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.

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.

Standardizing visual control devices for tsetse flies: east African Species Glossina fuscipes fuscipes and Glossina tachinoides.

BACKGROUND: Riverine species of tsetse are responsible for most human African trypanosomiasis (HAT) transmission and are also important vectors of animal trypanosomiasis. This study concerns the development of visual control devices for two such species, Glossina fuscipes fuscipes and Glossina tachinoides, at the eastern limits of their continental range. The goal was to determine the most long-lasting, practical and cost-effective visually attractive device that induces the strongest landing responses in these species for use as insecticide-impregnated tools in vector population suppression. METHODS AND FINDINGS: Field trials were conducted in different seasons on G. f. fuscipes in Kenya, Ethiopia and the Sudan and on G. tachinoides in Ethiopia to measure the performance of traps and 2D targets of different sizes and colours, with and without chemical baits, at different population densities and under different environmental conditions. Adhesive film was used to enumerate flies at these remote locations to compare trapping efficiencies. The findings show that targets made from black and blue fabrics (either phthalogen or turquoise) covered with adhesive film render them equal to or more efficient than traps at capturing G. f. fuscipes and G. tachinoides. Biconical trap efficiency varied between 25% and 33% for the two species. Smaller 0.25 m×0.25 m phthalogen blue-black targets proved more efficient than the regular 1 m2 target for both species, by over six times for Glossina f. fuscipes and two times for G. tachinoides based on catches per m2. Overall, targets with a higher edge/surface area ratio were more efficient at capturing flies. CONCLUSIONS/SIGNIFICANCE: Taking into account practical considerations and fly preferences for edges and colours, we propose a 0.5×0.75 m blue-black target as a simple cost-effective device for management of G. f. fuscipes and G. tachinoides, impregnated with insecticide for control and covered with adhesive film for population sampling.

Standardising visual control devices for tsetse flies: Central and West African species Glossina palpalis palpalis.

BACKGROUND: Glossina palpalis palpalis (G. p. palpalis) is one of the principal vectors of sleeping sickness and nagana in Africa with a geographical range stretching from Liberia in West Africa to Angola in Central Africa. It inhabits tropical rain forest but has also adapted to urban settlements. We set out to standardize a long-lasting, practical and cost-effective visually attractive device that would induce the strongest landing response by G. p. palpalis for future use as an insecticide-impregnated tool in area-wide population suppression of this fly across its range. METHODOLOGY/PRINCIPAL FINDINGS: Trials were conducted in wet and dry seasons in the Ivory Coast, Cameroon, the Democratic Republic of Congo and Angola to measure the performance of traps (biconical, monoconical and pyramidal) and targets of different sizes and colours, with and without chemical baits, at different population densities and under different environmental conditions. Adhesive film was used as a practical enumerator at these remote locations to compare landing efficiencies of devices. Independent of season and country, both phthalogen blue-black and blue-black-blue 1 m(2) targets covered with adhesive film proved to be as good as traps in phthalogen blue or turquoise blue for capturing G. p. palpalis. Trap efficiency varied (8-51%). There was no difference between the performance of blue-black and blue-black-blue 1 m(2) targets. Baiting with chemicals augmented the overall performance of targets relative to traps. Landings on smaller phthalogen blue-black 0.25 m(2) square targets were not significantly different from either 1 m(2) blue-black-blue or blue-black square targets. Three times more flies were captured per unit area on the smaller device. CONCLUSIONS/SIGNIFICANCE: Blue-black 0.25 m(2) cloth targets show promise as simple cost effective devices for management of G. p. palpalis as they can be used for both control when impregnated with insecticide and for population sampling when covered with adhesive film.

Standardizing visual control devices for tsetse flies: East African species Glossina swynnertoni.

BACKGROUND: Here we set out to standardize long-lasting, visually-attractive devices for Glossina swynnertoni, a vector of both human and animal trypanosomiasis in open savannah in Tanzania and Kenya, and in neighbouring conservation areas used by pastoralists. The goal was to determine the most practical device/material that would induce the strongest landing response in G. swynnertoni for use in area-wide population suppression of this fly with insecticide-impregnated devices. METHODS AND FINDINGS: Trials were conducted in wet and dry seasons in the Serengeti and Maasai Mara to measure the performance of traps and targets of different sizes and colours, with and without chemical baits, at different population densities and under different environmental conditions. Adhesive film was used as a simple enumerator at these remote locations to compare trapping efficiencies of devices. Independent of season or presence of chemical baits, targets in phthalogen blue or turquoise blue cloth with adhesive film were the best devices for capturing G. swynnertoni in all situations, catching up to 19 times more flies than pyramidal traps. Baiting with chemicals did not affect the relative performance of devices. Fly landings were two times higher on 1 m(2) blue-black targets as on pyramidal traps when equivalent areas of both were covered with adhesive film. Landings on 1 m(2) blue-black targets were compared to those on smaller phthalogen blue 0.5 m(2) all-blue or blue-black-blue cloth targets, and to landings on all-blue plastic 0.32-0.47 m(2) leg panels painted in phthalogen blue. These smaller targets and leg panels captured equivalent numbers of G. swynnertoni per unit area as bigger targets. CONCLUSIONS: Leg panels and 0.5 m(2) cloth targets show promise as cost effective devices for management of G. swynnertoni as they can be used for both control (insecticide-impregnated cloth) and for sampling (rigid plastic with insect glue or adhesive film) of populations.

Standardizing visual control devices for tsetse flies: West African species Glossina tachinoides, G. palpalis gambiensis and G. morsitans submorsitans.

Here we describe field trials designed to standardize tools for the control of Glossina tachinoides, G. palpalis gambiensis and G.morsitans submorsitans in West Africa based on existing trap/target/bait technology. Blue and black biconical and monoconical traps and 1 m(2) targets were made in either phthalogen blue cotton, phthalogen blue cotton/polyester or turquoise blue polyester/viscose (all with a peak reflectance between 450-480 nm) and a black polyester. Because targets were covered in adhesive film, they proved to be significantly better trapping devices than either of the two trap types for all three species (up to 14 times more for G. tachinoides, 10 times more for G. palpalis gambiensis, and 6.5 times for G. morsitans submorsitans). The relative performance of the devices in the three blue cloths tested was the same when unbaited or baited with a mixture of phenols, 1-octen-3-ol and acetone. Since insecticide-impregnated devices act via contact with flies, we enumerated which device (traps or targets) served as the best object for flies to land on by also covering the cloth parts of traps with adhesive film. Despite the fact that the biconical trap proved to be the best landing device for the three species, the difference over the target (20-30%) was not significant. This experiment also allowed an estimation of trap efficiency, i.e. the proportion of flies landing on a trap that are caught in its cage. A low overall efficiency of the biconical or monoconical traps of between 11-24% was recorded for all three species. These results show that targets can be used as practical devices for population suppression of the three species studied. Biconical traps can be used for population monitoring, but a correction factor of 5-10 fold needs to be applied to captures to compensate for the poor trapping efficiency of this device for the three species.

Antennal expression pattern of two olfactory receptors and an odorant binding protein implicated in host odor detection by the malaria vector Anopheles gambiae.

Odor-detection in the malaria mosquito Anopheles gambiae involves large families of diverse proteins, including multiple odorant binding proteins (AgOBPs) and olfactory receptors (AgORs). The receptors AgOR1 and AgOR2, as well as the binding protein AgOBP1, have been implicated in the recognition of human host odors. In this study, we have explored the expression of these olfactory proteins, as well as the ubiquitous odorant receptor heteromerization partner AgOR7, in the thirteen flagellomeres (segments) of female and male antenna. Expressing cells were visualized by adapting a whole mount fluorescence in situ hybridization method. In female mosquitoes, AgOR1-expressing olfactory receptor neurons (ORNs) were almost exclusively segregated in segments 3 to 9, whereas AgOR2-expressing ORNs were distributed over flagellomeres 2 to 13. Different individuals comprised a similar number of cells expressing a distinct AgOR type, although their antennal topography and number per flagellomere varied. AgOBP1-expressing support cells were present in segments 3 to 13 of the female antenna, with increasing numbers towards the distal end. In male mosquitoes, total numbers of AgOR- and AgOBP1-expressing cells were much lower. While AgOR2-expressing cells were found on both terminal flagellomeres, AgOR1 cells were restricted to the most distal segment. High densities of AgOBP1-expressing cells were identified in segment 13, whereas segment 12 comprised very few. Altogether, the results demonstrate that both sexes express the two olfactory receptor types as well as the binding protein AgOBP1 but there is a significant sexual dimorphism concerning the number and distribution of these cells. This may suggest gender-specific differences in the ability to detect distinct odorants, specifically human host-derived volatiles.

The Anopheles gambiae odorant binding protein 1 (AgamOBP1) mediates indole recognition in the antennae of female mosquitoes.

Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP-ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.

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.

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.

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.

The tick blood meal: from a living animal or from a silicone membrane?

An artificial feeding unit with a reinforced silicone membrane to replace host skin provides ticks with a perch over blood with a tick attachment rate of 75-100%. Some 5 mg of an acaricide like fipronil is sufficient to establish survival curves over different doses down to ppb levels in blood. This in vitro feeding assay for hard ticks is more advantageous than in vivo trials on animals.