Synergism between ammonia, lactic acid and carboxylic acids as kairomones in the host-seeking behaviour of the malaria mosquito Anopheles gambiae sensu stricto (Diptera: Culicidae)

Host odours play a major role in the orientation and host location of blood-feeding mosquitoes. Anopheles gambiae Giles sensu stricto, which is the most important malaria vector in Africa, is a highly anthropophilic mosquito species, and the host-seeking behaviour of the females of this mosquito is guided by volatiles of human origin. Ammonia, lactic acid and several carboxylic acids are known to be present in the human odour blend. We investigated the effect of these compounds on naive female mosquitoes using a dual-port olfactometer. Ammonia was an attractant on its own, whereas lactic acid was not attractive. Carboxylic acids, offered as a mixture of 12 compounds, were repellent at the concentration tested. The addition of ammonia to the carboxylic acid mixture overruled the repellent effect of the latter. Combining ammonia with either lactic acid or the carboxylic acids did not enhance the attractiveness of ammonia alone. However, a synergistic effect was found when ammonia, lactic acid and the carboxylic acids were applied as a blend. Our findings indicate that An. gambiae s.s. relies on the combination of ammonia, lactic acid and carboxylic acids in its orientation to human hosts. The role of lactic acid in this tripartite synergism differs from that reported for the yellow fever mosquito Aedes aegypti.     

Behavioural responses of Anopheles gambiae sensu stricto to components of human breath, sweat and urine depend on mixture composition and concentration

Host-seeking behaviour of the anthropophilic malaria vector Anopheles gambiae sensu stricto (Diptera: Culicidae) is mediated predominantly by olfactory cues. Several hundreds of odour components have been identified from human emanations, but only a few have been proven to act as attractants or synergists in the host-seeking behaviour of female An. gambiae. In previous work, aromatics, alcohols and ketones in human odours were found to elicit electrophysiological activity in antennal olfactory neurons of female An. gambiae. However, the behavioural effects of these compounds have not been investigated. In this study, behavioural responses of female An. gambiae to components of human breath, urine and sweat at a series of concentrations, or a single concentration in the case of acetone, were examined in combination with ammonia and L-lactic acid in a dual-choice olfactometer. The results showed that at specific concentrations 4-ethylphenol, indole, 3-methyl-1-butanol and two ketones inhibited the attractive effect of a mixture of ammonia and lactic acid. Acetone on its own was not attractive; however, when combined with lactic acid, the binary mixture was attractive. When combined with ammonia, acetone inhibited the attractiveness exerted by ammonia alone. Dodecanol and dimethyldisulphide did not affect the attraction exerted by ammonia and lactic acid at any of the concentrations tested. By contrast, a human-specific armpit odour, 7-octenoic acid, augmented the attraction exerted by the combination of ammonia and lactic acid at a specific dosage.     

Permethrin-impregnated bednet effects on resting and feeding behaviour of lymphatic filariasis vector mosquitoes in Kenya

The impact of permethrin-impregnated bednets on resting and feeding behaviour of mosquito vectors of Wuchereria bancrofti, causing human lymphatic filariasis was studied in six pairs of villages (treated and untreated) before and after intervention. The study villages were in Kwale District, near the coast of Kenya, where Bancroftian filariasis is highly endemic, transmitted by a combination of both anopheline and culicine mosquito vectors. Mosquitoes were collected weekly in each village, indoors (using pyrethrum spray catches) and outdoors (using pit traps) during 3–4 months following the long rainy season. Of the filariasis vector species of mosquitoes collected in 1994 before intervention, 33.6% were members of the Anopheles gambiae complex, 30% were An. funestus and 36.4% were Culex quinquefasciatus. PCR analysis of the An. gambiae complex species collected in 1995 demonstrated that 98.5% were An. gambiae sensu stricto, 1% An. arabiensis and 0.5% An. merus. Introduction of impregnated bednets in 1995 significantly reduced the number of indoor-resting An. gambiae s.l. by 94.6% and An. funestus by 96.7%, but there was no change in the number of Cx quinquefasciatus collected indoors. The number of outdoor-resting An. gambiae s.l. was significantly reduced, whereas densities of An. funestus and Cx quinquefasciatus remained unaffected outdoors. ELISA analysis of mosquito bloodmeals showed a shift from human to animal feeding after the introduction of treated nets. The human blood index (HBI) for indoor resting Cx quinquefasciatus was reduced from 93.1% to 14.4%. Vector potential based on the HBI and mosquito density was estimated to be reduced by 99% for An. gambiae s.l., 98% for An. funestus and 97% for Cx quinquefasciatus and vectorial capacity would be suppressed even more by the impact on the vector survival rates (not measured). These results suggest that permethrin-impregnated bednets give effective personal protection against transmission of W. bancrofti by An. gambiae, An. funestus and Cx quinquefasciatus in East Africa.     

Ammonia as an attractive component of host odour for the yellow fever mosquito, Aedes aegypti

Behavioural responses of Aedes aegypti mosquitoes to ammonia were investigated in a modified Y-tube olfactometer. Ammonia was attractive in concentrations from 17 ppb to 17 ppm in air when presented together with lactic acid. Aqueous solutions of ammonia salts in concentrations comparable to those found in human sweat also increased the attractiveness of lactic acid. The role of lactic acid as an essential synergist for ammonia became further apparent by the fact that ammonia alone or in combination with carbon dioxide was not effective, even though the synergistic effect of carbon dioxide and lactic acid was corroborated. An extract from human skin residues, which attracts approximately 80% of the tested mosquitoes, contains both lactic acid and ammonia. The combination of these compounds, however, attracts no more than 45%, indicating that other components on human skin also play a role in host finding. Preparative liquid chromatography of the skin extract yielded three behaviourally active fractions which work together synergistically. Fraction III contains lactic acid as the effective principle; the compositions of the other two have not been clarified yet. The attractiveness of fraction I was augmented considerably when ammonia was added, whereas the effect of fraction II was not influenced by ammonia. These results suggests that ammonia is part of the effective principle of fraction II and contributes to the attractive effect of host odours.     

d‐lactic acid production from renewable lignocellulosic biomass via genetically modified Lactobacillus plantarum

d ‐lactic acid is of great interest because of increasing demand for biobased poly‐lactic acid (PLA). Blending poly‐l ‐lactic acid with poly‐d ‐lactic acid greatly improves PLA's mechanical and physical properties. Corn stover and sorghum stalks treated with 1% sodium hydroxide were investigated as possible substrates for d ‐lactic acid production by both sequential saccharification and fermentation and simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Cellic CTec2) was used for hydrolysis of lignocellulosic biomass and an l ‐lactate‐deficient mutant strain Lactobacillus plantarum NCIMB 8826 ldhL1 and its derivative harboring a xylose assimilation plasmid (ΔldhL1‐pCU‐PxylAB) were used for fermentation. The SSCF process demonstrated the advantage of avoiding feedback inhibition of released sugars from lignocellulosic biomass, thus significantly improving d ‐lactic acid yield and productivity. d ‐lactic acid (27.3 g L^?1) and productivity (0.75 g L^?1 h^?1) was obtained from corn stover and d ‐lactic acid (22.0 g L^?1) and productivity (0.65 g L^?1 h^?1) was obtained from sorghum stalks using ΔldhL1‐pCU‐PxylAB via the SSCF process. The recombinant strain produced a higher concentration of d ‐lactic acid than the mutant strain by using the xylose present in lignocellulosic biomass. Our findings demonstrate the potential of using renewable lignocellulosic biomass as an alternative to conventional feedstocks with metabolically engineered lactic acid bacteria to produce d ‐lactic acid. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:271–278, 2016     

Electroantennogram and behavioural responses of the malaria vector Anopheles gambiae to human-specific sweat components

Afrotropical malaria vectors of the Anopheles gambiae complex (Diptera: Culicidae), particularly An. gambiae sensu stricto, are attracted mainly to human hosts. A major source of human volatile emissions is sweat, from which key human-specific components are the carboxylic acids (E)- and (Z)-3-methyl-2-hexenoic acid and 7-octenoic acid. Electrophysiological studies on the antennae of An. gambiae s.s. showed selective sensitivity to these compounds, with a threshold at 10(-6) g comparable to that of known olfactory stimulants 1-octen-3-ol, p-cresol, isovaleric acid, and lower than threshold sensitivity to L-lactic acid and the synthetic mosquito repellent N,N-diethyltoluamide (DEET). A combination of the acids released at concentrations > 10(-5) g in wind tunnel bioassays significantly reduced the response to CO2, the major attractant released by human hosts, for strains of An. gambiae s.s. originating from East and West Africa. Field trials with odour-baited entry traps (OBETs) in Burkina Faso showed that 7-octenoic acid significantly increased (by 1.7-fold) the catch of females of An. gambiae sensu lato (comprising two sibling species: An. arabiensis Patton and An. gambiae s.s.) in OBETs baited with CO2, whereas combinations of the acids significantly reduced the catch in CO2-baited traps (by 2.1-fold) and in whole human odour-baited traps (by 1.5-fold). The pure (E) and (Z) geometric isomers of 3-methyl-2-hexenoic acid gave comparable results to the (EIZ) isomer mixture. These results provide the first experimental evidence that human-specific compounds affect the behaviour of highly anthropophilic An. gambiae s.l. mosquitoes. The compounds appear to inhibit the upwind flight' response to known long-range attractants, and may serve either to mask' the attractants present or, more probably, to 'arrest' upwind flight when mosquitoes arrive at a host under natural conditions. In the final approach to hosts, vectors are known to reduce their flight speed and increase their turning rate, to avoid overshooting the source. In our experimental apparatus, these changes in flight behaviour would reduce the number of mosquitoes entering the ports of the collection devices.     

Identification of electrophysiologically-active compounds for the malaria mosquito, Anopheles gambiae, in human sweat extracts

Abstract. Human sweat samples were chemically fractionated into acid and non-acid components. The most abundant volatile compounds present in the fractions were identified by linked gas chromatography mass spectrometry. The acid fractions were found to be composed of a range of twenty aliphatic and three aromatic carboxylic acids ranging, on average, from 0.02 to 20 ig per ml of sweat sampled. Non-acid fractions were found to contain: 6-methyl-5-hepten-2-one, l-octen-3-ol, decanal, benzyl alcohol, dimethylsulphone, phenylethanol, phenol and 4-mefhylphenol, collectively amounting to 0.1 and 3 |ig per ml of sweat. The major component of sweat was found to be L-lactic acid which constituted from 1 to 5 mg/ml.
Using the intact antennae of the anthropophilic malaria vector mosquito Anopheles gambiae Giles, the peripheral olfactory activities of compounds identified in the sweat fractions were investigated by electroantennography (EAG). Short-chain saturated carboxylic acids, methanoic, ethanoic, propanoic, butanoic, pentanoic and hexanoic acids were found to elicit significantly larger EAG responses than longer chain saturated carboxylic acids from female An.gambiae. For a given dose the largest amplitude EAG response was elicited by methanoic acid. Pentanoic acid elicited larger EAG responses than either butanoic or hexanoic acids. Two non-acidic compounds, l-octen-3-ol and 4-methylphenol, were found to elicit significant dose-dependent EAG responses from female An.gambiae. 1 -Octen-3-ol elicited larger EAG responses than 4-methylphenol for a given dose, but both compounds elicited smaller EAG responses than the same dose of C_]-C₆straight-chain aliphatic carboxylic acids. The possible behavioural significance of the EAG-active compounds identified in human sweat samples is discussed.     

Mosquito responses to carbon dioxide in B West African Sudan savanna village

Abstract. Mosquito responses to carbon dioxide were investigated in Noungou village, 30 km northeast of Ouagadougou in the Sudan savanna belt of Burkina Faso, West Africa. Species of primary interest were the main malaria vectors Anopheles gambiae S.S. and An.arabiensis, sibling species belonging to the An.gambiae complex. Data forAn.finestus, An.pharoensis, Culex quinquefasciatus and Mansonia uniformis were also analysed. Carbon dioxide was used at concentrations of 0.04-0.6% (cf. 0.03% ambient concentration) for attracting mosquitoes to odour-baited entry traps (OBETs). The ‘attractiveness’ of whole human odour was also compared with CO₂, emitted at a rate equivalent to that released by the human bait. In a direct choice test with two OBETs placed side-by-side, the number of An.gambiae s. I. entering the trap with human odour was double the number trapped with CO₂, alone (at the human equivalent rate), but there was no significant difference between OBETs for the other species of mosquitoes. When OBETs were positioned 20 m apart, again CO₂, alone attracted half as many An.gambiae s.l. and only 40% Anlfunestus, 65% Ma.uniformis but twice as many An.pharoensis compared to the number trapped with human odour. The dose-response for all mosquito species was essentially similar: a linear increase in catch with increasing dose on a log-log scale. The slopes of the dose-response curves were not significantly different between species, although there were significant differences in the relative numbers caught. If the dose-response data are considered in relation to a standard human bait collection (HBC), however, the behaviour of each species was quite different. At one extreme, even the highest dose of CO₂, did not catch more An.gambiae s.1. than one HBC. At the other extreme, the three highest doses of CO₂, caught significantly more Ma.unifonnis than did one HBC. An.pharoensis and Cx quinquefasciatus showed a threshold response to CO₂, responding only at doses above that normally released by one man. An.funestus did not respond to CO₂, alone at any dose in sufficient numbers to assess the dose response. Within the An.gambiae complex, An.arabiensis 'chose' the CO₂,-baited trap with a higher probability than An.gambiae S.S. Also An.arabiensis, the less anthropophilic of the two species, was more abundant in CO₂,-baited OBETs than in human bait collections.     

Enhanced production of l‐lactic acid by Lactobacillus thermophilus SRZ50 mutant generated by high‐linear energy transfer heavy ion mutagenesis

The aim of this study was to improve l ‐lactic acid production of Lactobacillus thermophilus SRZ50. For this purpose, high efficient heavy‐ion mutagenesis technique was performed using SRZ50 as the original strain. To enhance the screening efficiency for high yield l ‐lactic acid producers, a scale‐down from shake flask to microtiter plate was developed. The results showed that 24‐well U‐bottom MTPs could well alternate shake flasks for L. thermophilus cultivation as a scale‐down tool due to its a very good comparability to the shake flasks. Based on this microtiter plate screening method, two high l ‐lactic acid productivity mutants, A59 and A69, were successfully screened out, which presented, respectively, 15.8 and 16.2% higher productivities than that of the original strain. Based on fed‐batch fermentation, the A69 mutant can accumulate 114.2 g/L l ‐lactic acid at 96 h. Hence, the proposed traditional microbial breeding method with efficient high‐throughput screening assay was proved to be an appropriate strategy to obtain lactic acid‐overproducing strain.     

Shading by Napier Grass Reduces Malaria Vector Larvae in Natural Habitats in Western Kenya Highlands

Increased human population in the Western Kenya highlands has led to reclamation of natural swamps resulting in the creation of habitats suitable for the breeding of Anopheles gambiae, the major malaria vector in the region. Here we report on a study to restore the reclaimed swamp and reverse its suitability as a habitat for malaria vectors. Napier grass-shaded and non-shaded water channels in reclaimed sites in Western Kenya highlands were studied for the presence and density of mosquito larvae, mosquito species composition, and daily variation in water temperature. Shading was associated with 75.5% and 88.4% (P < 0.0001) reduction in anopheline larvae densities and 78.1% and 88% (P < 0.0001) reduction in Anopheles gambiae sensu lato (s.l.) densities in two sites, respectively. Shading was associated with a 5.7°C, 5.0°C, and 4.7°C, and 1.6°C, 3.9°C, and 2.8°C (for maximum, minimum, and average temperatures, respectively) reduction (P < 0.0001) in water temperatures in the two locations, respectively. An. gambiae s.l. was the dominant species, constituting 83.2% and 73.1%, and 44.5% and 42.3%, of anophelines in non-shaded and shaded channels, respectively, in the two sites, respectively. An. gambiae sensu stricto (s.s.) constituted the majority (97.4%) of An. gambiae s.l., while the rest (2.6%) comprised of Anopheles arabiensis. Minimum water temperature decreased with increasing grass height (P = 0.0039 and P = 0.0415 for Lunyerere and Emutete sites, respectively). The results demonstrate how simple environmental strategies can have a strong impact on vector densities.     

Analysis of the sporozoite ELISA for estimating infection rates in Mozambican anophelines

Comparisons were undertaken to investigate cost‐effective methods of implementing the enzyme‐linked immunosorbent assay (ELISA) for sporozoite determination in anophelines when large numbers require processing. Comparisons between ELISA plate reader and visual assessments were performed with Anopheles funestus and Anopheles gambiae s.l. (Diptera: Culicidae), as were comparisons between whole‐body mosquito samples, heads and thoraces, and abdomens alone. Rates obtained from pools of five or 10 mosquitoes were compared with those for individual mosquitoes, as were rates obtained using different sampling methods. A total of 41 792 An. funestus and 9431 An. gambiae s.l. collected in light traps, and 22 323 An. funestus and 6860 An. gambiae s.l. from exit collections were analysed. Visual assessments gave results similar to those of machine readings. Sporozoite rates were similar in both species, as were rates by collection method. The use of whole mosquitoes increased estimates of infection rate by 0.6%. Pool size did not affect infection rates of An. gambiae s.l., but rates were higher among individually tested An. funestus than among those tested in pools. For large‐scale surveys, the use of whole mosquitoes in pools of 10 mosquitoes, with correction for overestimation, and the noting of results according to a simple three‐stage visual assessment of positivity is the most cost‐effective approach and is sufficient to obtain reliable data for comparative purposes.     

Crystallographic and mutational analyses of cystathionine β‐synthase in the H2S‐synthetic gene cluster in Lactobacillus plantarum

Cystathionine β‐synthase (CBS) catalyzes the formation of l ‐cystathionine from l ‐serine and l ‐homocysteine. The resulting l ‐cystathionine is decomposed into l ‐cysteine, ammonia, and α‐ketobutylic acid by cystathionine γ‐lyase (CGL). This reverse transsulfuration pathway, which is catalyzed by both enzymes, mainly occurs in eukaryotic cells. The eukaryotic CBS and CGL have recently been recognized as major physiological enzymes for the generation of hydrogen sulfide (H₂S). In some bacteria, including the plant‐derived lactic acid bacterium Lactobacillus plantarum, the CBS‐ and CGL‐encoding genes form a cluster in their genomes. Inactivation of these enzymes has been reported to suppress H₂S production in bacteria; interestingly, it has been shown that H₂S suppression increases their susceptibility to various antibiotics. In the present study, we characterized the enzymatic properties of the L. plantarum CBS, whose amino acid sequence displays a similarity with those of O‐acetyl‐l ‐serine sulfhydrylase (OASS) that catalyzes the generation of l ‐cysteine from O‐acetyl‐l ‐serine (l ‐OAS) and H₂S. The L. plantarum CBS shows l ‐OAS‐ and l ‐cysteine‐dependent CBS activities together with OASS activity. Especially, it catalyzes the formation of H₂S in the presence of l ‐cysteine and l ‐homocysteine, together with the formation of l ‐cystathionine. The high affinity toward l ‐cysteine as a first substrate and tendency to use l ‐homocysteine as a second substrate might be associated with its enzymatic ability to generate H₂S. Crystallographic and mutational analyses of CBS indicate that the Ala70 and Glu223 residues at the substrate binding pocket are important for the H₂S‐generating activity.     

Microbial production of d‐lactic acid from dried distiller's grains with solubles

d ‐Lactic acid production is gaining increasing attention due to the thermostable properties of its polymer, poly‐d ‐lactic acid . In this study, Lactobacillus coryniformis subsp. torquens, was evaluated for its ability to produce d ‐lactic acid using Dried Distiller's Grains with Solubles (DDGS) hydrolysate as the substrate. DDGS was first subjected to alkaline pretreatment with sodium hydroxide to remove the hemicellulose component and the generated carbohydrate‐rich solids were then subjected to enzymatic hydrolysis using cellulase mixture Accellerase® 1500. When comparing separate hydrolysis and fermentation and simultaneous saccharification and fermentation (SSF) of L. coryniformis on DDGS hydrolysate, the latter method demonstrated higher d ‐lactic acid production (27.9 g/L, 99.9% optical purity of d ‐lactic acid), with a higher glucose to d ‐lactic acid conversion yield (84.5%) compared to the former one (24.1 g/L, 99.9% optical purity of d ‐lactic acid). In addition, the effect of increasing the DDGS concentration in the fermentation system was investigated via a fed‐batch SSF approach, where it was shown that the d ‐lactic acid production increased to 38.1 g/L and the conversion yield decreased to 70%. In conclusion, the SSF approach proved to be an efficient strategy for the production of d ‐lactic acid from DDGS as it reduced the overall processing time and yielded high d ‐lactic acid concentrations.     

Malaria transmission dynamics in central Côte d'Ivoire: the influence of changing patterns of irrigated rice agriculture

The dynamics of malaria transmission was studied comparatively in the villages of Zatta and Tiemelekro, central Cote d'Ivoire, from February 2002 to August 2003. Prominent agroecosystems in these villages are irrigated rice growing and vegetable farming, respectively. Mosquitoes (Diptera: Culicidae) were collected on human bait at night and by pyrethrum knock-down spray sheet collections at four randomly selected sentinel sites in each village. In 2002, for a total of 96 man-nights per village, 7716 mosquitoes were collected in Zatta and 3308 in Tiemelekro. In 2003, with half the sampling effort, 859 and 2056 mosquitoes were collected in Zatta and Tiemelekro, respectively. Anopheles gambiae Giles s.l. was the predominant mosquito and the key malaria vector throughout, followed by An. funestus Giles. Anthropophily among adult female Anopheles exceeded 95% in both villages. Comparison between years revealed that the biting rate of An. gambiae s.l. in Zatta decreased several-fold from 49.3 bites per person per night (b/p/n) in 2002 to 7.9 b/p/n in 2003 (likelihood ratio test (LRT) = 1072.66; P < 0.001). Although the biting rate remained fairly constant in Tiemelekro, the difference between years was significant (16.1 vs. 18.2 b/p/n; LRT = 148.06; P < 0.001). These observations were paralleled by a marked decrease in the infective rate of An. gambiae s.l. in Zatta (4.6-1.2%), and an increase in Tiemelekro (3.1-7.6%). Meanwhile, the entomological inoculation rate of An. gambiae s.l. decreased 21-fold in Zatta, from 789 to 38 infective bites per person per year (ib/p/y), whereas it remained high in Tiemelekro (233 vs. 342 ib/p/y). The interruption of irrigated rice growing in Zatta in 2003, consequential to a farmers' conflict over land, might be the underlying cause for the significant reduction in malaria transmission, whereas more stable conditions occurred in Tiemelekro.     

Composition of human skin microbiota affects attractiveness to malaria mosquitoes

The African malaria mosquito Anopheles gambiae sensu stricto continues to play an important role in malaria transmission, which is aggravated by its high degree of anthropophily, making it among the foremost vectors of this disease. In the current study we set out to unravel the strong association between this mosquito species and human beings, as it is determined by odorant cues derived from the human skin. Microbial communities on the skin play key roles in the production of human body odour. We demonstrate that the composition of the skin microbiota affects the degree of attractiveness of human beings to this mosquito species. Bacterial plate counts and 16S rRNA sequencing revealed that individuals that are highly attractive to An. gambiae s.s. have a significantly higher abundance, but lower diversity of bacteria on their skin than individuals that are poorly attractive. Bacterial genera that are correlated with the relative degree of attractiveness to mosquitoes were identified. The discovery of the connection between skin microbial populations and attractiveness to mosquitoes may lead to the development of new mosquito attractants and personalized methods for protection against vectors of malaria and other infectious diseases.     

Age effects on the insemination rate ofAnopheles gambiae s.l. in the laboratory

Age effects on the insemination rate of the Galisua strain ofAnopheles gambiae s.s. (Diptera: Culicidae) and of the Nyanza strain ofAn. arabiensis were investigated in the laboratory. Batches of 7-day old males and females were kept together for 24 h with batches of mosquitoes of the opposite sex of ages ranging from 1–7 days. Males and females were also kept together continuously from emergence. The effect of increasing male/female ratios on the insemination rate was investigated as well. The insemination rate ofAn. arabiensis in the laboratory (96%) was similar to that found in nature, whereas that of our strain ofAn. gambiae s.s. was consistently low (maximum 72%), particularly after females had been together with males for a 24 h period only. The optimum age for insemination was 7 days for males and females of both species. The insemination rate of our strain ofAn. arabiensis was significantly higher at all ages than that of our strain ofAn. gambiae s.s.. The latter strain became inseminated at the earliest when 4 days old. An increase in the male/female ratio significantly enhanced the insemination rate in both strains. It was found that motility of spermatozoa inside the spermatheca, once in contact with saline, could be used as a marker for the approximate time of insemination. It is concluded that the relatively low insemination rate of the Galisua strain ofAn. gambiae s.s. is unlikely to be caused by the rearing conditions. Other factors that may be responsible are discussed.     

Characterization of mosquito larval habitats and assessment of insecticide-resistance status of Anopheles gambiae senso lato in urban areas in southwestern Ghana

The study was carried out to characterize potential larval habitats in the city of Sekondi with the aim of assessing the relative importance of anthropogenic and natural water bodies as larval habitats. Insecticide-resistance status of Anopheles gambiae senso lato in the southwestern part of the coastal savannah zone in Ghana was also assessed against four different classes of insecticides. Larval surveys were carried out in two communities that are separated by a lagoon. Although the lagoon was a potential mosquito larval habitat, we showed that it was not an important mosquito breeding site. The major larval habitats were anthropogenic, resulting from human behavior. Some of the organically polluted breeding sites were inhabited by both An. gambiae s.l. and Culex quinquefasciatus larvae. The data also showed that An. gambiae s.l. has currently developed a strong resistance to DDT and pyrethroid insecticides in southwestern Ghana, where the species was reported to be susceptible about a decade ago. The use of insecticides in households was implicated as a possible cause of the development of resistance among An. gambiae s.l. populations in the area. The management of insecticide resistance among malaria vectors needs urgent attention if insecticide-treated materials can continue to be used for malaria control.     

Field testing of different chemical combinations as odour baits for trapping wild mosquitoes in The Gambia

Odour baited traps have potential use in population surveillance of insect vectors of disease, and in some cases for vector population reduction. Established attractants for human host-seeking mosquitoes include a combination of CO(2) with L-lactic acid and ammonia, on top of which additional candidate compounds are being tested. In this field study in rural Gambia, using Latin square experiments with thorough randomization and replication, we tested nine different leading candidate combinations of chemical odorants for attractiveness to wild mosquitoes including anthropophilic malaria vectors, using modified Mosquito Magnet-X (MM-X) counterflow traps outside experimental huts containing male human sleepers. Highest catches of female mosquitoes, particularly of An. gambiae s.l. and Mansonia species, were obtained by incorporation of tetradecanoic acid. As additional carboxylic acids did not increase the trap catches further, this 'reference blend' (tetradecanoic acid with L-lactic acid, ammonia and CO(2)) was used in subsequent experiments. MM-X traps with this blend caught similar numbers of An. gambiae s.l. and slightly more Mansonia and Culex mosquitoes than a standard CDC light trap, and these numbers were not significantly affected by the presence or absence of human sleepers in the huts. Experiments with CO(2) produced from overnight yeast cultures showed that this organic source was effective in enabling trap attractiveness for all mosquito species, although at a slightly lower efficiency than obtained with use of CO(2) gas cylinders. Although further studies are needed to discover additional chemicals that increase attractiveness, as well as to optimise trap design and CO(2) source for broader practical use, the odour-baited traps described here are safe and effective for sampling host-seeking mosquitoes outdoors and can be incorporated into studies of malaria vector ecology.     

Pyrethroid resistance in Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya: phenotypic,metabolic and target site characterizations of three populations

Field and laboratory investigations revealed phenotypic, target site and metabolic resistance to permethrin in an Anopheles gambiae s.s. (Diptera: Culicidae) population in Bungoma District, a region in western Kenya in which malaria is endemic and rates of ownership of insecticide‐treated bednets are high. The sensitivity of individual An. gambiae s.l. females as indicated in assays using World Health Organization (WHO) test kits demonstrated reduced mortality in response to permethrin, deltamethrin and bendiocarb. Estimated time to knock‐down of 50% (KDT₅₀) of the test population in Centers for Disease Control (CDC) bottle bioassays was significantly lengthened for the three insecticides compared with that in a susceptible control strain. Anopheles arabiensis from all three sites showed higher mortality to all three insecticides in the WHO susceptibility assays compared with the CDC bottle assays, in which they showed less sensitivity and longer KDT₅₀ than the reference strain for permethrin and deltamethrin. Microplate assays revealed elevated activity of β‐esterases and oxidases, but not glutathione‐S‐transferase, in An. gambiae s.s. survivors exposed to permethrin in bottle bioassays compared with knocked down and unexposed individuals. No An. arabiensis showed elevated enzyme activity. The 1014S kdr allele was fixed in the Bungoma An. gambiae s.s. population and absent from An. arabiensis, whereas the 1014F kdr allele was absent from all samples of both species. Insecticide resistance could compromise vector control in Bungoma and could spread to other areas as coverage with longlasting insecticide‐treated bednets increases.     

Sweaty skin: an invitation to bite?

Anopheles gambiae sensu stricto and Aedes aegypti have a preference for human blood, which determines their importance as vectors of pathogens responsible for human diseases. Volatile organic chemicals are the principal cues by which humans are being located. Human sweat contains components that are attractive to anthropophilic mosquito species, and variation in sweat composition causes differential attractiveness to mosquitoes within and between individuals and also between humans and other mammals. Characteristics of skin glands and skin microbiota define the odorous organic compounds emitted by sweat, thereby the degree of attractiveness of the host to mosquitoes. Carboxylic acids in particular appear to characterize humans. Thus sweat-associated human volatiles are probably the primary determinant factor in the host preference of anthropophilic mosquitoes.