Insecticidal and repellent properties of novel trifluoromethylphenyl amides II.

This project focused on the design, synthesis, and testing of trifluoromethylphenyl amides (TFMPAs) as potential mosquitocides and repellents. Fourteen compounds were evaluated for toxicity against larvae and adults of Aedes aegypti. Several compounds were toxic against Aedes aegypti (larval, adult and feeding bioassays) and Drosophila melanogaster (glass-surface contact assay), but were much less toxic than fipronil, with toxicity ratios ranging from 100-fold in the larval assay to 100,000-fold for topical application to adult insects. In repellency bioassays to determine minimum effective dosage (MED), compound N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide (7b) repelled Ae. aegypti females at lower concentration, 0.017 (±0.006) µmol/cm2, than N, N-diethyl-meta-toluamide (DEET) 0.026 (±0.005) µmol/cm2. 2-Chloro-N-(3-(trifluoromethyl)phenyl)acetamide (6a) performed better than DEET against two species of mosquitoes: it repelled Ae. aegypti females at 0.013 (±0.006) µmol/cm2 and Anopheles gambiae females (in a warm body repellent assay), at a standard exposure of 2 nmol/cm2. These studies revealed novel active structures that could further lead to compounds with better repellent activity.

Behavioral response of the malaria mosquito, Anopheles gambiae, to human sweat inoculated with axilla bacteria and to volatiles composing human axillary odor.

The responses of Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) to odors from male and female axillary sweat incubated with human axilla bacteria were recorded in a dual-choice olfactometer. Staphylococcus epidermidis was selected for its low odor-producing pattern, Corynebacterium jeikeium for its strong Nα-acylglutamine aminoacylase activity liberating carboxylic acids including (R)/(S)-3-hydroxy-3-methylhexanoic acid (HMHA) and Staphylococcus haemolyticus for its capacity to liberate sulfur-containing compounds including (R/S)-3-methyl-3-sulfanylhexan-1-ol (MSH). Anopheles gambiae behavioral responses were evaluated under (i) its responsiveness to take off and undertake sustained upwind flight and (ii) its discriminating capacity between the two olfactometer arms bearing a test odor in either one or both arms. Experiments were conducted in the presence of carbon dioxide pulses as a behavioral sensitizer. Anopheles gambiae clearly discriminated for the olfactometer arm conveying odor generated by incubating any of the three bacteria species with either male or female sweat. Whereas An. gambiae did not discriminate between male and female sterile sweat samples in the olfactometer, the mosquito consistently showed a preference for male sweat over female sweat incubated with the same bacterium, independent of the species used as inoculum. Sweat incubated with C. jeikeium rendered mosquitoes particularly responsive and this substrate elicited the strongest preference for male over female sweat. Tested on their own, neither HMHA nor MSH elicited a clear discriminating response but did affect mosquito responsiveness. These findings serve as a basis for further research on the odor-mediated anthropophilic host-seeking behavior of An. gambiae.

Sugar-sensitive neurone responses and sugar feeding preferences influence lifespan and biting behaviours of the Afrotropical malaria mosquito, Anopheles gambiae.

Floral nectar is the main source of carbohydrates for many insects including mosquitoes. Nonetheless, the physiological mechanisms underlying feeding on carbohydrates by the Afrotropical malaria mosquito Anopheles gambiae remain poorly understood. Here, we tested whether sugar sensitivity and sugar feeding preferences correlate with longevity in A. gambiae. We also tested whether feeding females on different sugar diets influences their biting behaviours. Electrophysiological recordings show that sugar neurones on the labella of females are most sensitive to sucrose, mixtures of glucose and fructose, and to melezitose; other sugars tested, including glucose and fructose presented alone, only weakly activate these taste neurones. Mosquitoes survive longest on sucrose, the most preferred sugar. Whereas feeding on a mixture of glucose and fructose is preferred over fructose or glucose alone, fructose supports higher longevity than either glucose or the mixture of the two hexoses. Females that had previously fed on glucose show a stronger biting response than those fed on sucrose, perhaps in an effort to compensate for their lower energetic state. These findings contribute to our understanding of the physiological basis of sugar feeding in A. gambiae and indicate how the sugar diet can affect laboratory-reared A. gambiae biting behaviours.

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.

Plant volatiles enhance behavioral responses of grapevine moth males, Lobesia botrana to sex pheromone.

Plant volatiles play an important role in the lives of phytophagous insects, by guiding them to oviposition, feeding and mating sites. We tested the effects of different host-plant volatiles on attraction of Lobesia botrana males to the female-produced sex pheromone, in a wind tunnel. Addition of volatile emissions from grapevines or individual plant volatiles to pheromone increased the behavioral responses of L. botrana males over those to pheromone alone. At a low release rate (under-dosed) of pheromone, addition of (E)-ß-caryophyllene, (Z)-3-hexenyl acetate, 1-hexanol, or 1-octen-3-ol increased all behavioral responses, from activation to pheromone source contact, while addition of (E)-4,8-dimethyl-1,3,7-nonatriene, (E)-ß-farnesene, (Z)-3-hexenol, or methyl salicylate affected only the initial behavioral responses. Dose-response experiments suggested an optimal release ratio of 1:1000 (sex pheromone: host plant volatile). Our results highlight the role of plant volatiles in the sensory ecology of L. botrana.

Small cages with insect couples provide a simple method for a preliminary assessment of mating disruption.

Mating disruption by sex pheromones is a sustainable, effective and widely used pest management scheme. A drawback of this technique is its challenging assessment of effectiveness in the field (e.g., spatial scale, pest density). The aim of this work was to facilitate the evaluation of field-deployed pheromone dispensers. We tested the suitability of small insect field cages for a pre-evaluation of the impact of sex pheromones on mating using the grape moths Eupoecilia ambiguella and Lobesia botrana, two major pests in vineyards. Cages consisted of a cubic metal frame of 35 cm sides, which was covered with a mosquito net of 1500 µm mesh size. Cages were installed in the centre of pheromone-treated and untreated vineyards. In several trials, 1 to 20 couples of grape moths per cage were released for one to three nights. The proportion of mated females was between 15 to 70% lower in pheromone-treated compared to untreated vineyards. Overall, the exposure of eight couples for one night was adequate for comparing different control schemes. Small cages may therefore provide a fast and cheap method to compare the effectiveness of pheromone dispensers under standardised semi-field conditions and may help predict the value of setting-up large-scale field trials.

An in vitro assay for testing mosquito repellents employing a warm body and carbon dioxide as a behavioral activator.

We describe here an in vitro behavioral assay for testing mosquito repellents applied in a dose-based manner to a warm body (34 degrees C) in test cages. The system was used to assess the sensitivity of 4-6-day-old Anopheles gambiae to the insect repellent diethyl methyl benzamide (deet). These tests were made in the absence and presence of additional carbon dioxide (CO2) applied as a pulse to activate mosquitoes in the cages. In the absence of the CO2 pulse the mosquitoes hardly responded to the warm body. Increasing the CO2 level in the cage by 1,000 parts per million caused a 25-fold increase in the number of landings by mosquitoes on the warm body in 2-min tests. This mosquito activation allowed the measurement of a significant reduction in the number of landings to bite on the warm body with increasing doses of deet (0.4 to 3.8 microg/cm2). An asymptotic nonlinear model fitted to the repellency data in the presence of CO2 allowed estimation of the effective dose of deet that reduced landings to bite by 50% (ED50) at 0.95 microg/cm2 (5 nmol/cm2) and the corresponding ED95 at 4.12 microg/cm2 (21.5 nmol/cm2). This in vitro bioassay has the advantage of permitting a fast throughput of test products under standardized conditions and is suitable for screenings designed for the purpose of discovering lead products with as yet unknown human toxicological and dermatological profiles.

Fabrication of an olfactometer for mosquito behavioural studies.

BACKGROUND & OBJECTIVES: Olfaction is the major sensory modality involved in the resource searching behaviour of insects including vector mosquitoes (Diptera: Culicidae). To date, our current countrywide knowledge on the host-seeking behaviour of Iranian mosquitoes is mainly confined to host preference which has exclusively come from field studies. Olfactometer is a scientific tool by which more naive aspects of man-vector contact can be clarified under controlled and less biased conditions. METHODS: The wind tunnel and stimulus delivery system was constructed from acrylic materials based on previously introduced models with some modifications. Air supply and required light were ensured by a powerful compressor and incandescent bulbs, respectively. Desired level of temperature was maintained by controllable heating radiators. For humidity production a unique in-built piezo system was devised in the course of the air flow. Fine regulators facilitated the continuous generatation of the humidity at a preset level. RESULTS: Titanium tetrachloride smoke plus monitoring of the wind speed revealed that the flow of air was proper and invariable. A desired level of humidity and temperature could be set up in just 10 and 15-45 min, respectively. These physical parameters varied only +/-2% (humidity) and +/-0.15 degrees C (temperature) in a typical 20 min duration. CONCLUSION: The first sophisticated olfactometer in the field of medical entomology in Iran is reported here. Fast set up and stability of physical parameters are its salient features. It is expected that with the aid of this olfactometer further information on the physiological principles of the host-seeking behaviour of mosquitoes become available soon.

Deltamethrin Persistence on Textiles Used to Make Visual Targets for Tsetse Fly Control is Positively Correlated With Their Polyester Content.

Insecticide-impregnated traps and visual targets are used for tsetse (Diptera, Glossinidae) population control. Such devices are made with textiles and deltamethrin is frequently the insecticide of choice. However, persistence of an insecticide on textiles is affected by exposure to weather. Here we examine the effect of weathering on the capacity of four textiles with increasing proportions of polyester (0, 35, 65, and 100%) with cotton and viscose to retain deltamethrin. Textiles tested were those used to make visual targets in a pan-African program to maximize target efficiency for controlling tsetse vectors of African trypanosomiasis. Following impregnation in an aqueous suspension of deltamethrin at 1,000 mg/m2, textiles were weathered for 18 mo at Lambwe Valley, Kenya and sampled every 3 mo to make knockdown tests on the tsetse fly Glossina pallidipes Austen. Deltamethrin content of the textiles was established using gas chromatography mass-spectrometry at impregnation and after 9 mo of weathering. Textiles with higher proportions of polyester retained deltamethrin better: respectively, 100% polyester and 65:35 polyester/viscose textiles retained deltamethrin at 17 and 11 mg/m2 9-mo post-treatment that caused 100% knockdown in G. pallidipes after 1 h, and killed 67 and 47% of flies, respectively, after 24 h. Eighteen-month weathered 100% polyester treated textile still knocked down all tsetse exposed to it within 2 h. The LD50 for deltamethrin on filter paper for G. pallidipes was estimated at 28.8 mg/m2, indicating that deltamethrin is more available on polyester to kill tsetse.

Spectral sensitivity of L2 biotype in the Thrips tabaci cryptic species complex.

The onion thrips, Thrips tabaci (Lindeman, 1889), is a cosmopolitan pest of economic importance on a wide range of crops. Despite being one of the most studied thrips species, there is very limited knowledge available about its ability to perceive light. The T. tabaci cryptic species complex consists of a tobacco-associated (T) and two leek-associated (L1, L2) biotypes. We made electroretinogram recordings on the most widespread thelytokous (where unfertilized eggs produce females) T. tabaci L2 biotype and measured attraction to light sources in this biotype as a function of wavelength in behavioural experiments. The spectral sensitivity of the T. tabaci L2 biotype shows a unimodal curve peaking at λmax = 521 nm. Contrary to this spectral sensitivity curve, L2 biotype attraction in an arena is bimodal with local maxima at 368 nm (UV) and 506-520 nm (green) being practically of the same magnitude. Although being similar to the arrhenotokous (where unfertilized eggs produce males) L1 biotype in phototaxis, significant differences regarding photoreceptor cell responses emerged. This study contributes to our understanding of light perception in Thysanoptera as well as to the development of more effective monitoring tools for this economically important pest species.

Host plant volatiles serve to increase the response of male European grape berry moths, Eupoecilia ambiguella, to their sex pheromone.

The European grape berry moth is an important pest in vineyards. Males respond to the female-produced sex pheromone released from a piezo nebulizer in a dose-dependent manner in a wind tunnel: <50% arrive at the source at 5-50 pg/min (underdosed), 80% arrive at 100 pg/min to 10 ng/min (optimal) and <20% arrive at 100 ng/min (overdosed). Males responding to overdosed pheromone show in flight arrestment at 80 cm from the source. Host plant chemostimuli for Eupoecilia ambiguella increase the responses of males to underdosed and overdosed pheromone. (Z)-3-hexen-1-ol, (+)-terpinen-4-ol, (E)-beta-caryophyllene and methyl salicylate released with the underdosed pheromone cause a significant increase in male E. ambiguella flying to the source. Time-event analysis indicates a positive correlation between faster activation and probability of source contact by the responding males. The four host plant compounds added to the overdosed pheromone permitted males to take off faster and with a higher probability of flying to the source. This suggests that perception of host plant products with the sex pheromone facilitates male E. ambiguella to locate females on host plants, lending credence to the hypothesis that plant products can signal rendezvous sites suitable for mating.

Antiectoparasitic activity of the gum resin, gum haggar, from the East African plant, Commiphora holtziana.

The mechanism of ixodid tick (Acari: Ixodidae) repellency by gum haggar, a resin produced by Commiphora holtziana (Burseraceae), was investigated by evaluating activity against the cattle tick, Boophilus microplus. In an arena bioassay, a hexane extract of the resin of C. holtziana exhibited a repellent effect lasting up to 5h. The hydrocarbon fraction of the resin extract was shown to account for the repellent activity, and was analysed by coupled gas chromatography-mass spectrometry (GC-MS). Major sesquiterpene hydrocarbons were tentatively identified as germacrene-D, delta-elemene and beta-bourbonene. The identity and stereochemistry of the former compound was confirmed as the (+)-isomer by peak enhancement using enantioselective GC, whereas the latter 2 compounds, which are most likely degradation products of germacrene-type precursors, were identified through isolation by preparative gas chromatography followed by microprobe-NMR spectroscopy. GC comparison of gum haggar with another resin, C. myrrha, which was inactive in the tick bioassay, showed that the latter contained much lower levels of these hydrocarbons. To assess the suitability of the gum haggar resin as a general acarine repellent, further tests were made on a major acarine pest of European and US animal husbandry systems, the red poultry mite, Dermanyssus gallinae (Acari: Dermanyssidae). Gum haggar extract, and the isolated hydrocarbon fraction, showed strong repellent effects in an olfactometer assay, and again gum myrrh showed no effect. These findings provide a scientific basis for the observed anti-tick properties of gum haggar, and demonstrate the potential for its development as a general acarine repellent for use in animal husbandry systems.

Nutrient content of diet affects the signaling activity of the insulin/target of rapamycin/p70 S6 kinase pathway in the African malaria mosquito Anopheles gambiae.

Regulation of female mosquito feeding and reproduction plays a central role in their disease-vector competence. In this study we show that Anopheles gambiae mosquitoes engorged on albumin, amino acid and saline meals the same way as on blood, whereas sucrose evoked a typical plant nectar feeding response. Among the artificial diets, only the albumin-containing ones allowed follicular development. The target of rapamycin (TOR)/p70 S6 kinase (S6K) pathway has been identified as an essential nutrient-sensing tool controlling egg development in mosquitoes under the control of regulating inputs from the insulin pathway. We assayed the early response of TOR, S6K, tuberous sclerosis (TSC2), insulin receptor (INR) and two insulin-like peptides (ILPs) by quantitative real-time PCR assessment of mRNA levels and immunoblotting of phosphorylated active TOR and S6K in An. gambiae ovary and brain 3 h after engorgement. We show that transcript levels of s6k and members of the insulin pathway are readily affected by nutrients (especially one ILP in the head) and that the TOR/S6K phosphorylation is able to react quickly to a meal to an extent which depends on the true nutritive value.

Responses of Anopheles gambiae, Anopheles stephensi, Aedes aegypti, and Culex pipiens mosquitoes (Diptera: Culicidae) to cool and humid refugium conditions.

Like all terrestrial arthropods, mosquitoes must cope with the threat of desiccation. To gain insight into their survival strategies, we recorded the behavioral responses of Anopheles gambiae, Anopheles stephensi, Aedes aegypti, and Culex pipiens offered zones of different microclimatic conditions in laboratory cages. The cooled refugium was at 25.6 degrees C, 86% RH and the control was at 28.5 degrees C, 75% RH, i.e., a difference in saturation deficit of 3.9 mm Hg between the two zones. We show that newly-emerged adults, with no access to water or sugar, prefer the cooler and more humid refugium with a saturation deficit half that in the control and where the mosquitoes could reduce their metabolic rate. This response is delayed in Ae. aegypti, perhaps because the energy reserves accumulated as larvae are higher in this species. This study shows that mosquitoes under stress can use their thermohygroreceptor cells to guide them to locations that facilitate survival.

Vision and genesis of survival strategies in tsetse flies: A laboratory study.

Organisms respond to environmental stimuli in ways that optimize survival and reproduction. Tsetse fly life-history is characterized by high investment in progeny by the pregnant female and low birth rate. This places constraints on tsetse populations across the sub-Saharan biotopes they colonize where extreme climatic conditions militate against survival. Controlling metabolic rate is crucial in biotopes where daily swings in temperature can exceed 20 °C. Tsetse acquire their nutrient requirements from the blood meal. These diurnal flies are otherwise confined for most of their lives to perching sites in the shade. At these locations they are simultaneously threatened by vertebrate and invertebrate predators. Here we describe behaviours of the East African tsetse Glossina pallidipes Austen (Diptera: Glossinidae) that permit it to reduce risk daily. Newly-emerged flies remain immobile at emergence in the photophase but scotophase-emerging flies walk away within seconds to climb (negative geotaxis) vertical substrates to find a perch off the ground. Flies of all ages show the ability to fly in almost total darkness (1.10-5 lux) in the scotophase to perch on the upper side of horizontally suspended 1 cm diameter bars, simulating branches of vegetation, but perch under the same bars during the photophase. This underlines the predilection of tsetse for objects with a linear aspect that provide a vantage point and shade. Mature G. pallidipes can discriminate between horizontally suspended bars of different diameter and shape. Flicker fusion frequency values established by optomotor and retinogram recordings reveal a higher visual acuity in mature compared to newly-emerged tsetse.

Standardising visual control devices for Tsetse: East and Central African Savannah species Glossina swynnertoni, Glossina morsitans centralis and Glossina pallidipes.

BACKGROUND: This study focused on the savannah tsetse species Glossina swynnertoni and G. morsitans centralis, both efficient vectors of human and animal trypanosomiasis in, respectively, East and Central Africa. The aim was to develop long-lasting, practical and cost-effective visually attractive devices that induce the strongest landing responses in these two species for use as insecticide-impregnated tools in population suppression. METHODS AND FINDINGS: Trials were conducted in different seasons and years in Tanzania (G. swynnertoni) and in Angola and the Democratic Republic of the Congo (DRC, G. m. centralis) to measure the performance of traps (pyramidal and epsilon) and targets of different sizes, shapes and colours, with and without chemical baits, at different population densities and under different environmental conditions. Adhesive film was used to catch flies landing on devices at the remote locations to compare tsetse-landing efficiencies. Landing rates by G. m. centralis in both Angola and the DRC were highest on blue-black 1 m2 oblong and 0.5 m2 square and oblong targets but were not significantly different from landings on the pyramidal trap. Landings by G. swynnertoni on 0.5 m2 blue-black oblong targets were likewise not significantly lower than on equivalent 1 m2 square targets. The length of target horizontal edge was closely correlated with landing rate. Blue-black 0.5 m2 targets performed better than equivalents in all-blue for both G. swynnertoni and G. m. centralis, although not consistently. Baiting with chemicals increased the proportion of G. m. centralis entering pyramidal traps. CONCLUSIONS: This study confirms earlier findings on G. swynnertoni that smaller visual targets, down to 0.5 m2, would be as efficient as using 1 m2 targets for population management of this species. This is also the case for G. m. centralis. An insecticide-impregnated pyramidal trap would also constitute an effective control device for G. m. centralis.

Identification of novel bioinspired synthetic mosquito repellents by combined ligand-based screening and OBP-structure-based molecular docking.

In this work we report a fast and efficient virtual screening protocol for discovery of novel bioinspired synthetic mosquito repellents with lower volatility and, in all likelihood, increased protection time as compared with their plant-derived parental compounds. Our screening protocol comprises two filtering steps. The first filter is based on the shape and chemical similarity to known plant-derived repellents, whereas the second filter is based on the predicted similarity of the ligand's binding mode to the Anopheles gambiae odorant binding protein (AgamOBP1) relative to that of DEET and Icaridin to the same OBP. Using this protocol, a chemical library containing 42,755 synthetic molecules was screened in silico and sixteen selected compounds were tested for their affinity to AgamOBP1 in vitro and repellence against A. gambiae female mosquitoes using a warm-body repellent assay. One of them showed DEET-like repellence (91%) but with significantly lower volatility (2.84 × 10-6 mmHg) than either DEET (1.35 × 10-3 mmHg) or its parental cuminic acid (3.08 × 10-3 mmHg), and four other compounds were found to exhibit repellent indices between 69 and 79%. Overall, a correlation was not evident between repellence and OBP-binding strength. In contrast, a correlation between binding mode and repellence was found.

Odorant-binding protein-based identification of natural spatial repellents for the African malaria mosquito Anopheles gambiae.

There is increasing interest in the development of effective mosquito repellents of natural origin to reduce transmission of diseases such as malaria and yellow fever. To achieve this we have employed an in vitro competition assay involving odorant-binding proteins (OBPs) of the malaria mosquito, Anopheles gambiae, with a predominantly female expression bias to identify plant essential oils (EOs) containing bioactive compounds that target mosquito olfactory function. EOs and their fractions capable of binding to such OBPs displayed repellence against female mosquitoes in a laboratory repellent assay. Repellent EOs were subjected to gas chromatographic analysis linked to antennogram (EAG) recordings from female A. gambiae to identify the biologically active constituents. Among these compounds cumin alcohol, carvacrol, ethyl cinnamate and butyl cinnamate proved as effective as DEET at an equivalent dose in the repellent assay, and combinations of carvacrol with either butyl cinnamate or cumin alcohol proved to be significantly more effective than DEET in the assay. When tested as spatial repellents in experimental shelters housing sleeping humans in northern Nigeria a binary mixture of carvacrol plus cumin alcohol caused mosquitoes to leave shelters in significantly higher numbers to those induced by DEET in female Anopheles spp. and in numbers equivalent to that of DEET in Culex spp. mosquitoes. These findings indicate an approach for the identification of biologically active molecules of natural origin serving as repellents for mosquitoes.

In vitro feeding assays for hard ticks.

Prevention of tick bites and transmission of tick-borne pathogens requires the use of molecules that target physiological processes crucial to both tick and pathogen survival. These molecules are best tested in standardized in vitro assays. Because hard ticks require several days to feed to repletion, the development of in vitro feeding assays for these species is challenging. A standard and easily automated feeding assay has been developed for the tick Ixodes ricinus that involves feeding on blood through a membrane that mimics the elasticity of skin. The system can be adapted to feed other hard tick species in vitro. This assay permits, among others, investigations on the role of tick endosymbionts on tick survival, the identification of potential vaccine candidates and drugs, and the application of genomic tools in vitro, including RNA interference experiments.

An in vitro feeding assay to test acaricides for control of hard ticks.

Animal husbandry could not be practised over large areas of the planet without acaricides. The prevention of tick bite and the transmission of diseases requires the use of pesticides, but this contributes to the development of tick resistance against acaricides. This drives the quest for new molecules that target physiological processes crucial to tick survival. In vivo trials involve multiple repetitions because of inherent variations between host animals, requiring large amounts of test products and ticks. An in vitro alternative should permit the testing of the ability of a product to restrict attachment and feeding by ticks at precise doses. In this paper an in vitro feeding system is described where the European tick Ixodes ricinus L. feeds on blood through a cellulose rayon-reinforced silicone membrane. The membrane Shore hardness is modified to imitate the elastic retraction forces of skin that ensure the closing of tick penetration sites on the membrane to prevent bleeding. Tick attachment (75-100%) is achieved by adding chemical and mechanical stimuli to the membrane. Survival curves for different doses of fipronil and ivermectin tested with the method showed highly reproducible acaricide effects within 5-7 days. Significant effects are recorded down to ppb levels in blood. Standardised tests can be made with blood from the same donor animal or culture medium under the membrane.