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Mycamoeba gemmipara nov. gen., nov. sp., the First Cultured Member of the Environmental Dermamoebidae Clade LKM74 and its Unusual Life Cycle
Since the first environmental DNA surveys, entire groups of sequences called “environmental clades” did not have any cultured representative. LKM74 is an amoebozoan clade affiliated to Dermamoebidae, whose presence is pervasively reported in soil and freshwater. We obtained an isolate from soil that we assigned to LKM74 by molecular phylogeny, close related to freshwater clones. We described Mycamoeba gemmipara based on observations made with light- and transmission electron microscopy. It is an extremely small amoeba with typical lingulate shape. Unlike other Dermamoebidae, it lacked ornamentation on its cell membrane, and condensed chromatin formed characteristic patterns in the nucleus. M. gemmipara displayed a unique life cycle: trophozoites formed walled coccoid stages which grew through successive buddings and developed into branched structures holding cysts. These structures, measuring hundreds of micrometres, are built as the exclusive product of osmotrophic feeding. In order to demonstrate that M. gemmipara is a genuine soil inhabitant, we screened its presence in an environmental soil DNA diversity survey performed on an experimental setup where pig cadavers were left to decompose in soils in order to follow changes in eukaryotic communities. M. gemmipara was present in all samples, although related reads were uncommon underneath the cadaver.
Light and electron microscopy studies of the midgut and salivary glands of second and third instars of the horse stomach bot, Gasterophilus intestinalis
A morphological study of the midgut and salivary glands of second and third instars of Gasterophilus intestinalis (De Geer) (Diptera: Oestridae) was conducted by light, scanning and transmission electron microscopy. The midgut is anteriorly delimited by a proventriculus, without caeca, and is composed of posterior foregut and anterior midgut tissue from which a double-layered peritrophic matrix is produced. The midgut can be divided into anterior, median and posterior regions on the basis of the structural and physiological variations of the columnar cells which occur along its length. Two other types of cell were identified: regenerative cells scattered throughout the columnar cells, and, more rarely, endocrine cells of two structural types (closed and open). Different secretion mechanisms (merocrine, apocrine and microapocrine) occur along the midgut epithelium. Abundant microorganisms are observed in the endoperitrophic space of the anterior midgut. The origin and nature of these microorganisms remain unknown. No structural differences are observed between the second and third instar midguts. The salivary glands of G. intestinalis second and third instars consist of a pair of elongated tubular structures connected to efferent ducts which unite to form a single deferent duct linked dorsally to the pharynx. Several intermediate cells, without cuticle, make the junction with the salivary gland epithelium layer. Cytological characteristics of the gland epithelial cells demonstrate high cellular activity and some structural variations are noticed between the two larval stages.
Ultrastructure and receptor cell responses of the antennal grooved peg sensilla of Triatoma infestans (Hemiptera: Reduviidae)
Ultrastructural examination of grooved-peg (GP) sensilla on the antenna of fifth instar Triatoma infestans nymphs by scanning electron microscopy and transmission electron microscopy reveal that they are 8–18 μm long with a diameter of about 2–2.8 μm at the non-articulated base. Some pegs have a terminal pore. These double-walled wall-pore (dw-wp) sensilla have an outer cuticular wall with 13–18 longitudinal grooves at the distal part of the peg. Groove channels are present at the bottom of the grooves from which radial spoke channels lead into the inner sensillum-lymph cavity. A dendrite sheath connects the tip of the thecogen cell to the inner cuticular wall thus forming separated outer and inner sensillum-lymph cavities. Four or five bipolar receptor cells are ensheathed successively within the GP sensilla by the thecogen cell, trichogen and tormogen cells. The inner dendritic segments of each sensory cell give rise at the ciliary constriction to an unbranched outer dendritic segment which can reach the tip of the sensillum.
Electrophysiological recordings from the GP sensilla indicate that they house NH3, short-chain carboxylic acid and short-chain aliphatic amine receptor cells and can be divided into three functional sub-types (GP 1–3). All GP sensilla carry a receptor cell excited by aliphatic amines, such as isobutylamine, a compound associated with vertebrate odour. GP type 1 and 2 sensilla house, in addition, an NH3-excited cell whereas the type 2 sensilla also contains a short-chain carboxylic acid receptor. No cell particularly sensitive to either NH3 or carboxylic acids was found in the grooved-peg type 3 sensilla. GP types 1, 2 and 3 represent ca. 36, 10 and 43% of the GP sensilla, respectively, whereas the remaining 11% contain receptor cells that manifest normal spontaneous activity but do not respond to any of the afore mentioned stimuli.
Quinine and artesunate inhibit feeding in the African malaria mosquito Anopheles gambiae: the role of gustatory organs within the mouthparts
A membrane feeding assay in which the effects of the antimalarial drugs quinine and artesunate are tested on Anopheles gambiae Giles sensu stricto is described. In the present study, 87% of female A. gambiae are shown to feed on whole defibrinated bovine blood alone, whereas only 47% and 43.5% feed on saline and on saline + bovine serum albumin (BSA) solutions, respectively, suggesting that additional components in the blood stimulate mosquito feeding. The addition of 1 mm quinine or artesunate to the BSA solution results in a significant reduction in percentage engorgement to 16.2% and 14.1%, respectively. However, the feeding rate is higher when 1 mm artesunate and quinine are mixed in the blood because 67.8% and 78.4% of females engorge on these solutions respectively. Artesunate (10 mm) in the blood reduces percentage engorgement to 20%. Because circulating doses of quinine and artesunate affecting Plasmodium in humans are much lower than those affecting feeding by A. gambiae in the in vitro assay, these two antimalarial drugs should have no effect, or only a minor effect, on the infection rate of mosquitoes feeding on treated patients. Because only the stylets penetrate the membrane and not the labellar lobes, the results of the present study suggest that both blood phagostimulants and feeding deterrents are detected by internal gustatory organs in A. gambiae, namely sensory cells in the apical and subapical labral pegs, in sensilla on the inner face of the labellar lobes, or by cibarial receptor cells. The neuroanatomy of gustatory sensilla on the apical and subapical labral pegs and on the inner face of the labellar lobes of female A. gambiae is described in the present study.
Ultrastructure and receptor cell responses of the antennal grooved peg sensilla of Triatoma infestans (Hemiptera : Reduviidae)
Ultrastructural examination of grooved-peg (GP) sensilla on the antenna of fifth instar Triatoma infestans nymphs by scanning electron microscopy and transmission electron microscopy reveal that they are 8-18 mum long with a diameter of about 2-2.8 mum at the non-articulated base. Some pegs have a terminal pore. These double-walled wall-pore (dw-wp) sensilla have an outer cuticular wall with 13-18 longitudinal grooves at the distal part of the peg. Groove channels are present at the bottom of the grooves from which radial spoke channels lead into the inner sensillum-lymph cavity. A dendrite sheath connects the tip of the thecogen cell to the inner cuticular wall thus forming separated outer and inner sensillum-lymph cavities. Four or five bipolar receptor cells are ensheathed successively within the GP sensilla by the thecogen cell, trichogen and tormogen cells. The inner dendritic segments of each sensory cell give rise at the ciliary constriction to an unbranched outer dendritic segment which can reach the tip of the sensillum. Electrophysiological recordings from the GP sensilla indicate that they house NH3, short-chain carboxylic acid and short-chain aliphatic amine receptor cells and can be divided into three functional sub-types (GP 1-3). All GP sensilla carry a receptor cell excited by aliphatic amines, such as isobutylamine, a compound associated with vertebrate odour. GP type 1 and 2 sensilla house, in addition, an NH3-excited cell whereas the type 2 sensilla also contains a short-chain carboxylic acid receptor. No cell particularly sensitive to either NH3 or carboxylic acids was found in the grooved-peg type 3 sensilla. GP types 1, 2 and 3 represent ca. 36, 10 and 43% of the GP sensilla, respectively, whereas the remaining 11% contain receptor cells that manifest normal spontaneous activity but do not respond to any of the afore mentioned stimuli. (C) 2003 Elsevier Science Ltd. All rights reserved.
Behavioural and chemoreceptor cell responses of the tick, Ixodes ricinus, to its own faeces and faecal constituents
Ticks are ectoparasites of vertebrates and utilize a variety ofinfochemicals for host finding and acceptance as well as for intraspecific aggregation and mating responses. Individual male and female Ixodes ricinus, the vector of Lyme disease in Europe, readily arrest onfilter paper strips contaminated with their own faeces. I. ricinus also responds, but to a lesser degree, tofaeces-contaminated papers enclosed in metal mesh envelopes, i.e. without directly contacting the faeces, suggesting a role for volatiles in the arrestment response. The faecal constituents guanine, xanthine, uric acid and 8-azaguanine (a bacterial breakdown product of guanine) also caused arrestment of individual I.ricinus males and females. However, mixtures of these products induced arrestment of I. ricinus at doses one hundred fold lower than the lowest active dose of any of them tested singly. Saline extracts of faeces activated receptor cells in terminal pore sensilla on the first legtarsi of I. ricinus. One cell in these sensilla responded in a similar dose dependent manner to guanine and 8-azaguanine, whereas a second cell was more sensitive to lower doses of 8-azaguanine. The response threshold approached 100 fM for both cells. These findings suggest that faeces and faecal breakdown products are implicated in aggregation responses of I. ricinus. This may account for the clumped distribution of this ectoparasite on the ground and contribute to the high proportion of mated individuals recorded prior to host colonization.
The sugar meal of the African malaria mosquito Anopheles gambiae and how deterrent compounds interfere with it: a behavioural and neurophysiological study
In this study, we show that female African malaria mosquitoes Anopheles gambiae starved for 3–5 h start to engorge on sucrose at concentrations between 50 and 75 mmol l−1. Half of the feeding response (ED50) is reached at 111 mmol l−1 and the maximum response (0.4 mg) occurs at 250 mmol l−1. Two receptor cells in a trichoid sensillum of the labellum, called the ‘sucrose’ and ‘water’ neurones, are activated by sucrose and water, respectively. The electrophysiological response of the sucrose receptor cell starts well below the level of sugar necessary to induce engorgement. The sugar receptor cell is most sensitive to small increments in sucrose concentration up to 10 mmol l−1 with a response plateau from 25 mmol l−1. Fructose has a mild phagostimulatory effect on A. gambiae, whereas no significant differences in meal sizes between water and glucose were found. However, when 146 mmol l−1 fructose plus glucose are mixed, the same engorgement as on 146 mmol l−1 sucrose is observed. Likewise, even though the sucrose receptor cell is not activated by either fructose or glucose alone, equimolar solutions of fructose plus glucose activate the neurone. We conclude that there is a behavioural and neurophysiological synergism between fructose and glucose, the two hexose sugars of sucrose. We show that some bitter-tasting products for humans have a deterrent effect on feeding in A. gambiae. When 1 mmol l−1 quinidine, quinine or denatonium benzoate is added to 146 mmol l−1 sucrose, feeding is almost totally inhibited. The effect of berberine is lower and no significant inhibition on engorgement occurs for caffeine. The deterrent effect depends on the concentration for both quinine and quinidine. Capillary feeding experiments show that contact chemosensilla on the mouthparts are sufficient for the detection of sucrose and bitter products. The feeding assay findings with deterrents correlate with the neurophysiological responses of the sucrose and water labellar neurones, which are both inhibited by the bitter compounds denatonium benzoate, quinine and berberine between 0.01 and 1 mmol l−1, but not by the same concentrations of caffeine. In conclusion, sucrose stimulates feeding and activates the labellar sucrose neurone, whereas feeding deterrents inhibit both the sucrose and water neurones. This study provides an initial understanding of the physiological mechanisms involved in sugar feeding in A. gambiae and shows how some bitter products interfere with it.
Darkness induces mobility, and saturation deficit limits questing duration, in the tick Ixodes ricinus
The behaviour of Ixodes ricinus nymphs was recorded in 10-day experiments using computer-assisted video-tracking, in the absence of any host stimuli. These ticks switch spontaneously from questing in a desiccating atmosphere to quiescence in a water-saturated atmosphere after dark. Quantification of both questing and quiescence duration demonstrates that questing duration is inversely related to saturation deficit whereas quiescence duration is not. Distance walked after quiescence increased with desiccating conditions, while the distance walked after questing remained unchanged. Almost all locomotor activities of I. ricinus occurred during darkness under either a 14 h:10 h L:D or a 8 h:4 h L:D cycle. We established that all life stages of I. ricinus are equipped to sense shifts in light intensity with bilaterally placed strings of photoreceptors. This permits I. ricinus to use onset of darkness to trigger mobility when desiccation risk is reduced in nature.
Neurophysiological and behavioural evidence for an olfactory function for the dorsal organ and a gustatory one for the terminal organ in Drosophila melanogaster larvae
Multicellular electrophysiological responses from the dorsal organ on the cephalic lobes of third instar Drosophila melanogaster larvae (wild-type Canton S) stimulated with a cold-trapped banana volatile extract showed that this structure has an olfactory function in the fruit fly. Responses of the dorsal organ were also recorded to constituents of the banana volatile extract as they eluted from a gas chromatographic column (GC-coupled dorsal organ electrophysiology). The active chemostimulants were identified as 2-heptanone, isoamyl alcohol, hexyl acetate, hexanol and hexyl butyrate by gas chromatography-coupled mass spectrometry. Applying the same recording system to the terminal organ sensilla, no responses were obtained to either the banana volatile bouquet or its constituents. By contrast, high frequency multicellular responses were recorded in response to touching the terminal organ with the gustatory stimuli KCl and grapefruit juice; responses were absent on similar stimulation of the dorsal organ with either NaCl or KCl. This suggests a role for olfaction by the dorsal organ and for gustation by the terminal organ in Drosophila larvae.
In a 7-mm high wind tunnel with a thin 1.2% agar floor, the Drosophila larvae showed odour-conditioned upwind responses in an air stream of 0.1 m/s bearing banana volatiles. Drosophila larvae responded best to the odour of cut bananas. A 1:1 mixture of the banana odour constituents 2-heptanone and hexanol (at either 50 or 100 μg source dose each) proved as attractive as the known larval attractants propionic acid and isoamyl acetate on their own at 100 μg, whereas hexanol and 2-heptanone on their own at a 100 μg source dose were less attractive. The stronger behavioural response to the banana volatile bouquet and to the binary mixture serves to underline the multireceptor nature of the dorsal organ response to food odour in Drosophila.