Olfactory and behavioural responses of tsetse flies, Glossina spp., to rumen metabolites.

Herbivores provide tsetse flies with a blood meal, and both wild and domesticated ruminants dominate as hosts. As volatile metabolites from the rumen are regularly eructed with rumen gas, these products could serve tsetse flies during host searching. To test this, we first established that the odour of rumen fluid is attractive to hungry Glossina pallidipes in a wind tunnel. We then made antennogram recordings from three tsetse species (G. pallidipes morsitans group, G. fuscipes palpalis group and G. brevipalpis fusca group) coupled to gas chromatographic analysis of rumen fluid odour and of its acidic, mildly acidic and neutral fractions. This shows tsetse flies can detect terpenes, ketones, carboxylic acids, aliphatic aldehydes, sulphides, phenols and indoles from this biological substrate. A mixture of carboxylic acids at a ratio similar to that present in rumen fluid induced behavioural responses from G. pallidipes in the wind tunnel that were moderately better than the solvent control. The similarities in the sensory responses of the tsetse fly species to metabolites from ruminants demonstrated in this study testify to a contribution of habitat exploitation by these vertebrates in the Africa-wide distribution of tsetse.

Influence of soil quality in the larval habitat on development of Anopheles gambiae Giles.

Larval ecology is an important aspect of the population dynamics of anopheline mosquitoes (Diptera: Culicidae), the vectors of malaria. Anopheles larvae live in pools of stagnant water and adult fitness may be correlated with the nutritional conditions under which larvae develop. A study was conducted in Mbita, Western Kenya, to investigate how properties of the soil substrate of Anopheles gambiae breeding pools can influence development of this mosquito species. An. gambiae eggs from an established colony were dispensed into experimental plastic troughs containing soil samples from a range of natural Anopheles larval habitats and filtered Lake Victoria water. The duration of larval development (8-15 days), pupation rate (0-79%), and adult body size (20.28-26.91 mm3) varied among different soil types. The total organic matter (3.61-21.25%), organic carbon (0.63-7.18%), and total nitrogen (0.06-0.58%) levels of the soils were positively correlated with pupation rate and negatively correlated with development time and adult body size.

(1S,3S,7R)-3-methyl-alpha-himachalene from the male sandfly Lutzomyia longipalpis (Diptera: Psychodidae) induces neurophysiological responses and attracts both males and females.

Lutzomyia longipalpis adult males form leks on or near hosts and release (1S,3S,7R)-3-methyl-alpha-himachalene from their tergal glands to lure females to the same site for mating and feeding. Here we have examined whether the male-produced attractant could also serve as a male aggregation stimulus. High resolution chiral capillary gas chromatography analysis of male tergal gland extracts, synthetic (1S,3S,7R)-3-methyl-alpha-himachalene, and a synthetic mixture of all isomers of 3-methyl-alpha-himachalene, was coupled to electrophysiological recordings from ascoid sensillum receptor cells in antennae of male and female sandflies. Receptor cells of both sexes responded only to the main component of the male tergal gland extract that eluted at the same retention time as (1S,3S,7R)-3-methyl-alpha-himachalene. Furthermore, of the eight 3-methyl-alpha-himachalene isomers in the synthetic mixture only the fraction containing (1S,3S,7R)-3-methyl-alpha-himachalene, co-eluting with an isomer of (1S*,3S*,7S*)-3-methyl-alpha-himachalene, elicited an electrophysiological response from male and female ascoid sensillum receptor cells. Both males and females flew upwind in a wind tunnel towards a filter paper disk treated with either 4-6 male equivalents of the tergal gland extract, pure (1S,3S,7R)-3-methyl-alpha-himachalene or the synthetic mixture of eight isomers. This indicates that (1S,3S,7R)-3-methyl-alpha-himachalene derived from L. longipalpis males may have a dual function in causing male aggregation as well as serving as a sex pheromone for females.

The life-cycle of the bont tick Amblyomma hebraeum in vitro.

The life-cycle of the hard tick Amblyomma hebraeum was completed in vitro by feeding all life-stages of the tick through silicone membranes on bovine blood from an abattoir. Ticks were placed in a simple feeder membranes on bovine blood from an abattoir. Ticks were placed in a simple feeder consisting of a honey jar containing the blood with a glass tube insert (o.d. 42 mm) across the end of which the membrane was stretched. This feeding unit was held in a water bath (38 degrees C). Larvae and nymphs fed on a membrane (< 90 microns thick) made of silicone reinforced with Kodak lens cleaning paper, and adults on a silicone membrane (0.5 mm thick) reinforced with Terylene netting. To control microbial growth, gentamicin (5 micrograms/ml) and nystatin (100 i.u./ml) were added to the weekly open-collected blood, which was manually defibrinated. The blood was changed twice daily for nymphs and three times for adults and larvae. Attachment of ticks was induced with combinations of host hair, tick faeces, a bovine pelage extract and a synthetic aggregation-attachment pheromone mixture. The in vitro life-cycle started with unengorged "natural" adults, which had moulted from nymphs fed on steer. The life-cycle closed with unengorged, first in vitro generation adults which had moulted from nymphs fed in vitro. Although the feeding and development of larvae and nymphs were similar to in vivo controls, females fed and developed poorly in vitro. The toxicity of the systemic acaricide Ivermectin for nymphs of A. hebraeum was confirmed using the in vitro feeding method.

Sex pheromone ofAdoxophyes orana: Additional components and variability in ratio of (Z)-9- and (Z)-11-tetradecenyl acetate.

Twelve products related to the sex pheromone main components (Z)-9- and (Z)-11-tetradecenyl acetate (Z9-14∶Ac andZ11-14∶Ac, respectively), were identified in female pheromone gland extracts of the laboratory-reared summerfruit tortrix moth,Adoxophyes orana F.v R. These are the geometric isomers and the alcohols of the main components, (Z)-9-dodecenyl acetate, (Z)-11-hexadecenyl acetate, and saturated acetates of 12-22 carbons. The ratio ofZ9-14∶Ac toZ11-14∶Ac in individuals varied from 3.5∶1 to 11∶1 with an average of 6.2; their total added up to 462 ng/female with an average of 182 ng for 2- to 7-day-old individuals. No qualitative or quantitative differences were observed between laboratory and field insects.Z9-14∶Ac,Z11-14∶Ac and the corresponding alcohols were also found in female effluvia. Addition of either of the two alcohols to a blend of the two acetates augmented trap catch in the field. The same was true for (Z)-9,(E)-12-tetradecadienyl acetate which was not detected in gland extracts.

Tsetse flies are attracted to the invasive plant Lantana camara.

In tsetse both sexes feed exclusively on the blood of vertebrates for a few minutes every 2-3 days. Tsetse flies seek cover from high temperatures to conserve energy and plants provide shelter for tsetse in all the biotopes they occupy. Recently, tsetse have taken cover in plantations and under the invasive bush Lantana camara that has invaded large areas of the tsetse fly belt of Africa. Flies from such refugia are implicated in sleeping sickness epidemics. In a wind tunnel we show that both foliage and an extract of volatiles from foliage of L. camara attract three tsetse spp. from different habitats: Glossina fuscipes fuscipes (riverine), G. brevipalpis (sylvatic) and G. pallidipes (savannah). Gas chromatography analysis of volatiles extracted from leaves and flowers of L. camara coupled to electroantennograme recordings show that 1-octen-3-ol and beta-caryophyllene are the major chemostimuli for the antennal receptor cells of the three tsetse spp. studied. A binary mixture of these products attracted these flies in the wind tunnel. The gas chromatography linked electroantennograme analysis of the L. camara extracts also show that the antennal receptor cells of the three tsetse spp. respond similarly to groups of volatiles derived from the major biosynthetic and catabolic pathways of plants, i.e. to mono- and sesquiterpenes, to lipoxidation products and to aromatics. Mixtures of these plant volatiles also attracted tsetse in the wind tunnel. These findings show that tsetse flies have conserved a strong sensitivity to volatile secondary products of plants, underlining the fundamental role of vegetation in tsetse survival.

Contrasting effects of sera from rabbits and cattle infested with ticks on the in vitro feeding performance of the tick Rhipicephalus appendiculatus.

The in vitro feeding activity of partially engorged Rhipicephalus appendiculatus (Neumann) (Acari: Ixodidae) females fed on sera from uninfested hosts was compared to that of ticks fed serum of hosts which had previously been infested with ticks. Although ticks fed best on sera from bovid hosts which had no prior exposure to this ectoparasite, two infestations of a bovid had no significant effect on the acceptability of its serum. In contrast, ticks fed sera from rabbits which had twice been infested with ticks gained significantly less weight than those fed serum obtained from the same animals before the infestations. Clearly there is a difference between natural host resistance and that of laboratory animals.

Chemostimuli implicated in selection of oviposition substrates by the stable fly Stomoxys calcitrans.

Horse and cow dung were tested as substrates for oviposition by the stable fly Stomoxys calcitrans (L) (Diptera: Muscidae) in laboratory cages. Odour alone from either horse or cow dung was sufficient to attract flies for oviposition. This was confirmed in wind tunnel experiments, where both horse and cow dung were shown to attract gravid stable flies. However, when S. calcitrans was offered a choice between these two oviposition substrates, flies always chose horse dung over cow dung, both when allowed to contact the substrates and when relying on dung odour alone. Analyses of volatile compounds emanating from horse and cow dung by gas chromatography linked antennogram recordings from S. calcitrans antennae revealed no differences in the chemostimuli released from the two substrates. The predominant chemostimulant compounds in both substrates were carboxylic acids (butanoic acid), alcohols (oct-1-en-3-ol), aldehydes (decanal), ketones (octan-3-one), phenols (p-cresol), indoles (skatole), terpenes (beta-caryophyllene) and sulphides (dimethyl trisulphide). Higher levels (20-40 p.p.m.) of carbon dioxide were recorded over horse dung compared with cow dung, a factor that may contribute to the preference exhibited by S. calcitrans for this substrate for oviposition.

Sensory and behavioural responses of the stable fly Stomoxys calcitrans to rumen volatiles.

Analysis of volatiles from rumen digesta by gas chromatography linked antennogram recordings from Stomoxys calcitrans (L) (Diptera: Muscidae) antennal receptor cells revealed about 30 electrophysiologically active constituents, the most important of which is dimethyl trisulphide with a sensory threshold in the femtogram range. The behavioural responses of S. calcitrans to five chemostimulants (dimethyl trisulphide, butanoic acid, p-cresol, oct-1-en-3-ol and skatole) were tested in a wind tunnel where activation and attraction of hungry flies to rumen volatiles were recorded. Dimethyl trisulphide, butanoic acid and p-cresol were found to attract S. calcitrans. This sensitivity to rumen volatile constituents, that also occur in animal wastes used for oviposition by Stomoxys spp., as well as in flowers used by stable flies as sources of nectar is discussed in the context of the behavioural ecology of these flies.

Perception of breath components by the tropical bont tick, Amblyomma variegatum Fabricius (Ixodidae). II. Sulfide-receptors.

Wall-pore sensilla in the capsule of Haller's organ on foreleg tarsi of the tick, Amblyomma variegatum, show multicellular responses upon stimulation with human and bovine breath. Filtering breath through charcoal removes the stimulant for some of these receptors. Analysis by gas chromatography coupled with olfactory sensillum electrophysiological recordings indicates that an ethanol extract of the breath components trapped on charcoal contains a major stimulant eluting at the same retention time as H2S. Two types of H2S-sensitive receptors have been identified. They are housed in separate sensilla, and are called sulfide-receptor 1 and 2. Although, both receptor types are characterized by a high sensitivity to H2S with an estimated threshold of ca. 0.1 ppb and a response range covering 5-6 log orders of magnitude, their overall response to sulfides and mercaptans is nevertheless dissimilar. The type 1 receptor fires slightly more upon stimulations with H2S than type 2, whereas ethylmercaptan induces a stronger response from type 2, and dimethyl sulfide activates only receptor 2. In a bioassay, H2S tested at concentrations of ca. 0.02 ppm and 1 ppm equally arouses 60% of resting ticks. Two-thirds of these ticks quest the air with their first pair of legs, and the remainder start active search. By contrast, H2S at ca. 1 ppm in a mixture with CO2 severely diminishes the locomotor stimulating effect of CO2.

Perception of breath components by the tropical bont tick, Amblyomma variegatum Fabricius (Ixodidae). I. CO2-excited and CO2-inhibited receptors.

Wall-pore olfactory sensilla located in the capsule of Haller's organ on the tarsus of Amblyomma variegatum ticks bear cells responding to vertebrate breath: one of these sensilla contains a CO2-excited receptor and a second sensillum has a CO2-inhibited receptor. Each of these antagonistic CO2-receptors, which display typical phasic-tonic responses, monitors a different CO2-concentration range. The CO2-inhibited receptor is very sensitive to small concentration changes between 0 and ca. 0.2%, but variations of 0.01% around ambient (ca. 0.04%) induce the strongest frequency modulation of this receptor. An increase of just 0.001-0.002% (10-20 ppm) above a zero CO2-level already inhibits this receptor. By contrast, the CO2-excited receptor is not so sensitive to small CO2 shifts around ambient, but best monitors changes in CO2 concentrations above 0.1%. This receptor is characterized by a steep dose-response curve and a fast inactivation even at high CO2-concentrations (greater than 2%). In a wind-tunnel, Amblyomma variegatum is activated from the resting state and attracted by CO2 concentrations of 0.04 to ca. 1%, which corresponds to the sensitivity range of its CO2-receptors. The task of perceiving the whole concentration range to which this tick is attracted would thus appear to be divided between two receptors, one sensitive to small changes around ambient and the other sensitive to the higher concentrations normally encountered when approaching a vertebrate host.

Olfactory and behavioural responses of the blood-sucking bug Triatoma infestans to odours of vertebrate hosts.

Olfactory receptors in basiconic and grooved-peg sensilla on the antenna of fifth-instar Triatoma infestans nymphs respond to host odours. Gas chromatography analyses of host odour extracts coupled to electrophysiological recordings from basiconic sensillum receptors indicate that nonanal is a constituent of sheep wool and chicken feather odour that stimulates one of the receptors in this type of sensillum. Similar analyses revealed isobutyric acid in rabbit odour to be a chemostimulant for one of the receptors in grooved-peg sensilla. The response of the aldehyde receptor was higher to heptanal, octanal and nonanal than to other aliphatic aldehydes, and the response of the acid receptor was higher to isobutyric acid than to other short-chain branched and unbranched acids. The behavioural responses of fifth-instar T. infestans nymphs to nonanal and isobutyric acid in an air-stream on a servosphere indicate that, whereas nonanal causes activation of the bugs, isobutyric acid induces an increase in upwind displacement, i.e. odour-conditioned anemotaxis. Binary mixtures of these compounds did not improve the attraction obtained with isobutyric acid alone. A comparison of the behavioural and electrophysiological responses of the bugs to different amounts of isobutyric acid in air suggests that attraction is obtained at concentrations that causes low-to-moderate increases in the firing rate of the acid-excited receptor in the grooved-peg sensilla, whereas at a dose that evokes relatively high firing rates (>40 Hz) no attraction is obtained.

Identification of vertebrate volatiles stimulating olfactory receptors on tarsus I of the tick Amblyomma variegatum Fabricius (Ixodidae). I. Receptors within the Haller's organ capsule.

Gas chromatography-coupled electrophysiological recordings (GC-EL) from olfactory sensilla within the capsule of Haller's organ of the tick Amblyomma variegatum indicate the presence of a number of stimulants in rabbit and bovine odours, and in steer skin wash. Some of these stimulants were fully identified by gas chromatography-mass spectrometry analysis and by matching electrophysiological activity of synthetic analogues as: 1) hexanal, 2-heptenal, nonanal, furfural, benzaldehyde, and 2-hydroxybenzaldehyde (in all extracts); 2) heptanal, 2-, 3-, and 4-methylbenzaldehyde, and gamma-valerolactone (only in bovine and rabbit odour). Careful examination of the electrophysiological responses permit characterization of 6 receptor types: 1) a benzaldehyde receptor, 2) a 2-hydroxybenzaldehyde receptor, 3) three types of receptors responding differently to aliphatic aldehydes, and 4) a lactone receptor.

Responses of the tropical bont tick, Amblyomma variegatum (Fabricius), to its aggregation-attachment pheromone presented in an air stream on a servosphere.

Male Amblyomma variegatum ticks feeding on a host release a mixture of o-nitrophenol and methyl salicylate which serves to attract conspecifics. The behavioural responses of A. variegatum on a servosphere to these volatiles presented in an air stream are detailed here. In still air, ticks walked on all eight legs, but with long halts. In contrast, the air stream caused continuous walking and induced a reaching response where the forelegs actively sampled the air. Such reaching increased the angular velocity and reduced walking speed, effects that were amplified in the presence of vapours from o-nitrophenol and methyl salicylate in the air flowing over the ticks. Vapour from a 1:1 mixture of o-nitrophenol and methyl salicylate was attractive over a 10(4)-fold concentration range providing an increase in upwind displacement of 20-40%, significantly higher than the natural ratio where o-nitrophenol vapour predominates. Although the responses to o-nitrophenol vapour were variable when presented alone, this chemical was consistently attractive when delivered with steer hair odour unattractive on its own. Moreover, the upwind walk to this combination did not cause a change in speed or angular velocity. This supports the hypothesis that the response to the pheromone is enhanced by host odour.

Inadvertent introduction of squalene, cholesterol, and other skin products into a sample.

Recent developments in analytical techniques permit the chemical ecologist to achieve identification of naturally occurring compounds with relatively small amounts of the products of interest. However, the microanalytical techniques employed frequently require the handling of sample vials and other transferral instruments such as syringes and micropipets, where the analyst's hands come into close contact with the sample. Here we show how inadvertent contamination of a sample with skin lipids can occur simply by catching a 1-ml sample vial by the neck rather than the base or by activating a syringe by holding the plunger extension between the fingers rather than taking it by the head. Squalene, cholesterol, and, to a lesser extent, hydrocarbons and fatty acids from fingers are easily introduced into the sample in this manner. These findings are particularly relevant for a parasitology laboratory such as ours, investigating the function of vertebrate-derived products in hematophagous arthropods.

Attraction of the tropical bont tick, Amblyomma variegatum, to human breath and to the breath components acetone, NO and CO2.

Ticks are of medical and veterinary importance and employ several cues in search of a host. Olfaction is one modality by which ticks locate a blood-meal and breath is the major vent of gaseous and volatile metabolites from the host that could contribute to this search. We studied the responses of a hunter tick, Amblyomma variegatum, to diluted human breath and five of its components (acetone, CO2, NO, isoprene and NH3) while walking in an air stream on a locomotion compensator. Diluted breath elicited the greatest responses of all treatments in terms of time to onset of upwind walk, attraction, speed and local search behaviour after stimulus off. Acetone, NO and CO2 also attracted, but with a reduced speed in the case of acetone and NO. Neither isoprene nor NH3 induced any response. Our study indicates that breath was the most adequate stimulus tested. It also attracted two other ixodid tick species, Rhipicephalus sanguineus and Ixodes ricinus, as well as the argasid tick, Ornithodorus moubata. It appears that the evolution of resource tracking in ticks included sensory and behavioural adaptations for recognition and orientation to host metabolites that are regularly expelled in breath.

Ixodid ticks avoid contact with liquid water.

Larvae of the cattle tick Boophilus microplus and all life stages of the European sheep tick Ixodes ricinus avoid walking on a wet membrane surface surrounding a dry patch. Of 170 reactions made at a border with liquid water by 22 B. microplus larvae, 40% consisted of immediate turns to the opposite side to bring all the legs back onto a dry patch, 41% were walks along the border, during which the ticks maintained contact with both the dry and wet zones, and 19% were returns to the dry patch after a short excursion onto the wet surround. Since contact with one front leg tip was sufficient to cause return reactions from the wet surface in most of the border contacts, the water receptor(s) that enable ticks to perceive the wet surface are probably located in terminal pore sensilla on the first-leg tarsi. Observations on the return reactions of ticks with different groups of chemosensilla masked confirmed this. Ticks have an ambiguous relationship with water: they appear to avoid direct contact with it, but they need a high humidity to compensate for any deficit in body water.

Contact chemostimuli in the mating behaviour of the cattle tick, Boophilus microplus.

Mating of the cattle tick Boophilus microplus is mediated by chemical stimuli on the cuticle of females. Males are arrested on the dorsum of females attached to the host, frequently sample the substrate, and then tip-over to the ventrally located gonopore. These behaviours are also observed in vitro when males are placed on a small glass bead treated with a female extract. Time spent and tip-over by male ticks on dummies is used in an assay to test the behavioural significance of fractions of the extract. TLC separation yields one apolar fraction that arrests males, though much less so than the whole extract, but lost tip-over behaviour. This apolar fraction contains a series of cholesteryl esters that, when tested individually, show no arrestment activity at levels present in the extract but, when combined, are as active as the fraction. When a small silica column is used for fractionation, all biological activity is reproduced after recombining the fractions. In addition to the early eluting apolar fraction containing cholesteryl esters, a set of highly active more polar fractions is isolated. Electrophysiological recordings from gustatory sensilla on the pedipalps of male B. microplus, which are regularly brought into contact with the cuticle of the female during mating, provide evidence for receptors in two of them responding to the whole extract and to the behaviourally active polar fractions. Mating behaviour involving arrestment and tip-over is clearly initiated by a mixture of chemical stimuli, and tip-over behaviour is associated with the more polar material.

Antennal responses of the two host races of the larch bud moth, Zeiraphera diniana, to larch and cembran pine volatiles.

The larch bud moth (LBM) Zeiraphera diniana Guenée causes defoliation on larch in the Alps at 8- to 10-year intervals, after which populations crash. There are two LBM host races, one on larch and the other on cembran pine. These host races are morphologically indistinguishable as adults but they differ genetically in larval color types. Furthermore, females of each host race produce distinct pheromone blends and show oviposition preferences for their respective hosts. It is not clear to what extent host choice contributes to assortative mating in the LBM. Here, we compare the olfactory sensitivities of the two host races to the odors of fresh foliage of the host plants using the electroantennogram (EAG) technique, and the responses of the two host races to volatiles collected from the two host plants as analyzed by gas-chromatography-linked antennographic detection (GC-EAD). Both sexes of the larch and cembran host races show the same EAG responses to vapors of fresh larch and cembran pine foliage. Fifteen plant volatiles identified as chemostimuli by GC-EAD from larch and cembran pine odors elicited the same antennogram responses from the two host races. However, the GC-EAD analyses indicate that the number and quantity of chemostimuli emanating from each host plant is different. It is, therefore, most probably the array of olfactory receptors responding to the bouquet of volatiles unique to each host plant that underlies the host preferences of the two races. What remains open is the extent to which the similarity of the olfactory systems may contribute to cross-attraction. The fact that LBM individuals with intermediate characteristics between the two host races exist, suggests that olfactory perception does not hinder gene flow and contributes to sustained genetic diversity within the species Z. diniana.

In vitro assays for repellents and deterrents for ticks: differing effects of products when tested with attractant or arrestment stimuli.

Most in vivo and in vitro tests with repellents or deterrents against ticks have not considered which sensory channel is being targeted. We have recorded the responses of two hard tick species (Acari: Ixodidae) in vitro to determine if such products can disrupt the perception of an attractant in a repellent assay or the perception of an arrestment stimulus in a deterrent assay. Ethyl butylacetylaminopropionate (EBAAP), N,N-diethyl-methyl-benzamide (deet), permethrin and indalone were chosen to test their capacity to inhibit the attraction of Amblyomma variegatum Fabricius to its aggregation-attachment pheromone. Vapours of each test product plus those from a synthetic blend of the pheromone were delivered to the walking tick in an air stream on a locomotion compensator. Neither EBAAP, deet, permethrin nor indalone could inhibit attraction of A. variegatum even when each of the test products was delivered at 106 times the pheromone. Indalone did decrease the attraction of A. variegatum to the pheromone and induced repulsion of A. variegatum when presented on its own in the air stream. The effect of permethrin, a sodium channel blocker, was also tested in a deterrent assay measuring the arrestment of Ixodes ricinus (L.) adults on its own faeces and faecal constituents. Permethrin deterred arrestment at doses of 670 fg/cm2 to 67 ng/cm2, i.e. at levels five times lower than the dose of chemostimuli present in the arrestment stimulus. This sensitivity to permethrin suggests that it acts via the contact chemoreception channel.