick, i.e., larvae locate and colonise the host and nymphs moult into adults on the host so that in the latter life-stages there is no host seeking and host colonising phase prior to the meeting of the sexes. It has also been shown that males are capable of recognising potential mates while they are still in the nymphal stage (Falk-Vairant et al., 1994), and form pairs of venter to venter feeding ticks. Under the conditions described by these authors, when a steer is infested once with a large quantity of larvae, the males emerge 12 days later whereas females appear on day 13. By day 14 most ticks are in pairs but fertilisation starts only on day 15. As in other tick species,, fertilisation.is.followed by rapid feeding and weight increase of the females, resulting in thè first engorged ticks dropping off the host on day 18. These results are briefly summarised in Fig. 1. 100 C O CO '•tE a> co S (D 50 I I First drop I/ 3 Q. O U Pairing 12 13 / 14 15 16 Days after infestation / 200 150 Ol 100 \ 50 17 FIGURE 1. Development of B. microplus females on a young steer and the incidence of important events in their life history (modified after Falk-Vairant et al.). Female engorgement is indicated by weight (*•—•) and bars represent the percentage of sampled females found to contain a spermatophore. Black marks on the x-axis indicate scotophase. It has been shown in choice experiments on the host that B. microplus males mate indiscriminately with B. microplus and B. decoloratus females even though the two species do not produce offspring after interspecific mating (Spicket and Malan, 1978). Likewise, under on-host experimental conditions, B. microplus males copulate with B. ammlaius females (Graham et al., 1972). This lack of specificity in mating preference has also been observed in the two Dermacentor species and even between D. variabilis and Rhipicephalus sanguineus (Sonenshine et ai, 1974). It is questionable whether behavioural and/or pheromonal mechanisms of reproductive isolation are present in ticks. Allan et al. (1989) argue that in D. variabilis and D. andersoni a 21 RESULTS 1 chemical signal at the level of the gonopore can provide species recognition. In the three Australian reptile ticks the chemical signal causing males to detach in the presence of females is apparently species specific (Bull and Andrews, 1984). According to Bull (1986) ecological mechanisms can also explain the maintenance of parapatric boundaries that exist between species without wasteful interspecific mating. I have observed mating behaviour on rabbit ears to determine the ability of male ticks to locate females and to characterise the different behaviours involved in mating. I have also studied mating under various in vitro conditions and developed a bioassay for isolating chemical cues mediating this behaviour. In order to understand the development of sex-ratio and tick density on the bovine host I also report on some population sampling during the days before copulation occurs. MATERIALS AND METHODS Ticks and tick sampling from a steer Boophilus microplus (Canestrini) of the Biarra strain was reared on young Simmental steers at the Ciba-Gcigy Agricultural Research Centre, St Aubin, Switzerland, under conditions corresponding to those described by Falk-Vairant et al. (1994). For studying tht development of the tick population on the bovine host, all attached ticks in square areas (55 cm2) located on the shoulder of a steer roughly 20 cm down from the centre or on the back were sampled with forceps. Male mobility was estimated by brushing the back, flank and neck area on one side of a steer with a paintbrush and collecting ticks in a plastic tray working on alternate sides of the steer between days. It was assumed that in this way only unattached ticks were collected: Some fully engorged females (<20) were also collected with this method on day 19 but not counted. In order to obtain newly moulted unfed ticks, engorged nymphs were collected by carefully removing them from the host with forceps and transported to the laboratory in a humidified container. They were kept in glass vials in an incubator at 28°C and 90% r.h. and, after moulting, adult males and females were put separately on the ears of New Zealand White rabbits enclosed in cotton bags where they readily attached. In addition, males were collected on different days from the bovine host and transferred to rabbit ears in the laboratory. Behavioural observations on rabbit ears and in an artificial feeding system Observations of male and female behaviour was filmed at magnifications of 5x or 2 Ij? with a Canon CI-20P colour CCD video camera attached to a Zeiss operational microscope (working distance: 25 cm) equipped with a coaxial cold-light source. Light intensity in these and other observations was between 2000 and 7000 lux. Recordings were made on a Panasonic super VHS video recorder (AG- 7350) and played back for analysis on a Sony Trinitron colour monitor. A rabbit was held (90 min max.) in a closed wooden restraincr leaving the head and ears exposed. The ears were flattened out and lightly restrained. A male B. microplus was released on the car between 3 and 12 mm away from an attached male or female tick and allowed 3 min to commence walking (i.e., displace itself >2 mm). Attached female ticks were classified according to sire: I) undistcnded (3 mm), 2) semi-engorged (5 mm) and 3) gorging (8 mm) The following behaviours were scored: male contact with attached tick, first tip-over of male, duration of dorsal exploration until first tip-over (latency), location of first tip- over, and incidence of female body lift (see results for definitions) Using the same observation method, male behaviour was observed vis-a-vis females feeding in an artificial feeding chamber. Female B. microplus had been allowed to moult in the laboratory and were placed on a silicon membrane treated with an extract of steer pelage according to the method described by Kuhnert et al. (1995). The ticks were allowed to attach and fed for 2 days on dcfibrillated bovine blood enriched with ATP and glutathione, while non attached females were removed. Individual males were released and their behaviour was scored as above. 22 RESULTS 1 12 13 14 15 16 17 18 19 days after infestation 100 ta -y Ü 80 - S. 60 - a, vi O O O &0 C O O kl a. 40 - 20 - B shoulder 1 140 159 _n T 1 I 12 13 14 C shoulder 121 * i r ^r 12 13 14 days after infestation FIGURE 2. Density of B. microplus males and females and their mobility on the host. Samples were taken from the poulation of ticks that developed from single infestations of two young steers with ca. 10,000 larvae. Larvae were deposited in a 30 cm line from between the shoulders on day 0. A: Unattached (mobile) ticks collected from infestation 1 by brushing neck, shoulder, flank and back on one side of the host, alternating between sides from day to day. B, C and D: AU ticks from an area of 55 cm2 on the shoulder or back were removed with forceps (A: infestation 1, B and C: infestation 2). The location of the areas sampled is indicated in the drawing top right. Absolute number of females are indicated and asterixes indicate days without sampling. Observations in a host-simulating arena and destructive treatment of females To further reduce the presence of possible naturally occurring chemical and mechanical stimuli, female ticks were presented to males in a circular arena (40 mm dia.) consisting of a Baudruche membrane (Joseph Long Inc., USA) stretched over a 0.9% NaCl solution at 35±1°C on a warm plate. A 40 ram high plastic tube placed around this arena and the permeability to water of the membrane assured a constant r.h. (> 80%). This set-up is here referred to as "host-simulating" arena and is being used in this laboratory for studying the responses of B. microplus larvae to host derived chemicals (Kröber and Guerin personal communication, Guerin et ai, 1992). In three separate experiments, one semi-engorged female B. microplus collected from the steer on day 19 was placed in the centre of the arena either 1) attached to the membrane ventrally with 23 RESULTS 1 double-sided adhesive tape, 2) lying loose or 3) the same female as in 2 but positioned ventral side up. In order to inhibit movement of the female the legs were cut off at the level of the genu two hrs before the experiment. A single male tick was released from a fine paintbrush directly on top of the female. In addition to recording the same events as above, the time the male spent in contact with the female was also recorded, i.e., from the first moment all legs were in contact with her till the last leg lost contact. Males were allowed to contact the female during a maximum observation time of 180s. In order to characterise the movements of males over the body of the females, palpal tracks were recorded and related to defined areas of the female cuticle. In frame-by-frame video observations the position of the male's capitulum was traced on transparent plastic sheets from the moment of first contact to tip-over, or loss of contact with the female. Such tracks were also made for the observations in the feeding chamber mentioned above. In an attempt to remove or destroy chemical information on the cuticle, individual semi-engorged female B. microplus collected from the steer on day 19 were immediately frozen at -200C, kept there for 2 days and subsequently treated in one of the following ways after which they were stored again at -200C: I) control, 2) rinsed in 2ml hexane for 30 min, vortexed and dried under He, 3) extracted in chloroform !methanol 1:1 for 24 hrs, vortexed and dried under He, 4) as 3 and then rinsedlO times for 2 min in 1ml chloroform and heated to UO0C for 12hrs. These females were presented to males as above, lying loose on the membrane and after being kept at 300C for 4hrs. Behavioural observations using glass beads as dummy females A glass bead (ca. 5 mm dia., 3 mm high, 0.1-O.2 g), roughened with a wet-stone and flattened on one side to inhibit rolling, was placed in the centre of the host-simulating arena. Two such arenas were used simultaneously on the same warm plate, one bearing a bead treated with an extract of female ticks in solvent applied with a micro-pipette, the second treated with solvent alone as control. The extract was prepared by submerging freshly collected females (<15 min after removal from the host, 50-500 at a time) for 5 - 15 days at -2O0C in small volumes (0.5-5 ml) of chloroform or chloroform: methanol (1:1). The extract was collected in a syringe and evaporated to dryness under a gentle stream of nitrogen, immediately redissolved in chloroform at 0.5 or 1 tick equivalent/ ul and stored at -2O0C. A single male tick, 2-14 days after moult, was released from a fine paintbrush onto the top of the bead. All males in a given experiment were tested on both the control and treated bead, half first on the control the other half first on the test. Different behaviours were quantified using The Observer 2.0 event recorder (Noldus Inf. Tech., Netherlands). The tick was recorded as either being on the bead, i.e., from the first moment all legs were in contact with it till the last leg lost contact, or on the membrane. Ticks were allowed to descend and remount the bead but a maximum of 180 s was allotted to each tick or observations ended when the male crossed the edge of the arena. The total time spent on the bead (contact time) was dien taken as a parameter for statistical analysis with the Wilcoxon signed ranks test on paired replicates (test versus control). In addition, note-was made of typical tip-over behaviour while on the bead (see results for definition). RESULTS Tick population development and mobility on the host Under the rearing conditions mentioned above, ticks hardly migrate across the host from the area of infestation and most adults probably attach immediately where they moult or do not detach at all. Some males and females were found unattached on the host on day 12, 13 and 14 (Fig. 2A). However on day 15, males seem to be much more mobile than females and by day 16 all females are attached whereas an increased proportion of the male population is mobile. A large number of males is found actively walking on the host on day 19 when female drop-off is at its optimum. Results from several collections from the bovine host are shown in Fig. 2B,C and D. The number of ticks is less on the back of the steer further away from the area of infestation where development of nymphs to adults is also slightly delayed. On day 12 only a few females are present but by day 13 most females have moulted. The average sex ratio for days 13 and 14 is close to 1:1, though slightly in favour of males, and density of females for day 14 ranges from 1 to 3 per cm . 24 RESULTS 1 Observations on mate finding and mating behaviour in vivo and in vitro The sequence of behavioural events in mating of B. microplus as observed in vivo and in vitro is summarised (Fig. 3A). The diagram includes detachment of the male as an event that precedes the searching phase. This is not what was actually observed as forcibly detached males were used in all our experiments. When male B. microplus were released on the ear of a rabbit, in the immediate vicinity of an attached conspecific tick, about half of the released males showed activity and started to walk, irrespective of the sex of the attached tick (Table 1). The other half did not move at all within the set period of 3 min or made movements resulting in males descending in the rabbit's fur. There is no correlation between activation of males and distance from the female within the range I tested. Most of the males that did walk, quickly located the female (70-90%). TABLE 1. Behavioural:responses oi.B. m;'cro/»/«5jnales-placed 3-12 mm from a male or female tick attached on rabbit '-tarsr-fin vivo) or in aifeedingxhamberfîn vitro). Results are the number of males (n) observed walking, contacting the attached tick and tipping-over out of a total of N males. In addition, the part of the female body (as indicated in Fig. 4) where the first tip-over was attempted and the female's body lift response were scored. attached tick males tip-over location female total walking contact tip-over back front/side body lift N n n n n n n female undistended female semi-eng. female gorging male 14 22 18 10 on rabbit ears (tn vivo) 6 4 4 10 7 6 8 7 7 5 5 3 4 3 2 1 0 3 5 2 3 4 0 female undistended 9 in feeding chamber (in vitro) 9 9 9 4 5 6 First contact was generally made with one of the front legs and immediately followed by a short exploration of the dorsal cuticle of the female. Depending on the size of the female and the thickness of the rabbit pelage, a male had to descend or mount to reach^e^female;'doKum:-.andididiso.with^roping, scraping movements of the legs. Once fully on the female, he continued these movements while keeping his body in close contact with her. Since males {ca. 2 mm) are always smaller than females (ca. 3-10 mm) most legs were in contact with the female cuticle at this stage. The mouthparts engaged in movements up-down and backward-forward with respect to his body (flexing) while the legs at times made rapid vertical taps. Upon reaching an edge of the dorsum, males continued to slide along it, actively pushing forward. At some point during this process the female tick lifts her body at an angle to the host's skin facilitating male access to the ventral side of her body via the posterior end. The male, following the curvature of the body, would then tip-over and rapidly disappear under the female. This event in male behaviour is therefore termed 'tip-over'. Almost all males which had explored a female dorsum attempted this, and the first tip-over followed fairly rapidly after the male had mounted the female; only one male out of 17 observed took longer than 20s. However, the location of tip-over was not always chosen correctly for many males were engaged for from several seconds up to 1 min in efforts to pass via the side or the front of the female. This was physically impossible since at the front her capitulum was embedded in the host's skin and on the side her legs practically blocked the passage. Only on undistended females did all four males observed immediately find their way to the passage at the back of the idiosoma. Furthermore, the female does not always show the body lift response, in fact in none of the observations made on gorging females was she able or willing to lift her body. 25 RESULTS 1 A detachment searching unoritntcd? directed? contact female dorsal exploring close body contact tarsal tapping and scraping rostrum probing up-over ventral positioning thigmotaxis? probe gonopore gonopore exploring spermatophore transfer descent on control dorsal exploring tip-over membrane probing FIGURE 3. Schematic representation of male behaviour leading to copulation. A: Sequence of behavioural elements observed during in vivo and in vitro mating. Behavioural phases arc characterised in squares whereas the events separating the phases are encircled. B: Typical male poses characterising behavioural elements observed on a glass bead Part of the behaviour described here is not a specific response to females. In the observations of male responses to attached males, all males that walked located the attached male (table 1), all five spent more then 20 seconds in contact with him and three of them tried to tip-over. However, it is hard to compare male behaviour vis-à- vis males with that on females because the male body is so small that most of the legs of the mounting male are either in contact with the attached male's legs or the rabbit's pelage and the behaviour tends to be confused by entangled legs. Interestingly, at some point after testing several males, the two attached males that were used in these observations detached and walked off. Males that were released in the feeding chamber, where females had been feeding //; vitro for 2 days, all immediately walked and located one of the females (table 1). The 26 RESULTS 1 density of 17 females on the 12 cm membrane.was such that this high encounter rate was not a surprise. Mating proceeded as described above and could be observed easier than on the rabbit. It was confirmed in this in vitro situation that most males first contacted the female's body with the tarsus I (only one male made first contact with tarsus II). Which cuticle zone was first contacted did not seem to matter: 3 males first came into contact with the female scutum, 4 with the lateral zone and 2 with the festoon area (see Fig 4B). All males mounted, tipping-over within 20s with ca. 50% immediately reaching the venter via the back. Males that first tried tip-over on the side or front all eventually reached the venter by moving sideways while continuing to probe and push forward until a free passage to the venter was possible. Most males then continued to slide forward positioning themselves venter to venter with the female and became immobile, only making minor adjustments in the position of the legs which were clasped around the female body in between her coxa. The actual insertion of the chelicera into the gonopore could not .be observed from above but, after folding the female forward, the mouthparts of the male could be seen, flexed at 90° to the body axis, and positioned on the gonopore. Mating behaviour and orientation on detached females in a host-simulating arena Female ticks that were removed from the host and placed in a host-simulating arena still induced males to investigate the dorsum and tip-over (Table 2). Moreover, placing males directly on the females did not seem to disturb normal courtship behaviour. Tip-over latencies on artificially attached, loose normal and loose inverted females were of the same order of magnitude as mentioned above and exploration of the cuticle seemed to involve the same scraping and probing movements of legs and mouthparts. In all three experiments some males left the female without tipping-over but on the artificially attached female more males had relatively short contact times, sometimes after fruitless attempts to tip-over. Tip-over was possible on females lying loose on the membrane, dorsal side up, but the female was moved around by the male under her. None of these males was able to complete ventral positioning and locate the gonopore and many males.crawled back up on.top of the female only to tip-over again, leading to long contact times: Jnterestingly^allrmales also-readily tipped-over on inverted females. Some males actually contacted the area of the gonopore before tip-over but did not seem to be arrested by it at all. Movements of males over the female cuticle could be observed more precisely in these in vitro experiments. I did not observe any consistent pattern in the palpal tracks on any of the live females (Fig 4), i.e., there was no obvious order in male orientation on artificially attached, loose normal or loose inverted females. As far as the palps are concerned, there was no evidence for contact with specific cuticular zones before tip-over nor was there any particular zone which was never contacted before tip-over. The tracks also give no evidence for any single cuticular zone guiding males to the correct location for tip-over (Fig. 4A1B). The first tip-over attempts of males on semi-engorged females lying loose on the membrane are located more or less randomly around the edge of the body (Fig. 4C). Male behaviour on a dead female in the host-simulating arena was not considerably different (table 2) except for the fact that in this case 5 out of 10 males managed to locate the gonopore and became immobile under the female. Our attempts to remove the factor(s) inducing male arrestment by females and tip-over by various destructive treatments, were largely unsuccessful. After a 30 min hexane wash there was no apparent change in the colour or texture of the female tick. The chloroform:methanol extraction changed her colour to a deep red/orange which faded in subsequent chloroform rinses but all aspects of the cuticle appeared unchanged. Even after heating 27 RESULTS 1 at 1100C for 12 hrs the structure of the cuticle was completely preserved, including the numerous folds characteristic for female ticks, though it had collapsed due to evaporation of the contents and turned to a dark brown colour. None of these treatments completely removed arrestment but contact time was considerably reduced after heating the female to 11O0C. Tip-over behaviour was not affected even after this harsh treatment and latencies remained short. TABLE 2. Behavioural responses of B. microplus males when placed on females in a host-simulating arena. Females were semi-engorged and placed either dorsal side up, attached with double-sided adhesive tape (attached), or lying loose with dorsal side up (normal) or ventral side up (inverted) in the centre of a host-simulating arena. In an attempt to remove or destroy chemical information on the cuticle, such females were killed at -2O0C and treated as follows: control = untreated, HEX. washed - rinsed in 2ml hexane for 30 min, vortexed and dried under He; CH:M extracted = extracted in chloroform rmethanol 1:1 for 24 hrs, vortexed and dried under He; CH:M & heated = as previous, then rinsed 1Ox for 2 min in ImI chloroform and heated to 1100C for 12hrs. Results are.the number of males (n) observed tipping-over out of a total of N males and the median latency of this response. In addition, the part of the female body (as indicated in Fig. 4) where the first tip-over was attempted and the contact time with the female were scored. males total tip-over latency tip-over location back front/side contact time <20s 20-180s >I80s females N n median (i 0 n n n n n live females attached normal 13 10 9 5 5 3 6 4 loose normal 17 14 7 9 5 0 8 9 loose inverted 18 17 4 9 dead females 8 0 6 12 control 10 9 5 - - 1 1 8 HEX washed 10 10 5 5 5 0 1 9 CH:M extracted 10 10 7 2 8 0 2 8 CH:M & heated 10 9 6 3 6 1 6 2 Arrestment and tip-over on glass dummies Males were also arrested on glass beads treated with 3 female equivalents of a chlorofornvmethanol (1:1) extract of female B. microplus as is showm|^Lthe ratio between their contact times on extract-treated beads and solvent-treated controls (Fig. 5A). On test beads they engage in exploratory behaviour similar to that seen on live and dead females (see Fig 3B). Conversely, when placed on control beads, males generally leave within 20 seconds and very often raise their body and wave the first pair of legs in the air just before loosing contact with the bead. In addition to being arrested on test beads, some males descend toward the membrane while keeping their body in close contact with the surface of the bead. Such males then try to push themselves in between the bead and the membrane. Characteristically, as viewed from above, the tick's venter becomes visible as the mouthparts and first pair of legs disappear under the bead, and the bead is sometimes moved. This behaviour was observed only on treated beads and is comparable to the tip-over observed on females. Other males stop all locomotion and pierce the membrane with their mouthparts often attaching perpendicular to the membrane, with six legs on the bead and the first pair resting on the membrane. This behaviour was termed 'membrane probing1 and was occasionally observed on the control beads. Normally, on control beads, males simply continue walking, reaching away from the bead for the membrane as they descend, hence the venter is never seen. 28 RESULTS 1 FIGURE 4. Orientation of male B. microplus movements relative to locations on the female body during dorsal exploration and tip-over in a host-simulating arena or artificial feeding chamber. A: Some examples of tracks made by the palps of males across the body surface of semi-engorged female ticks prior to male tip-over,-Open .circle .marksürst point of palpal contact after the male was placed on the female. An arrow marks the direction in which-one male left the female without tipping-over. Cuticular zones referred to in the text are separated by dotted lines; Sc, scutum & capitulum; La., lateral zones; Fo., foveae dorsales zone; Me., medial zone; Fe., festoon zone B: One such track on a female in the artificial feeding chamber where the male contacted the female by himself. C: Location (arrow heads) of first tip-over of males on a semi-engorged female attached with double sided sticky tape (left), a loose female (middle) and this same female placed ventral side up (right). The locations used in table 1 and 2 are indicated by lines. When both test and control bead were treated with solvent, males descended generally within 20 s and the logarithm of the arrestment ratio was normally distributed around the mean 0 (Fig. 5C). The influence of a certain variability in bead size was negligible; males took only slightly longer descending a bigger bead (Fig. IB), but this was far from significant. Their seemed to be no influence of features around the arena on the orientation of males, i.e., under these circumstances they were not attracted by stimuli associated with the experimenter (Fig. 5D). Temperature is apparently an important factor in male behaviour (Fig. 5A). Males were significantly arrested when temperatures are around those found on the host (35°C) as opposed to ambient temperature (220C). Humidity, however, had no effect on arrestment but high relative humidity seemed to reduce the number of males showing tip-over behaviour. This is especially apparent at ambient temperature and 90% r.h. (cold & humid) when no tip- over was observed. 29 RESULTS 1 CQ O) E *-» 80%) and a membranous surface. Various odours and contact chemostimuli normally present on the host were absent as were mechanical stimuli such as hairs. Under all these circumstances, in vivo and in vitro, both live and dead females induced dorsal exploring, tip-over and ventral positioning. Attempts at removing activity from dead females with solvent extraction were largely unsuccessful and even heating such an extracted female to 1100C did not completely remove activity though arrestment was reduced. 35 RESULTS 1 In spite of the fact that I did not succeed in removing activity from females by solvent extraction some of the chemical stimuli responsible must have been extracted since males spend more time investigating glass beads treated with such an extract as compared to solvent treated control beads. In addition, the typical tip-over behaviour can be reproduced only on extract treated beads and not on controls. The fact that mating responses can be induced on glass beads with such an extract which is comprised of a mixture of chemical constituents from different zones of the female cuticle, supports the fact that male tip-over need not be guided by chemicals though it is clearly induced by them. In view of the difficulty of removing them, these chemical stimuli must be intimately associated with the cuticle of females and not too volatile or thermolabile. Males of different ages, fed and unfed, are arrested and tip-over on extract-treated glass beads, but newly moulted unfed females do not respond this way. Since this is normally the only mobile stage for females they can be assumed not to show these male specific mating responses. Moreover; the fact that unfed males respond in this way to female extracts would explain the pairing before copulation as mentioned above. The results also demonstrate that the temperature gradient in the host-simulating arena is essential for normal mating responses to occur. Chilton and Andrews ( 1991 )aJso found that the body temperature of their reptile host is an important factor in mating of A. hydrosauri and A. limbatvm. The mating behaviour of B. microplus, described here as a process that can be devided in four phases, is clearly mediated by chemical as well as mechanical factors. Males are capable of locating females on the host, pair with them and remain attached under them before and after copulation. Chemical stimuli play a crucial role during male exploration of the dorsal surface of female ticks and initiate tip-over behaviour that brings the male into a position to probe the female gonopore. Extracts of female ticks, when applied to glass dummies, induce arrestment and tip-over in male ticks. 36 RESULTS 2 /55¾^ J !wo Physio! Vol. 40. No. 2, pp. MJ-IM. 1994 SSf ) Pcrgamon „ . 0^Tf1 °'?" B^^cnee Ud KoC/ / Pnnlcd in Grcai Bniain, All nghis reserved ^QU/ O02M91O/94 S6.00 + 0.00 Isolation of 2,6-Dichlorophenol from the Cattle Tick Boophilus microplus: Receptor Cell Responses but No Evidence for a Behavioural Response MARIEN DE BRUYNE,* PATRICK M. GUERIN* Received 13 May 1993: revised 19 July 1993 2,6-Dichlorophenol, a compound known as a sex pheromone for several met astria te tick species, was isolated from different life-stages of the cattle tick Boophilus microplus. Receptor cells in two wall-pore single-walled scnsilla on the tarsus I of male ticks responded to this compound in a dose-dependant manner. Using these receptors as specific detectors for compounds in the effluent of a gas Chromatograph, we detected 2,6-dichlorophenol in extracts of females, males, engorged nymphs and larvae of this one-host tick, but not in an extract of eggs. No other components of the extracts elicited responses from these olfactory sensi! I a. However, male B. microplus were not arrested on a glass bead treated with 2,6-dichlorophcno! and placed on a membrane in a host-simulating arena, whereas a bead treated with a female extract did evoke a strong arrestment response. In addition, no odour-conditioned anemotaxis, change in angular velocity or speed of males walking on a locomotion compensator was observed in response to this compound in a conditioned air-stream. We could therefore not establish a role for 2,6-dichlorophenol on its own as a semiochemical in males of this species. Boophilus microplus Tick Pheromone 2,6-Dichlorophenol Walking-behaviour Arresimcni INTRODUCTION Berger et al. (1971) found that one fraction of a dichloromethane extract of female ticks excited males of three species, resulting in responses typical of mating behaviour. The compound responsible for this behaviour was subsequently identified as 2,6- dichlorophenol (referred to hereunder as 2,6-DCP) from Amblyomma americanum (L.) (Berger, 1972), and has since been isolated from at least 14 species of metastriate ticks (Sonenshine, 1985). It has remained the only positively identified volatile sex pheromone common lo the Ixodidae, though other phenols have also been suggested (Wood et al., 1975). However, the precise behavioural role of 2.6-DCP has not been fully investi- gated in any species. The fovcal glands, with terminal ducts ending in the fovea dorsalis on the lick's dorsal cuticle, are thought to be the source of 2,6-DCP pro- duction (Sonenshine et al., 1981). Foveae dorsales are apparently present in all life-stages of metastriate ticks but not in proslriates such as Ixodes ricinus L (Schulze. 1942; Dinnik and Zumpt, 1949). "Insultile of Zoology. University of Ncuchàicl. Charnemerlc 22. CH-2007 Neuchâtel. Switzerland. 2,6-DCP causes detachment of males of different species of ticks and induces displacement towards fe- males in experiments on the host (Sonenshine, 1985). However, in Hvalomma dromedarii Koch it acts only as a male attractant at relatively short distances on the host and appears not to be attractive when offered m an air-stream off the host (Khalil et ai. 1981). Attraction was observed in both male Dermacentor andersoni Stiles and Dermacentor variabilis (Say) to a wide range of concentrations of this product (Sonenshine et ai, 1976). thus providing no basis for species specific concentration dependent responses. Sex pheromones acting over a distance, till now unidentified, have also been described in Hvalomma asiaticum Schulze and Schlottke (Leonovich, I981) and three Australian reptile ticks (Bull and Andrews, 1984). In ihe latter three species the excitant volatile appears to be species specific. A different class of pheromones. emitted by male licks and mediating aggregation and attachment on the host, has been described for several Amblyomma species (Gladney et ai, 1974). These phero- mones generally consist of a mixture of phenols and short chain fatty acids (Schoni et ai. 1984; Apps et ai, 1988). 37 RESULTS 2 Receptor ceils responding (o 2,6-DCP were investi- gated with tungsten electrodes by Haggart-and Davis (1981) in A. americanum (L.) and responses were thought to originate from a wall-pore single-walled (wp-sw) sensillum in the anterior pit of Haller's organ on the tarsus of the first leg pair: thed II I of Hess and Vlimant (1986). A study in Rhipicephalus oppendiculatus Neu- mann and A. variegatum (Fabricius), using the tip recording technique, confirmed the presence of a similar receptor in the corresponding sensillum (Waladde, 1982). The latter also showed that responses to 2,6-DCP can be obtained from another wp-sw sensillum posi- tioned more distally on the dorsal surface of (he tarsus: the d I I of Hess and Vlimant. B. microplus (Canestrini) (Acari: Ixodidae) is a one- host ixodid tick. Contrary to other tick species pre- viously investigated for sex pheromoncs, this tick passes through all its life-stages on the same bovine host. Widely distributed in the tropical- and sub-tropical regions, it is a vector for anaplasmosis and babesiosis and causes severe economic losses to cattle ranching. Very little is known about the mating behaviour and chemical ecology of this species. Chow et al. (1972) isolated a fraction from extracts of female B. microptus with similar chromatographic properties as that found active by Berger et al, (1971), bui did not go so far as to identify what they called "a phenolic compound". The olfactory wall-pore sensilla of B. microplus, hom- ologous to those bearing 2,6-DCP receptors in other Ixodidae, arc highly innervated: (he more distal d I I sensillum by five neurones and the d IM by 15 neurones. organized in three separate bundles of 5 (Hess and Vlimant, 1986; Waladde, unpublished). Our results demonstrate responses to 2,6-DCP by sensory cells in both of these sensilla and we use these receptors as specific detectors to demonstrate the presence of 2,6- DCP in different life-stages of this species. Behavioural bioassays were then used to detect possible responses of adult males to this compound. MATERIALS AND METHODS Animals Ticks were obtained from a laboratory colony at the Ciba-Geigy Agricultural Research Station, St Aubin. Switzerland and belong to the organophosphorus-resist- ant strain Biarra from southern Queensland, Australia. This strain has been reared on the backs of young Simmeniaf steers for 31 generations in closed stables at 23'C and 60-70% r.h. Under these circumstances males appear on the 12th day after infestation and copulation peaks at the end of the 15th day (Falk-Vairant et al., 1994). Engorged nymphs or adult males were collected by carefully removing individuals from the host with for- ceps, and were transported to the laboratory in a humidified container. Engorged nymphs were kept in an incubator at 32*C and about 100% r.h. until they moulted, and adults were put on the ears of New Zealand White rabbits enclosed in cotton bags where they readily attached. Electrophysiological experiments were made with males that had moulted in the incubator, whereas behavioural experiments were done with males collected from the host before mating. When not on the rabbits, ticks were kept at room temperature (22-28°C) over water in closed containers to assure high relative humidity. Chemicals and extracts Phenol, 2-nitrophenol. 4-meihylphcnoI, 2,6-DCP and other halogenated phenols (all >98%, GC), were ob- tained from Supelco, USA; methyl salicylate (>99%) and benzaldehyde (>98%, GC) from Fluka, Switzer- land; all solvents (analytical, grade) were from Merck. U.S.A. Ticks were extracted by submerging them for 2-6 h in small volumes (0.5-5 ml) of either dichloromethane or hexane/dichloro-methane (1:1) and sonicating for 15 min (see Table 1 for details). The extract was removed with a syringe and stored at -200C. Air surrounding semi-engorged females on the host was collected by holding a glass cap (4.5 cm dia. I cm high) lightly against a steer's skin over a group of ticks. Air was sucked in over a charcoal filter via a 3 mm i.d. inlet with a portable air sampling pump (model 222-5, SK.S Inc., U.S.A..) at 200 ml/min and volatiles were collected on Porapak-Q (Waters Inc., Framingham, U.S.A.) conditioned accord- ing to Byrne et o/.(l975) and packed into a 2 ml glass cartridge. A control air collection was made, by repeal- ing the same procedure, from the steer's skin alone after removing the females. Electrophysiology #^ A male tick (2-14 days olSTwas fixed with adhesive tape on a glass plate with an anterior tarsus extended in such a way that its sensilla were visible in the transmitted beam of light on the stage of an inverse microscope (Nikon. Diaphoi-TMD at 60Ox. working distance: 15 mm). Two olfactory wall-pore single-walled sensilla on the tarsus of the forelegs were studied (Fig. I]. the d II I in the anterior pit of Haller's organ (20/^m long. 5pvn dia at base) and the d I I on the knoll dista! from the Haller*s organ (36 ft m long. 5 \i m dia at base) (Hess and Vlimant, 1986). To facilitate electrical contaci, the tip of the sensillum was cut using the lip of an oscillating glass stylet (Gödde, 1989). and a glass electrode filled with 0.05% polyvinylpyrroüdon K90 (Fluka) in 0.15 M KCl was immediately placed over it with the aid of a micro-manipulator. The reference electrode filled with 0.15 M NaCl was inserted into the coxa of the same leg and put to earth. The recording electrode was connected via a chlorinated silver electrode to a high impedance preamplifier, mounted on the micro-manipulator, and io a universal a.c./d.c. amplifier (UN-03, Syntech. The Netherlands) and signals amplified 1000 x . a.c. and d.c. Signals were recorded separately on two channels of a DAT recorder (DTR-1200. Biologic. France} and the 38 RESULTS 2 a.c. channel was played back via a DAS 16 analogue- digiial card (Melrabyle Corp., U.S.A.) into an IBM compatible PC equipped with the spike analysis pro- gramme sapid (Smith et at., 1990). Known amounts of chemicals dissolved in CH2CI2 were pipetted onto filter paper strips (45 mm') and, after evaporation of solvent, inserted into 5 ml plastic syringes serving as stimulus cartridges. Charcoal -filtered humidified air at 26-280C and 85-95% r.h. was blown over the tick preparation al 60 cm/s from a 6 mm i.d. glass lube whose orifice was at 2 mm from the preparation. A second air-stream (1 ml/s) from a blank cartridge was added to the main air-stream al 45 mm from the preparation and solenoid valves were used to switch for I s to the cartridge containing the stimulus. Since it was not possible to consistently associate responses to stimuli with distinct olfactory units in the sensilla studied here, increase in action potential frequency was calculated from the total number of spikes counted in the second after stimulus arrival minus ihe number in lhe second before stimulus delivery. Mainly due to travel time in the glass air delivery tube, a delay of 100 ms existed between solenoid valve activation and arrival of lhe stimulus at the sensillum as determined from the response to I /Jg of synthetic 2.6-DCP on filter paper; the relatively high dose was used to produce a sharp rise in spike activity. Only recordings where signal lo noise raiio was at least 2:1 were analysed. GC, GC coupled eleclrophystology and GC-MS Separation of extracts and comparison of peaks with known amounts of synthetic standards was done with cold on-column injection on a 30 m high resolution fused silica capillary column (DB-wax, J&W Scientific, U.S.A., 0.25//m film thickness, 0.25 mm i.d.) in a Carlo Erba HRGC 5160 gas Chromatograph (GC) with H2 as carrier gas at 1.5mf/min (0.5 m/s) temperature pro- grammed from 6O0C aficr I min to 2000C at 25°C/min, 200 to 23O0C at 5°C/min and held at 230°C for at leasl 5 min. ECD (Ni") and FID detectors were installed in series. Quantification was by peak area integration using a Spectra-physics SP-4270 integrator and by comparing with known amounts of standards injected in the same session; A splitter was installed allowing 60% of the capillary column effiueni to pass io the detectors and the remain- der was led through a heated transfer-line in the oven wall (at 250°C) into the conditioned airflow mentioned above al 30 cm from the preparation. A level discrimina- tor incorporated in the amplifier allowed us to sort impulses from noise in the a.c. signal recorded from the sensillum and impulse frequency was convened into a d.c. voltage with a frequency to voltage converter (lime constant 1 s). This voltage and the d.c. potential drop recorded from the sensillum upon stimulation were used as indicators of biological achvity of eluting products and printed simultaneously with ECD and FlD re- sponses on a chart recorder. A water-jacketed glass lube TABLE I. Gas chromatography linked cleciron capture and olfaciory sensillum detection, and gas chromatography linked mass selective detection of 2.6-dichlorophenol in different life stages of B. .microplus (amounts of 2.6-DCP,detected are-'rounded off to one significant digit) Also detected by- Sample 2.6-DCP (number extracted in Extraction pg/tick (ECD Olfactory parentheses) method* peak area) sensillum MS Immature Stages Eggs I? days old (10.000) CH .a, 6 h 0 n.i n I Larvae 16 weeks old (1500) CH .Cl. 6 h 2 d I I n 1 Engorged nymphs (50) CH,Cl, oh Adult Females 30 d I 1 n 1 Pharate (45) CH,ClyTiex 5 h 20 d Il I n.t Newly moulted in vitro (80) CH,CU 6 h 300 d I I n i I day old (100) CH3CVhCJt 3 h 600 d Il 1 + d 1 I n i 2 days old (200) CH;CI,/hex 5 h 500 d I 1 — j- 3 days old (20) CH3CI2,hex 5 h 300 n.i. Ii I Fertilized 5 days old (67) CH,Cli/hex 2 h 100 n.i. ~ Unfertilized 5 days old (50) CH,Cl,,'hex 2 h Adult Males 400 n.i + +¦ Pharate (215) CH,Clj/hcx 5 h 10 n.t. n.t. Newly moulted m vitro(76) CH1C], 6 h 200 d Il I +d I I n I I day old (200) CH.Cyhex 3 h 300 d M I n I 2 days old (247) CH,a,/hex 5 h Air Sample 200 d I I n i 31 air o«cr 25 host-atlached CH,CI,/hex to n.i n I Unfertilized females in 20 min On Porapak* *AII extractions terminated with 15 mm somcalion. n.t.. Not tested; hex., hexane: ECD. electron capture detector; MS. mass selective deieciot 39 RESULTS 2 distal W 1 mV W 1 ng 2,6-Dichlorophcnol IO ng 2,6-Dichlorophcnol 100 ng 2.6-Dichlorophcnol 100 ng 2-Nitrophcnol FIGURE 1. Characterization of oiractory receptors in two wall-pore single-walled sensilla on the dorsal side of tarsus I of male B microplus ticks. (A) Dorsolateral abaxial view of Haller"s organ and surrounding sensilla. showing the d I 1(1) and d Il I (2) sensilla. (8) and (C) Electrical signals obtained by lip recordings of these two sensilla, the d 1 I (B) and the d II 1 (C) upon stimulation with different volatiles Stimulus dose is in ng of substance on filter paper tn an odour cartridge from which air was displaced at I ml/s into a humidified air stream of 60cm/s flowing over the preparation. Horizontal bar represents I s stimulus period. In (C) the frequency of the largest amplitude spite is irregular but was not modified by any of the volatiles tested 40 RESULTS 2 (8 mm i.d.), circulating water from a bath (28°C), served to ensure constant conditions of the airflow (26^-28°C, 80-90% r.h.) right up to the preparation. Any decline in (he activity of the preparation was monitored with a 100 ng dose of 2,6-DCP as stimulus which was added to the air-stream as described above, before and after each GC run and the responses calibrated. Gas chromatography-mass spectrometry (GC-MS) analyses were conducted with an HP-597IA mass selec- tive detector (ionization energy 7OcV, temperature 1800C) linked to a HP-5890 series Il GC equipped with the DB-wax column described above and programmed from 600C after 5 min to 23O0C at 8°C/min and held at 2300C for 15 min with helium as carrier gas at a flow rate of 1.2 ml/min. Mass spectra of unknowns were analysed and compared with standards held in a library using the . HP Chemstation program; on.an HP IBM compatible computer. Behavioural bioassay I: dummy female on a membranous subs i rale A 0.1-0.2 g glass bead (5 mm dia), roughened with a wet-stone and flattened on one side to inhibit rolling (3 mm high), was placed in the centre of a round arena (40 mm dia) consisting of a Baudruche® membrane (Joseph Long Inc., U.S.A.) stretched over a 0.9% NaCI solution held at 36 ± 2°C on a warm plate. A 40 mm high plastic tube placed around this arena and the permeability of the membrane assured a constant r.h. of >95%(Krobcr, unpublished). Two such arenas were used simultaneously on the same warm plate, one bear- ing a bead with a tick extract or 2,6-DCP applied with a micro-syringe, the second treated with just solvent alone as control. When 2,6-DCP was applied, both beads were treated afresh for each tick having been washed in solvent and heated, to .420C for 1 min.:. The treated bead was left on the membrane for 2-5 min before introducing the tick to allow bead temperature to rise to that of the membrane. A single male tick was released from a fine paintbrush onto the lop of the bead. Behaviour was viewed from above and filmed at magnifications of 5 x or 21 x with a Canon CI-20P colour CCD video camera attached to a Zeiss operational microscope (working distance: 25 cm). Recordings were made on a JVC super VHS video recorder (HR-S5500E) and played back for analy- sis on a Sony Trinitron colour monitor. All males in a given experiment were tested on both the control and treated bead, half of them first on the control the other half first on the test. Behaviour was quantified using The Observer event recorder (Noldus Information Technol- ogy, The Netherlands) (Noldus, 1991). A maximum time of 180 s was allotted to each tick on the bead and/or arena. The total time spent on the bead (contact time) and on the arena around it (searching time) before the tick's first crossing of the edge of the arena to leave were taken as parameters for statistical analysis with the Wilcoxon signed ranks test on paired replicates (test vs control). Behavioural .,bioassay 2; locomotion compensator and wind-borne odours To study the walking behaviour of male B. microplus and its responses to wind-borne 2,6-DCP we used a servosphere apparatus which serves to keep the animal in a fixed position while permitting free displacement in the horizontal plane (Kramer, 1976). A perspex sphere (50 cm dia) with a rough painted surface is mounted between two low-inertia servo motors capable of moving it along two orthogonal axes. A lick is supplied with a ca 1.5 mm2 piece of reflective foil (No. 7610, 3M, Switzerland) attached to its dorsum and placed on the sphere. A filtered incandescent light beam (40 mm dia, filter cut-off at 780 nm) is projected on the upper pole of the sphere. Light, reflected by the foil, hits a position sensor-which continuously generates information about the displacement of the tick and this is used to drive the servo-motors that compensate for the displacement, thus holding the animal on the apex of the sphere. Two incremental pulse generators supply information about all 0.1 mm displacements of the sphere in the A'and Y directions every 0.1 s and this is fed to a SAM II 68K computer (KWS Inc. Ettlingen, Germany) for track recording. Temperature- and humidity-conditioned air was con- tinuously blown from a water jacketed aluminium tube (35 mm i.d.) fitted with an aluminium foil tube at its mouth (70 mm long) supporting a honeycomb baffle to reduce turbulence and ending in a rectangular mouth (18 mm high, 35 mm wide) 3 cm from the sphere's apex. This air-stream (28°C, for humidity and velocity sec Table 2) arrived tangentially at the top of the sphere where the tick walked- Stimuli were introduced to the air-stream from a 25 ml gas-wash bottle via a silicone tube and syringe needle inserted through a rubber septum in the wall of the aluminium tube 23 cm from its mouth. 2,6-DCP in solution was pipetted onto a 20cm- piece of filter paper and soaked in ca 0.5 ml paraffin oil after evaporation of the solvent; a blank was made up in an identical way with solvent only. Voltage/pressure converters controlled the flow (240 ml/min) of the char- coal filtered air through the gas-wash bottle, and solenoid valves permitted air-stream switching from the blank to the bottle containing the stimulus. MaIe ticks were placed on the sphere and allowed to adapt to the conditions for 8 min before testing. The area around the sphere was kept dark with black curtains. TABLE 2. Treaiments delivered io male 8. mieroptus on the loco- motion compensator and percentage upwind displacement {mean + SD) in control and lest periods Air condii ions Upwind displacement (V0) 2,6-DCP Wind speed (V0) r.h. dose (ng) Control Test n 15 70 5000 14+14 19 + 25 12 15 90 5000 19 + 19 9+12 10 30 90 5000 7+9 II ± 12 9 15 90 500 9 ±12 15 ±27 9 30 90 50 9 ±8 4+3 7 41 RESULTS 2 Each test consisted of a 60 s blank nan followed by 60 s with 2,6-DCP. The tracks were analysed on an IBM compatible computer. Mean displacement of B. mi- croplus males (2.5 mm body length) in 0.1 s was relatively low compared to the sphere's base resolution (0.1 mm), leading frequently to inaccurate description of angles associated with displacement segments. Displacement, deviation angle from wind direction and turn angle (difference between deviation angles of successive seg- ments) were calculated instead for each 0.6 s segment (or 100 segments/min). Additionally, records of animals that Walked less than 0.5 mm/s for more than 50% of either the test or control period were discarded. The following statistics were calculated for control and test walk of each animal: mean speed (displacement/time), median angular velocity (absolute turn angle/time), circular mean of the deviation angles (Batschelet, 1981), and upwind displacement (sum of all segment lengths with a deviation angle between 60° and —60° upwind, as a percentage of the total displacement). Differences be- tween test and control responses were evaluated with a permutation test on paired replicates. RESULTS Elect rophysiolggy of 2,6-dichlorophenol receptive sensilla The spontaneous activity of cells in the d I I and d II 1 sensilla was highly variable and appears to be due to the absence of certain cells in some recordings. Whether this was due to cutting the tip of the sensillum is not clear. A consistent separation of action potentials into different cell classes was not possible. The overall spon- taneous activity of olfactory cells in sensillum d I 1 was generally lower and a clear response to stimulation was obtained, whereas responses from the d II 1 were more difficult to analyse (Fig. 1). Responses were obtained to a range of synthetic 2,6-DCP loads on filter paper from both the d I I and the d II 1 sensilla (Fig. 2). Higher doses tended to distort the signal and cause long-lasting excitation, indicating satu- ration. This effect occurred at lower doses in the d 1 1 than in the d II 1. Though increases in global activity of cells from the d I 1 in response to increasing doses of 2,6-DCP tended to be slightly higher, the regression lines do not differ except for the fact that variation was somewhat higher in responses from the d II I. Olfactory cells in both sensilla responded to five tick equivalents of a total extract of either females or males (Fig. I). Recordings from the d II I showed responses to 2-nitrophenol in the same dose range as 2,6-DCP (Fig. 1 ) o C CD CT 100 ng). Both of these products only excited the d I I sensory cells at very high doses (>1000ng). Both sensilla also re- sponded to 2,6-dibromophenol and 2,6-difiuorophenol (>!00ng). GC, GC-electrophysiology and GC-MS analysis of extracts The retention times and elution characteristics on the DB-wax column were determined for various com- pounds known from ticks such as bcnzaldehyde, phenol, FIGURE 3 (Opposite.) FIGURE 3. Capillary gas chromatography linked single sensillum tip recordings of dichloromcthane/hcxane extracts of females, males and larvae of B. microplus. Separation was done on a 30 m DB-wax fused silica column, temperature programmed from 600C after 1 min to 2O0°C at 25"C/min and to 2300C at 5"C/min with ^carrier gas at 0.S m/s, ECD detector. Recordings ofd.c. and a.c. signals were made from the d I I wall-pore single-walled sensillum on lhe anterior tarsus of a male tick (cf. Fig. I). Frequency to voltage conversion (time constant [ s) was applied to the a.c. signal impulses while the d.c. drift was compensated with an automatic base line return (time constant I s). Note presence of one chromatographic peak cluting at 2130C which evokes an olfactory response from wiihin this sensillum. 42 RESULTS 2 ty&^Wf&^VtmfmHMi'+^+W+^iWf+d 30 imp./f 0.3m V imputi e frequency DC liguai larval extract: 150 equivalenti 100 150 200 210 Elution temperature (0C) FIGURE 3. (Caption opposite.) 220 230 43 RESULTS 2 4-mcthylphenol (/7-cresol), 2-nitrophenol (o-nitro- phenol), methyl salicylate and 2,6-DCP, as well as for the related products 2,4-DCP, 2,5-DCP, 2,6-difiuoropheno! and 2,6-dibromophenoI. One peak at the retention time of 2,6-DCP in extracts of B. microplus larvae, nymphs and adults (male and female) consistently caused a d.c. potential drop and an increase in spike frequency of receptor cells in the d I 1 or d II 1 sensillum (Fig. 3) (see also Table I). The characteristically higher response of the ECD compared to that of the FID (not shown here) suggested a halogenated compound. The positional iso- mers of 2,6-DCP and 2,6-dibromophenol elute later whereas 2,6-difluorophenoI elules earlier than 2,6-DCP on this GC phase. Using either the d I 1 or d II 1 sensillum as biological detectors, no consistent responses have been observed to any other products eluting from the DB-wax column in GC-electrophysiology analysis of extracts of the different life-stages of B. microplus. Identification of 2,6-DCP was based on the match between the mass spectrum of the peak at the retention time of 2,6-DCP in three different extracts and that of the synthetic product. The higher amounts of 2,6-DCP for females over males, as determined by ECD peak integration, can well be ascribed to lheir higher average body weight (Londt and Arthur, 1975), so thai the amounts of 2,6-DCP per gram body weight are approxi- mately the same for both sexes (Table I). Pharate adult extracts contain this compound in quantities identical to that of engorged nymphs but freshly moulted adults already contain near aduli quantities. Larval and nymphal extracts also contain 2,6-DCP bui clearly less than in adults. 2,6-DCP was not present in detectable amounts in an extract of eggs (1000 equivalents injected). Behavioural bioassay on a dummy female Male B. microplus placed on top of the control dummy walked around on it for a brief period while periodically raising their front legs, but left the bead generally within 20 s. Treating the dummy with different concentrations of 2,6-DCP did not increase the total duration of male contact with the dummy, nor did it appear to influence the time spent searching in the arena aficr leaving the dummy (Fig. 4). Consequent visits to the bead did occur but this was evidently not related Io the treatment. When the dummy was coated with a dichloromethane extract of 10 female ticks however, duration of contact was drastically increased (Fig. 4). In addition, the front legs were kept in close contact with the substrate and a behaviour typical of the first stages of mating in this species (Guerin er ai, 1992) could be observed with some males even crawling under the dummy. Toial duration of search time on the arena was noi analysed here since it was considerably reduced by the long slay of (he male on the dummy. Walking behaviour in wind carrying 2,6-dichloropheno! After the adaptation period on the servospherc most males walked downwind in controls (Fig. 5). though occasional loops and short upwind walks were observed. A D) O I 3 OJ o 2 extract 0.05 0.5 5 SO 148 55 20 I 7.4 2.7 FIGURE A. Box plots of the lime spent by male B. microplus on a ca 0.5 mm glass bead (A) and the area around il (B) on a host-simulating arena. Open boxes are controls, hatched boxes are tests; the horizontal lines of each box represent from top to'.bottom the 90th. 75th. 50th. 25th and 10th percentiles of the data distribution. Other datapoints arc those outside the 10th and 90th percentile range (open circles controls. solid circles tests). The horizontal dotted tine indicates the maximum time allotted to each tick (i.e. 180 s). Treatments arc: a CH1Oj extract of IO two-day-old females [n = 18. but this is reduced for (B) to display only those ticks that left the experiment within 180 s. n = 8|. and four doses of 2,6-dichlorophenol indicated in ng/bead [n = 16 for each dose). Males were observed at different wind speeds and hu- midities, and at three different 2,6-DCP concentrations (Table 2). The distance walked upwind was not influ- enced by 2,6-DCP in any one of these treatments (/>>0.05). In addition, no change in overall walking direction was observed {P > 0.05) (Fig. 5). Walking speed was relatively low in most individuals, ranging from 0.6 to 3.4 mm/s, but usually less than one body length/s, and this was not significantly changed by switching on the stimulus (P >005) (Fig. 6)- Turn angles were normally distributed around zero. indicating no preference for a certain turning direction. and median angular velocities were also not altered by any of the 2,6-DCP concentrations offered (P > 0.05) (Fig. 6). 44 RESULTS 2 DISCUSSION A remarkable uniformity seems to exist among melas- triate ticks both in the production and perception of 2,6-DCP. In the one-host tick B. microplus, investigated here, this compound is extractable from all life-stages except eggs. However, we do not know whether it is also released by all life-stages. It is possible that 2,6-DCP is the phenolic product Chow et ai, (1972) could not identify because of the low quantities they obtained in B. microplus female extracts. Quantities reported here are low compared to the ca 60 ng found in A. variegatum and A. americanum females (Kellum and Berger, 1977), but compare better to the levels found for D. variabilis (Sonenshine et ai, 1984). Adult production in B. micro- plus is clearly higher than that of unfed larvae or engorged nymphs but differences between-males and" females are only marginal. A. maculatum Koch and D. variabilis males also produce 2,6-DCP in roughly the same quantities as females (Kellum and Berger, 1977; Sonenshine et ai, I984). Presence of 2,6-DCP in extracts of larvae and absence from eggs is also reported for R. appendiculatus (McDowell and Waladde, 1986). It would seem therefore that 2,6-DCP is widely present in differ- ent life-stages of metastriate ticks. Production of the aggregation attachment pheromone blend containing 2-nitrophenol and methyl salicylate in A. kebreaum and A. variegatum only starts after feeding has taken place (Diehl et ai, 1991). This direct relation with feeding is not valid for the production of 2,6-DCP in B. microplus since unfed adults, less then 12 h after moulting, already contain nearly the same quantities of 2,6-DCP as fed adults. Its occurrence in unfed adults has also been reported in other tick species but production commences only several days after the moult (Sonen- shine et ai, !982, 1984). Development of B. microplus from larva to adult on the same host is relatively fast so production can be considered more or less continuous. An increase in synthesis associated with adults may already start in pharates. That the amount of 2,6-DCP extracted from pharates was comparable to that of engorged nymphs in this study might be due to the inability of the solvent to reach the foveal glands of the adult ticks, still enveloped in nymphal cuticle. Our results also show thai 2,6-DCP evokes responses of sensory cells in two olfactory sensilla on the tarsus of B. microplus males, the d M and d II 1, with similar sensitivities. Responses of cells in the two homologous sensilla of R. appendiculatus and A. variegatum compare well with our results (Waladde, 1982). The increase in spike amplitude with increase in stimulus concentration reported by the latter author is also present in our recordings. Receptors for phenolic compounds seem to be widespread in ticks but olfactory receptor responses 40mm B 180' 180° FIQURE 5. Walking behaviour of male B. micropluj ticks on a locomotion compensator in a constarli flow of air (I5cm/s. 90% r.h.). Displacement was recorded during 2 min with a resolution of O.l mm. The air over a 5|ig source of 2.6-dichlorophenol on filler paper under paraffin oil was introduced into the air-stream during the second minute (test period). (A) Four examples of tracks: the starting point is indicated by an open square, the size of a male tick. Cross line indicates start of stimulus delivery. (B) and (C) Scatter diagrams of the circular means of deviation angles (0" is upwind) of 0.6 s samples of the track for control (B) and test (C) period of (0 walks. 45 RESULTS 2 speed (m m/s) B 40 O 30 60 angular velocity (Vs) ROURE 6. Frequency distributions of speed (A) and angular velocity (B) of 0.6 s segments of male B. microptus walking tracks on a locomotion compensator. Displacement of ticks was first recorded for I min io the conditioned air-stream (I5cm/s, 90% r.h.) with air from a blank stimulus bottle added (controls with hatched bars) followed for a second minute with air over 5 fig 2,6-dichlorophenol in a stimulus bottle added to the conditioned air-stream (tests with dark bars). Pooled data of walks by IO males. do not necessarily imply behavioural activity. Haggart and Davis (1981) for example observed no difference in responses from receptors of male and female A. ameri- canum to 2,6-DCP, yet only males show behavioural responses to this product (Keilum and Berger, 1977). Even Ixodes ricinus L., a prostriate tick species which does not possess foveal glands and is known to show no oriented responses to 2,6-DCP (Graf, 1975), bears a receptor for this product in the homologous d I ! sensillum (cf. Guerin et ai, 1992). Similarly, the d Il I sensillum in B. microplus showed responses to 2-nitro- phenol, a component of the aggregation attachment pheromone of Amblyomma spp., but neither this nor indeed any product other than 2,6-DCP was detected by GC coupled electrophysiology in any of the extracts investigated here. Since 2,6-DCP is both produced and perceived by B. microptus a role in the behaviour of this species would seem plausible. However we could not find any evidence to support this hypothesis in the responses of aduli males. Although the total female extract containing 2,6-DCP caused male arrestment on a glass dummy, 2,6-DCP alone on the bead failed to induce this be- haviour. It could be argued that 2,6-DCP may function as an attractant rather than an anrestant. As a concen- tration gradient can be assumed to exist immediately around the treated bead, males leaving the bead would be expected to turn back to higher concentrations of an attractant but this was not observed. It cannot be excluded that 2,6-DCP may play a role in courtship in combination with other tick related compounds but on its own it clearly does not contribute to arrestment of males near or on potential mates. We also could not demonstrate an oriented response on the part of walking male Bi-jnicroplus to 2,6-DCP in an air-stream on the servosphere. Other tick species, Bruchid beetles and Triatomìne bugs do show oriented responses to semiochemicals in the same experimental set-up (Guerin unpublished; Taneja unpublished). The air-stream can apparently be perceived by the ticks since they show an overall downwind walking behaviour, a phenomenon also noted for some of the other arthropods cited above. We therefore conclude that 2,6-DCP does not evoke anemotactic responses in male B. microplus ticks guiding them to the stimulus source. Some kind of anemotactic response however, is likely to be involved in the orientation of A. hebraeum and A. variegatum males to a combined source of CO1 and 2,6-DCP in the field (Norval et al., 1991) though the role of CO1 in the blend could be decisive. The doses tested on the locomotion compensator were equivalent to those evoking strong responses in electrophysiology. Unori- ented responses such as a change in walking speed or angular velocity (rate of turning) indicating kinetic orientation mechanisms (ortha&and klinokinesis, re- spectively) were also excluded'by our experiments. Orientation and/or arrestment of other tick species to 2,6-DCP have been demonstrated in off-host exper- iments with a Petri-dish bioassay (Leahy and Booth, 1983) and a four choice olfactometer (Yunker et ai, 1992). The behavioural mechanisms underlying the orientation in these non-discriminating experiments should have been detected in our experiments had they been part of a response to 2,6-DCP by B. microplus males. Experiments demonstrating attraction to 2,6- DCP for a number of other lick species with doses of 2,6-DCP applied on hosts (Berger, 1972; Keilum and Berger, 1977; Khalil et ai, 1981) include factors such as host odour and certain mechanical stimuli not included in our laboratory experiments. In a single experiment we did aim to register any major influence of these factors on the responses by B. microplus to 2,6-DCP. Two rubber septa treated with 1 mg 2,6-DCP and another two with dichtoromethane alone (solvent) were stapled on the hips of a young steer. The animal was heavily infested with B. microplus males and females', and the dispensers were placed on the 14th day of the infesta- tion—just prior to when fertilization of females begins 46 RESULTS 2 (Falk-Vaìrant et al., 1993). The area around the dis- pensers was investigated after 24, 48 and 96 h but no newly attached or moving males were observed in the vicinity of the dispensers. The apparent absence of a behavioural response to 2,6-DCP in males of B. microplus is contradictory to conventional knowledge about the role of this product as a pheromones in ticks. Though behavioural responses have been reported in a number of species of metastriate ticks from various genera, no convincing evidence has been presented for any one-host species. The detachment response of male B. microplus and Rhipicephalus san- guineus reported by Chow.cr al. (1972) to a "phenolic" compound eluting from the GC is not fully convincing since the conditions of the air-stream in which the compound was delivered from the chromatograrruto the -, attached ticks were not described and.data-on adequate controls was not presented. Moreover, when 2,6-DCP was subsequently identified by Chow et al. (1975) in extracts of R. sanguineus no further reference was made to B. microplus. It could be that males of this species show behavioural responses to 2,6-DCP only during a specific physiological state not present in any of our test males. It can also not be excluded that 2,6-DCP in combination with other volatiles perceived by receptors other than those tested here may play a role in male courtship behaviour. Finally, since all life stages of B. microplus succeed each other on the same bovine host, aggregation of larvae and nymphs could account for females normally being in the immediate vicinity when males moult, thus reducing the need for a long range altractant in this species. 2,6-DCP may have an ad- ditional function in ticks other than that of sex phero- monc. In conclusion, although 2,6-DCP is produced by B. microplus and can be perceived by olfactory receptors in- males of this species, we have no evidence that it plays a role in their behaviour. The total extract of females on a glass dummy did evoke a strong arrestment response from males. It is likely therefore that other chemical constituents of this extract play a crucial role in male behaviour. REFERENCES Apps P. J.. Viljocn H. W. and Prelorius V. (1988) Aggregation pheromones of the boot tick Amblyomma hebraeum: identification of candidates for bioassay. Onderslepoon J. Vet. Res. 55, 135-137. Batschelet E. (1981) Circular Statistics in Biology. Academic Press, London. Berger R. S. (1972) 2,6-Dichlorophenol, sex pheromone of the lone star tick. Science 177, 704-705. Berger R. S., Dukes J. C. and Chow Y. S. (1971) Demonstration of a sex pheromone in lhree species of hard licks. J. med. Eni. 8, 84-S6. Bull C. M. and Andrews R. H. {1984) Two different mating signals used by female reptile ticks, in Acarotogy Vl (Eds Griffiths D. A. and Bowman C. E.). pp. 427-429. Ellis Horwood. Chichester. Byrne K., Gore W E.. Pearce G. T. and Silverstcin R. M. (1975) Porapak-Q collection of airborne organic compounds serving as models for insect pheromones. J. chem. Ecol. I, 1-7. Chow Y. S., Lu F. M.. Peng C. T. and Cheng P. C. (1972) Isolation of lipids and sex pheromone from hard licks. Bull. Inst. Zool. Acad. Sin. (Taipei) II, 1-8. Chow Y. S.. Wang C. B. and Lin L. C. (1975) Identification of a sex-pheromone of lhe brown dog tick Rhipicephalus sanguineus. Ann. em. Soc. Am. 68, 485-488. Diehl P. A.. Guerin P. M-. Vlimant M. and Steullet P. (1991) Biosynthesis, production site, and emission rates of aggregation- attachment pheromone in males of two Amblyomma licks. J. chem. Ecol. 17, 833-848. Dinnik J. and Zumpt F. (1949) The integumentary sense organs of the larvae of Rhipicephalinae (Acarina). Psyche (Comb.) 56, 1-17. Falk-Vairant J., Guerin P. M., de Bruyne M. and Rohrer M. (1994) Some observations on mating and fertilisation in the caule lick Boophilus microplus. Med. Vet. Eni. 8. In press. Gladncy W. J., Grabbe R. R., Ernst S. E. and Oehler D. D. (1974) The gulf coast lick: evidence of a pheromone produced by males. J. med. Eni. 11.303-306. Gôdde J.. (1989) Vibrating glass stylets: tools for precise microsurgery on cuticular structures. J. Neurosci. Meth. 29, 77-83. Graf J.-F. (1975) Ecologie et etnologie à'Ixodes ricinus L. en Suisse (Ixodoidca; lxodidae), cinquième note: mise en évidence d'une pheromone sexuelle chez Ixodes ricinus. Acarologia 17, 436-441. Guerin P. M., Steullet P., Krôber T.. Diehl P. A.. Vlimant M.. de Bruyne M, Cordas T.. Falk-Vairant J.. Kuhnert F. and Lösel P M (1992) The Chemical ecology of licks at the host vector interface. In Proc. 1st Int. Conf. Tick-borne Pathogens at the Host-vector Inter- face: an Agenda for Research (Eds Munderloh U. G. and Kurtii T. J.), pp. 314-323. Minnesota University, Saint Paul, MN. Haggart D. A. and Davis E. E. (1981) Neurons sensitive to 2,6- dichlorophenol On the tarsi of the tick Amblyomma americanum (Acari. lxodidae). J. Med. Eni. 18, 187-193. Hess E. and Vlimant M. (1986) Leg sense organs of ticks. In Morphology, Physiology, and Behavioural Biology of Ticks ( Eds Sauer J. R. and Hair J. A.), pp. 361-390. Eilis Horwood, Chichester. Kellum D. and Berger R. S. (1977) Relationship of the occurence and function of 2,6-dichlorophenol in two species of Amblyomma. J. med. Eni. 13, 701-705. Khalil G. M., Nada S. A. and Soncnshine D. E. (1981) Sex pheromone regulation of the mating behaviour in lhe camel tick liyalomma dromedari! (Ixodoidca: lxodidae). J. Parasit. 67, 70-76. Kramer E. (1976) The orientation of walking honeybees in odour fields ¦ with «mall concentration gradients. Physiol. Eni. 1, 27-37. Leahy M. G. and Booth K. S. (1983) Attraction of metastriate ticks (Acari: lxodidae) to sex pheromone 2,6-dichlorophenol and to substituted phenols. J. med. Eni. 1, 104-105. Leonovich S. A. (1981 !Occurence of a sex pheromone in the Ixodidtick Hyalomma asialicum (lxodidae). Parasitologiye (Leningr.) 15, 159-156. Londt J. G. H. and Arthur D. R (1975) The structure and parasitic life cycle of Boophilus microplus (Canestrini, 1888) in South Africa (Acarina: lxodidae). J. Ent. Soc. S. Afr. 38, 321-340. McDowell P. G. and Waladde S. M. (1986) 2,6-Dichlorophenol in the tick Rhipicephalus appendiculaius Neumann: a reappraisal. J chem Ecol. 17, 69-82. Noldus L. P. J. J. (1991) The observer: a software system for collection and analysis of observational data Behav. Res. Meth. Insirum. Comput. 23,415-429. Norval R. A. 1., Peter T., Yunker C. E.. Soncnshine D. E. and Burridge M. J. (1991) Responses of the ticks Amblyomma hebraeum and A. variegatum to known or potential components of the aggregation-at- tachment pheromone. I. Long-range attraction. Exp. Appi. Acaro!. 13, 11-18. Schöni R., Hess E., Blum W. and Ramstein K. (1984) The aggregation- atlâchement pheromone of the tropical bont tick Amblyomma variegatum Fabricius (Acari: lxodidae): isolation, identification and action of its components- J. Insect Physiol. 30, 613-618. Schulze P. (1942) Die Rûckensinnesfelder (Foveae dorsales) der Zecken. Z. Morph. Oekol. Tiere 39, I -20. Smith J. J. B., Mitchell B. K.. Rolseth B. M., Whitehead A. T. and Albert P. J. (1990) SAPID tools: microcomputer programs 47 RESULTS 2 for analysis of multi-unit nerve recordings. Chem. Senses 15, 253-270. Sonenshinc D. E. (1985) Pheromones and other semiochemicals of the Acari. A. Rev. Ent. 30, 1-28. Sonenshinc D. E., Silverstein R. M., Plummer E. C1 West J, R. and McCullough T. (1976) 2,6-Dichlorophenol, the sex pheromonc of the Rocky Mountain wood tick, Dermacentor andersoni Stiles and the American dog tick, Dermacentor variabilis (Say). J. chem. Ecol. 2, 201-209. Sonenshinc D. E., Gainsburg D. M., Rosenthal M. O. and Silverstein R. M. (1981) The sex pheromonc glands of Dermacentor variabilis (Say) and Dermacentor andersoni Stiles, sex pheromonc stored in neutral lipid. J. chem. Ecol. 7, 345-357. Sonenshinc D. E., Silverstein R. M. and Rechav Y. H. (1982) Tick pheromonc mechanisms. In Physiology of Ticks (Eds Obcnchain F. D. and Galun R. L.), pp. 439-468. Pergamon. Oxford. Sonenshinc D- E., Silverstein R. M: and West J. R. (1984) Occurrence of the sex at tractant pheromonc, 2,6-dichlorophenol, in relation to age and feeding in the American dog tick, Dermacentor variabilis (Say) (Acari, Ixodidac). J. chem. Ecol. 10, 95-100. Waladdc S. M. (1982) Tip recording from ixodid lick olfactory sensilla: responses to tick related odours. J. comp. Physiol. 148, 4IM18. Wood W. F., Leahy M. C, Galon R., Preslwich G. D., Meinwald J., Purneil R. E. and Payne R. C. (1975) Phenols as pheromones of ixodid licks: a general phenomenon? J. chem. Ecol. I, 501-509. Yunker C E., Peter T., Norval R. A. I.. Sonenshinc D. E., Burridge M. J. and Butler J. F. (1992) Olfactory responses of adult Ambtyomma hebraeum and A. variegaium (Acari: Ixodidac) to attractant chemicals in laboratory tests. Exp. Appi. Acarol. 13, 295-301. Acknowledgements—Wc are indebted to the Hasselblad, Roche and Sandoz Foundations as well as to the Swiss National Science Foundation (Grant Nos 3.609-0.87 and 31-28654.90), the Gba-Geigy- Jubilaeums-Stiftung, Schweizerische Mobiliar and the Swiss Office for Education and Science for funding studies on tick sensory physiology al Neuchâtel. We thank Messrs Bouvard, Rohrer, Jonczj and Cesari of the Gba-Geigy Agricultural Research Station, St. Aubin. Switzerland for supplying us with ticks. We are grateful for the programming expertise ofMrT. Beyens, University of St EtienneTFrance and of DrE. Kramer, Max-Plank-lnstitute, Secwiesen, Germany. We are thankful to Mrs KLnutti for taking care of the rabbits and we acknowledge the input from Mr Falk-Vairant in some initial work on this project. This paper is part of the Ph.D, thesis of Marien de Bruyncal the University of Neuchâtel. 48 RESULTS 3 Cholesteryl Esters and other Contact Chemostimuli in the Mating Behaviour of the Cattle Tick Boophilus microplus. MARIEN DE BRUYNE, PATRICK M. GUERIN manuscript intended for submission, to. Arcfulnsect BioçhenuPhysiol INTRODUCTION Ticks (Acari: Ixodida) are obligate blood-sucking ectoparasites of vertebrates. All life- stages feed but females take a big blood meal that is used to produce a large quantity of eggs. Fertilisation of eggs is a necessity and mating is necessary for engorgement by females (Pappas and Oliver, 1972). Copulation takes place on the host in metastriate ticks and mating behaviour is relatively similar in different species (Feldman-Muhsam, 1986, Sonenshine, 1985). Males detach and search for females. Upon contact, the male mounts the attached female and investigates her dorsum, probing the surface with his tarsi and mouthparts. He then proceeds to the ventral side, aided by the female who lifts her body, locates the gonopore and inserts his chelicerae. After some time, a spermatophore is produced and transferred to the gonopore. Volatile pheromones have been.shown to stimulate aggregation on the host in several Amblyomma species (Gladney et al, 1974b) and have recently been suggested for Hyalomma truncatum (Dongus and Gothe, 1995). Such aggregation-attachment pheromones are produced by feeding male ticks to attract other conspecifics to a host and induce attachment. They generally consist of a mixture of phenols and short-chain fatty acids (Schöni et al, 1984, Apps et al, 1988). On the host, 2,6-dichlorophenol has repeatedly been suggested as a volatile sex pheromone in many tick species which induces males to detach and move towards females (Sonenshine, 1985). A potent non-volatile pheromone mediates mating behaviour once the male contacts the female (Hamilton and Sonenshine, 1988) and cholesteryl oleate and other cholesteryl esters have been implied for Dermacentor variabilis and Dermacentor andersoni (Hamilton et aLt 1989, Sonenshine et al, 1991). In these species 2,6-dichlorophenol is needed in addition to induce these mating responses, whereas a second sex pheromone containing fatty acids mediates the probing of the gonopore (genital sex pheromone, Allan et al, 1988). Cholesteryl esters have also been reported to induce mating responses in Rhipicephalus appendicular (Hamilton et al, 1994). Other non-volatile compounds (assembly pheromones) are involved in off-the-host aggregation in several argasid and ixodid tick species (Leahy et al, 1973, Hâkové et al, 1980, Petney and Bull, 1981). Guanine, an excretory product of ticks, has been identified as an arrestant for Argas persicus and Rhipicephalus sanguineus (Otieno et 49 RESULTS 3 aLt 1985) and other purines are thought to play a role in this behaviour as well (Dusbâbek^ûi, 1991b). Boophilus microplus (Canestrini) is a major pest of cattle throughout the tropical and subtropical regions causing extensive losses to the industry by weakening cattle as well as transmitting babesiosis and anaplasmosis. This is a one-host species, Le., whereas most tick species drop from the host to moult, this species goes through all life-stages on the same bovine host. In spite of its obvious importance very little is known about the mating behaviour and chemical ecology of B. microplus. It has been shown that males moult one day earlier, are more mobile than females and are able to recognise engorged female nymphs as potential mates and attach underneath them (Falk-Vairant et al.t 1994), Females moult in situ and their fertilisation starts in the night of the third day after male moult when females are well attached and have started feeding. During rapid engorgement of the female the male stays attached to the host underneath her. Males start moving around on the host again after females have detached and dropped to the gound to start oviposition (2-4 days after copulation). The males can live on the host for up to 40 days after moulting and are able to fertilise several females (Thompson, etaL, 1980). Ticks bear gustatory terminal pore sensilla on the palps and legs (see appendix II). The fourth segment of the palps in Ixodid ticks is a modified movable organ that can be folded back and inwards. On the apical surface of this palpal organ, Amblyomma americanum bears 10 sensilla of two types. Four are Tp A type with a double lumen and electron dense sensillum liquid around the dendrites (upto 4) in the inner lumen and six areTp B with a single lumen and its dendrites (7-12) enclosed in a cuticular sheath, one of which is not innervated by a tubular body at the base (Foelix and Chu Wang, 1972). Ivanov and Leonovich (1983) describe the same for Hyalomma asiaticum and some other Ixodid ticks. However B. microplus adults have only nine such terminal pore sensilla, six Tp B and three Tp A (FIG 8B), one of which is placed a little off the apical surface of the palp (Waladde, 1978). All are innervated by several dendrites, the cell bodies of which lie deep down in the palpal organV On the tarsus of the first pair of legs B, microplus carries twelve more gustatory^sensilla (Hess and Vlimant, 1986) one pair of Tp B is located distally just behind the claws, ten other Tp sensilla on tarsus I are of the A type: one pair distally below the Tp B, two pairs placed ventrally and more proximal and two dorsally behind Haller's organ. Little is known about the physiology of these sensilla, though Balashov et al /(1976) noticed electrical differences between Tp A and B sensilla of the palpal organ of H. asiaticum, and reported responses to mechanical stimuli and NaCI solutions. Palpal chemoreceptors are suggested to perceive assembly pheromones in Argassid ticks (Leahy et al, 1975a). Masking experiments suggest that terminal pore sensilla on the tip of the tarsi of the first pair of legs perceive a contact sex pheromone in Dermacentor (Phillips and Sonenshine, 1993). Other gustatory sensilla are present on the chelicerae (Waladde and Rice, 1977) and it has been shown in D. variabilis that they respond to 20-hydroxyecdysone which has been proposed as another component of the genital sex pheromone (Taylor et al., 1991). Wall-pore olfactory sensilla are exclusively located on the tarsi of the first pair of legs especially in Haller's organ (Hess and Vlimant, 1986). Their olfactory function has been demonstrated in several species, most recently in Amblyomma variegatum (Steullet and Guerin, 1994a&b). Two wall-pore sensilla on the tarsi of B. microplus mates house receptors for 2,6-dichlorophenoI, and this compound was isolated from 50 RESULTS 3 various life stages of this tick (de Bruyne and Guerin, 1994). However no behavioural responses could be observed and the role of 2,6-dichlorophenol in the biology of B, microplus remains unclear. Here we present evidence for behaviourally and electrophysiologically active components isolated from Ä microplus ticks which mediate mating in this species. Males are arrested in vitro on a substrate treated with these extracts and behavioural elements strongly resemble mating behaviour. One active fraction is identified as containing cholesteryl esters but other components are required to inducing the full mating behaviour. MATERIALS AND METHODS Ticks Ticks were obtained from a laboratory colony at the Ciba-Geigy Agricultural Research Station, St Aubin, Switzerland and belong to the organophosporous resistant strain Biarra from southern Queensland, Australia. They were reared on the backs of young Simmental steers for more than 30 generations in closed stables at 23°C and 60-70 %RH. The life-cycle and occurrence of mating under these conditions are described elsewhere (Falk-Vairant et al, 1994). Ticks were collected by carefully removing individuals from the host with forceps. In addition, unattached males were readily collected, after female drop-off had started, by brushing them off the pelage with a paintbrush. Males or engorged nymphs were transported to the laboratory in a humidified insulated container. Pharate females were kept in an incubator at 32°C and ecu 100 %RH until they moulted, and males were put on the ears of New Zealand White rabbits enclosed in cotton bags where they readily attached. Males were removed from the rabbit 20-60 min before bioassays and kept at ecu 300C over water in a closed container to assure high relative humidity. Males were kept in an incubator at 18°C on humid tissue paper for 1-4 days before electrophysiological recordings. Behavioural Bioassay A glass bead (ca. 5 mm dia.. 3 mm high, 0.1-0.2 g), roughened with a wet-stone and flattened on one side to inhibit rolling, was placed in the centre of a round arena (40 mm dia.) consisting of a Baudruche® membrane: (Joseph long Ine, rrUS A).stretched .over, a 0.9% NaCI solution at 35±1°C on a warm plate. A 40 mm. high-plastic tube:placed,around>lhis arena and the permeability to water of the membrane assured a constant r.h. (>80%). Two such arenas were used simultaneously on the same warm plate, one bearing a bead treated with an extract or synthetic product in solvent applied with a micro-pipette, the second treated with solvent alone as control. A single male tick (2-14 days after moult) was released from a fine paintbrush onto the top of the bead. Behaviour was viewed from above and filmed at magnifications of 5x or 21x with a Canon CI-20P colour CCD video camera attached to a Zeiss operational microscope (working distance: 25 cm). Recordings were made on a Panasonic super VHS video recorder (AG-7350) and played back for analysis on a Sony Trinitron colour monitor. All males in a given experiment were tested on both the control and treated bead, half first on the control the other half first on the test. Different behaviours were quantified using THE Observer 2.0 event recorder (Noldus Inf. Tech., Netherlands). The tick was recorded as either being on the bead, i.e., from the first moment all legs were in contact with it till the last leg lost contact, or on the membrane. Ticks were allowed to descend and remount the bead but a maximum of 180 s was allotted to each tick or observation was ended when it crossed the edge of the arena. The total time spent on the bead (contact time) was then taken as a parameter for statistical analysis with the Wilcoxon signed ranks test on paired replicates (test versus control). In addition it was noted whether a tick showed typical 'tip-over behaviour while on the bead (see results for definition). Chemicals and extracts All cholesteryl esters, lipid standards, cholestanol (dihydrocholesterol), ecdysone (2,3.14,22,25-penta- hydroxycholest-7-ene-6-one), caffeine (l,3,7-trimethyl-2.6-dioxopurine) and MSTFA (N-Methyl-N- trimethylsilyl-trifluoroacetamide) were obtained form Sigma, USA. Fatty acid methyl esters and 2,6-dichlorophenol were obtained from Supelco, USA. Palmitoleic acid was purchased from Larodan 51 RESULTS 3 AG, Germany. All other fatty acids. cholest-4-en-3-one and Iecithine (l,2-dioleoyl-sn-glycero-3- phosphocholine) were from Fluka, Switzerland. Cholesterol and all solvents (analytical grade) were from Merck, USA. The 9.10,16-trihydroxypalmitic acid (aleuritic acid) was kindly supplied to us by Dr S- Schulz, University of Hamburg, Germany. Tick extracts were obtained by submerging freshly collected ticks (<15 min after removal from the host, 50-500 at a time) for 5-15 days at -200C in small volumes (0.5-5 ml) of chloroform or chloroform:methanol (1:1). The extract was collected in a syringe and evaporated to dryness under a gentle stream of nitrogen, immediately red isso! ved in chloroform at 0.5 or 1 tick equivalent/u.1 and stored at -200C. Tick washes were obtained by extracting them for only 30 min at room tempereature (ca. 22°C), using either hexane. chloroform:methanol or methanol:water (1:1). A bovine hair extract was obtained by repeatedly washing hair shaved off ca, 0.3 m of the flank of a Simmental steer in dichloromethane. The filtered extract was concentrated and contained ca. 10 pg/I of low volatile mass (Krober, unpublished). Thin layer chromatography and solid phase extraction Comparative chemical analysis and preparative chromatography was carried out on 20 x 20 cm thin layer chromatography (TLC) plates with 0.25 mm layers of silica gel 60 including a 2.5 cm concentration zone (Merck, Germany). The plates were first washed twice with chloroform:methanol (1:1) and conditioned for 60 min at 1100C. Extracts and standards were applied in <1 cm dia. spots and concentrated thrice to the bottom of the silica layer using chloroform:methanol (1:1). The plate was subsequently developed in one of the following solvent systems: I) hexane to 17 cm, toluene to 17 cm and twice hexane:diethyl ethenacetic acid (70:30:1) to 12 cm or ID chloroform:methanol:water (69:27:4) to 12cm. After drying, the plates were sprayed with 50% H2SO4 in water and heated to 1500C in an oven. For preparative TLC 100 female equivalents of the extract was applied in a 5 cm band and this part of the plate was not sprayed. Fractions of the resolved extract were scraped from the plate as indicated by the visualised spots on the sprayed part (see FIG 3). The silica gel was subsequently eluted over glass wool in a pasteure pipette with 2 ml chloroform for each fraction, then dried under nitrogen and redissolved in a smaller volume of eh loro from. Preparative solid phase extraction (SPE) was done on 500 mg Silica gel in a glass Chromabond* column (Machery-Nagel, Switzerland) conditioned consecutively with 2 ml each of methanokwater (1:1), methanol, chloroform:methanol (1:1), chloroform, hexanexhloroform (75:25). The extract was applied as 150 female equivalents in 100 u.1 chloroform and subsequently eluted at ca. 1 ml/min with 4 ml hexanexhloroform (75:25), 3 ml chloroform, 3 ml chloroform:methanol (1:1), 2 ml methanol and 2 ml methanol:water (1:1). The ten 1 ml serial fractions, the methanol (FIl), and methanol:water (F12) fractions were dried under nitrogen and redissolved in chloroform. These fractions were tested as A(Fl+2+3+4), B (F5+6+7) and C (F8+9+10+11+12) in the behavioural bioassays. Isolation of cholesteryl esters, cholesterol and fatty acids Cholesteryl esters were transmethylated at 85°C for 60 min in flame sealed 1 ml glass ampoules with 200 pi of 1% H2SO4 in methanol after adding 1 ug of tetradecane as internal standard. Fatty acid methyl esters were solvent-solvent extracted into hexane and analysed with cold on-column injection on a 30 m DB-wax capillary column (J&W Scientific, USA, 0.25 pm film thickness, 0.25 mm ID) in a Carlo Erba HRGC 5160 gas Chromatograph (GC) with H2 as carrier gas at 1.5 ml/min (0.5 m/s) temperature programmed from 700C after I min to 900C at 15°C/min, to 1600C at 20°C/min, to 2400C at 5°C/min and held there for 10 min. Identification was by comparing retention times with standards and matching mass spectra (see below). Quantification was made by peak area integration of the FID detector signal using a Spectra-physics SP-4270 integrator and comparison with known amounts of standards injected in the same session. Cholesterol and free fatty acids were isolated from 70 female equivalents of extract fractionated on an SPE column in a separate procedure: After conditioning with 3 ml hexane and applying the extract, the column was extracted consecutively with 1 ml hexane, 1 ml hexane:dichloromethane (9:1), 2 ml hexane:dichloromethane (1:1), 2 ml dichloromethane, 2 ml dichloromethaneimethanol (1:1), 2 ml methanol. After identifying the presence of Cholesterol and free fatty acids in the dichloromelhane:methanol fraction by TLC evaluation, an aliquot of this fraction was evaporated to dryness in an ampoule and sylilated as described by Grenacher and Guerin (1994) after being flame- sealed. 52 RESULTS 3 ìzm 0.03 0.1 0.3 Ss * m *•> O X as UJ fi X X o UJU.U-U.U-ll.U.U.U.U.*-~ 0.14 0.05 FIGURE 3. Preparative thin layer chromatography (TLC) and bioassay of the fractions of a chloroform extract of female B. microplus. A: TLC separation on 0.25mm silica gel of extract (EXT) and lipid standards (LIP). FR indicates fractions of the silica gel scraped off the plate, solvent extracted, and subsequently bioassayed (below). Solvent system I was used: hexane to 1st front, toluene to 1st front and twice hexane:diethyl ethenacetic acid (70:30:1) to 2nd front Spots that could only be observed under UV light (36ónm) are open, the asterisk marks those spots that tum up orange in UV and the grey shading indicates intensity after charring. Standards are 1. cholesterol, 2. oleic acid. 3. triolein, 4 methyl oleate, 5 cholesteryl oleate. B: Behavioural responses of individual male B. microplus ticks to a glass bead treated with the female extract and its TLC fractions at 10 equivalents. EXT. extract before separation, POS, positive control, Le., all fractions recombined, NEG, negative control, Le., all fractions except F8. For details on presentation of data see FIG 1. 56 RESULTS 3 A second group of more polar fractions restores total activity. After separation of 150 female equivalents of a chloroformrmethanol (1:1) extract on a small SPE column, recombination of all fractions restores the full activity (FIG 5). Some arrestment is associated with the early eluting, apolar, A set of fractions as might be expected from the behavioural responses to the apolar TLC fraction (compare FIG 3 and FIG 5). However, considerable arrestment is also obtained with the more polar C fractions, whereas the fractions in set B are not active. Of the different combinations, most activity is found after combining A with C. The tip-over on the bead appears to be associated with C and particularly with CA. Analytical TLC using the more polar solvent system II (FIG 6) revealed the presence of several components in the material remaining at the origin with solvent system I (FIG 5A), whereas all spots above cholesterol elute close to the solvent front. 1st fron 2nd fronl ongir FEM CH:M MAL LAR CH:M CH:M IRX CHL BOV DCM sy sy Ir-- I \mmm FIGURE 4. TLC separations on 0.25mm silica gel of different extracts and synthetics. For details on solvent system I employed and presentation of data see FIG 3A. FEM, females, MAL, males, LAR, unfed larvae, IRX, Ixodes ricinus exuviae, BOV, Bovine hair, CHL, chloroform, CH:M, chloroform:methanol (1:1). DCM. dichloromethane. Synthetics (sy) are 1. ecdysone, 2. 9,10,16- trihydroxy-palmitic acid, 3. monoolein, 4. cholestanol, 5. cholesterol, 6. diolein, 7. cholest-4-en-3-one, 8. oleic acid, 9. cholesteryl acetate. 10. triolein, U. methyl oleate, 12. oleoyl oleate, 13. cholesteryl oleate and 14. squalene. 57 RESULTS 3 TABLE 1 Fatty acid moieties of cholesteryl esters identified by gas chromatographic analysis after transmethylation of an apolar fraction from a TLC separation of chloroform extracts. Synthetic mixtures of these products tested in behavioural bioassays with male ticks are indicated on the right._________________________________________________________ cholesteryl esters fatty acid methyl ester (FID peak area) synthetic mixtures _____________________pmol/tick_____________________ nmol trivial name code_______female %______pharate % larva %_______I_______n_______III saturated fatty acid moieties caprate 10:0 29 2 107 20 l tf _ _ _ laurate 12:0 28 2 24 4 1 5 — — 3 myristate 14:0 325 2? 134 25 S 26 AO — 30 palmttate 16:0 166 14 76 14 7 35 20 — 15 stéarate 18:0 127 11 49 9 4 19 20 — 15 arachidate 20:0 205 17 81 75 — — 20 — 15 behenate 22:0 25 2 57 _ _ _ _ 5 lignocerate 24:0 54 5 16 3 — — — ^ — 5 ceratale* 26:0 137 12 285 _-— _ — _ unsaturated fatty acid moieties palmitoleate 16:1 12/ — — — — — 33 3 oleate 18:1 49 4 17 3 2 9 — 33 3 linoleate 18:2 19 2 __ _ _ _ 33 3 totals 1182 99 536 99 19 100 100 99 97 —, not detected or not present, *,tentative identification of this fatty acid TABLE 2 Arrestment of male B. microplus on a glass bead treated with cholesteryl «sters and mixtures*. Data are medians of the arrestment ratios, Le,, the ratio between time spent on the test versus control bead for individual males (130.05) 58 RESULTS 3 Cholesteryl esters in the apolar TLC fraction cause male arrestment GC-MS analysis of the active TLC fraction F8 on a high temperature (35O0C) nonpolar capillary column indicated the presence of a series of compounds with molecular weight of 500 and higher, co-eluting with some cholesteryl ester standards. Their mass spectra contain m/z 368 and other ions characteristic of the cholesterol moiety but their molecular ions could not be determined. No other major peaks were observed under these conditions in this fraction. Transmethylation and subsequent analysis of methyl esters with an FID detector on a DB-wax capilary column indicated the presence of several fatty acid moieties (TABLE 1). Recovery of various synthetic cholesteryl esters from TLC plates with this method was above 80%. A comparison between extracts shows that in both the female and pharate female extracts straight chain saturated fatty acids predominate, notably myristic, palmitic, stearic and arachidic. The pharate. female extract. is quite comparable with the female B. microplus extract. We could not confirm the presence of methyl cerotate (FAME 26:0) by comparing with the synthetic analogue but it was inferred from its retention time as an additional peak in a regular series from 14:0 to 24:0. The identification of other unknown peaks in the chromatogram was not attempted but none exceeded 1% of the most abundant peak (methyl myristate). The larval cholesteryl ester fraction shows relatively high amounts of cholesteryl oleate and absence of the longer chain fatty acids. IsI Iron A 2nd Iron > ^^^m * = I ¦ 'I ,—.—, rrr^a, i-----------1 I- ,| ^ "^ i i EXT A B C O ¦«-» c E B 1 I i I 20.09 7.39 2.72 .w (O C 1.00 <" «-I Vi a> FIGURE 5. Preparative solid phase extraction (SPE) on a small silica gel column of a chloro- form:methanol (1:1) extract of female B. microplus and bioassay of fractions. SPE eleution was with 4 ml hexanexhloroforni (75:25). 3 ml chloroform. 3 ml chloroform:methanol (1:1), 2 ml methanol and 2 ml methanol:water (1:1). The ten 1 ml serial fractions and methanol (FIl) and methanol:water (F12) fractions were recombined in A (Fl+2+3+4), B (F5+6+7) and C (F8+9+10+11+12) A: Thin layer chromatography (TLC) of whole extract (EXT) and SPE fraction sets A B and C. For details on solvent system I employed and presentation of data see FIG 3A. B: Behavioural responses of individual male B. microplus ticks to a glass bead treated with the female extract and its SPE fractions at 3 equivalents. EXT. extract before separation. POS, positive control, i.e.. all fractions A, B and C combined. For details on presentation of data see FIG 1. 59 RESULTS 3 When the most important cholesteryl esters were tested individually, none of them arrests male ticks at doses comparable to those present in the extract (TABLE 2). Activity was observed only at very high doses (1000 nmol) of individual products. Of the three different mixtures tested, only the most complete, including both saturated and unsaturated fatty acid esters, was active at the physiologically relevant level of 10 nmol, corresponding to ca. 10 tick equivalents. Cholesterol, free fatty acids ana 2,6-dichlorophenol inactive at natural levels GC-MS analysis of the dichioromethane:methanol fraction form an SPE column indicated the presence of cholesterol and free fatty acids in a chloroforrmmethanol (1:1) extract of female & microplus at ca. 3 nmol cholesterol and ca. 0.5 nmol of total free fatty acids per female. A small peak at the retention time of cholesterol was also observed in the SPE fraction F8 used for the bioassays as were free fatty acids. This could be expected from the TLC analysis (FIG 5A). Cholesterol induces arrestment in male B. microplus at doses just above the level present in 10 female equivalents (TABLE 3). A synthetic mixture of fatty acids corresponding to that found in the extract (myristic:palmitic:paImitoleic: stearic:oleic:linoleic acids at 6:10:2:6:50:30) was not active at doses near natural levels. No tip-over behaviour was observed in any of these tests. Adding fatty acids and cholesterol to the cholesteryl esters at doses comparable to 3 tick equivalents did not significantly increase arrestment (FIG 7), nor did it influence tip-over. In addition, no effect was observed for 2,6-dichIorophenol when added to this complex mixture at naturally occurring amounts {i.e., 1.5 ng for 3 equivalents, see de Bruyne & Guerin, 1994). TABLE 3 Arrestment of male B. microplus on a glass bead treated with cholesterol and fatty acid mixture isolated from a chloroform:methanol extract of females. Data are medians of the arrestment ratios ratios, i.e., the ratio between time spent on the test versus control bead for individual B. microplus males (160.05) Electrophysiological responses of gustatory sensilla on male palps: response to the more polar SPE fractions With the tip recording method used here we could not obtain noticeable electrical contact with palpal sensilla 3, 4 and 9 (FIG.8B). Sensillum 6 was difficult to reach and therefore not studied. A few recordings were made from sensilla I1 5 and 8 but no consistent responses were obtained to the control [Wo ethanol in 0.1 M KCl), and since this could therefore not be used as a test for the condition of the preparation, further analysis of these sensilla was abandoned. However, sensilla 2 and 7 house a receptor which consistently responded with a tonic spike train of 30 - 60 spikes/s to the control (FIG.8C). This response is similar in both sensilla and dose dependent for KCl (0.001, 0.003, 0.01, 0.03, 0.1, 0.3 and 1 M tested). In both sensilla there was a clear change in this pattern, with an increase in the total number of action potentials recorded when stimulated with 0.1 equivalent/nl of SPE fractions F8, F9 and FIl. 60 RESULTS 3 Fraction FIO was active but to a lesser extent, whereas some inhibition occurred with fraction F5 (FIG.9). Responses to different doses of fraction F8 were obtained (0.001, 0.003, 0.01, 0.03 and 0.1 equivalents/ul) and the response to F9 resembles that to 0.01 equivalent of F8 (FIG-8C). Clearly a receptor or receptors other than the one responding to KCl is/are involved in the perception of these fractions. However, responses are complicated by superposition of spikes, the similarity of spike sizes within single recordings and variability of spike size between recordings (FIG.8) from the same sensîllum. A clear response was also obtained to 0.01 and 0.1 equivalents/uj of the unseparated extract but not to 0.1 ug/u.1 of bovine hair extract FIGURE 6. TLC separation on 0.25mm silica gel of a chlorofornvmethanol (1:1) extract (EXT), the polar SPE fractions (that make up C the set in FIG 5A) and some synthetics (sy) with solvent system II (chloroform:methanol:water 69:27:4 to 12cm). Spots that could only be observed under UV light (366nm) are open, the asterisk marks those spots that turn up orange in UV and the grey shading indicates intensity after charring. Standards are 1. caffeine. 2. lecithine, 3. 9,10,16-trihydroxy-palmitic acid, 4. ecdysone, 5. monoolem, 6. cholesterol, 61 RESULTS 3 DISCUSSION Male behavioural responses to chemical stimuli on the cuticle of females The mating behaviour of Boophilus microplus is clearly mediated by chemical stimuli associated with the cuticle of female ticks. The behaviour of males in vitro on a glass bead treated with extracts of females in various solvents is similar to that observed on females in vivo (Falk-Vairant et at, 1994) but distincly different from that on an untreated bead. Responses are obtained to doses of extracts near naturally occurring levels. This response is obtained to extracts of whole ticks and of exuviae. Several elements are present in the behaviour observed on the glass beads which also occur during mating on the host such as the strong drive to tip-over and crawl under the bead. 20.09 O "+3 CO l— +-* C Q) £ +-< W Q) (G Q) Ö) O -2h -3 -1 3¾ m 7.39 O 2.72 £ +-» C 1.00