Cedrol, a malaria mosquito oviposition attractant is produced by fungi isolated from rhizomes of the grass Cyperus rotundus.

BACKGROUND: Cedrol, a sesquiterpene alcohol, is the first identified oviposition attractant for African malaria vectors. Finding the natural source of this compound might help to elucidate why Anopheles gambiae and Anopheles arabiensis prefer to lay eggs in habitats containing it. Previous studies suggest that cedrol may be a fungal metabolite and the essential oil of grass rhizomes have been described to contain a high amount of different sesquiterpenes. RESULTS: Rhizomes of the grass Cyperus rotundus were collected in a natural malaria mosquito breeding site. Two fungi were isolated from an aqueous infusion with these rhizomes. They were identified as Fusarium falciforme and a species in the Fusarium fujikuroi species complex. Volatile compounds were collected from the headspace above fungal cultures on Tenax traps which were analysed by gas chromatography-mass spectrometry (GCMS). Cedrol and a cedrol isomer were detected in the headspace above the F. fujikuroi culture, while only cedrol was detected above the F. falciforme culture. CONCLUSION: Cedrol an oviposition attractant for African malaria vectors is produced by two fungi species isolated from grass rhizomes collected from a natural mosquito breeding site.

Gravid Anopheles gambiae sensu stricto avoid ovipositing in Bermuda grass hay infusion and it's volatiles in two choice egg-count bioassays.

BACKGROUND: A number of mosquito species in the Culex and Aedes genera prefer to lay eggs in Bermuda grass (Cynodon dactylon) hay infusions compared to water alone. These mosquitoes are attracted to volatile compounds from the hay infusions making the infusions effective baits in gravid traps used for monitoring vectors of arboviral and filarial pathogens. Since Bermuda grass is abundant and widespread, it is plausible to explore infusions made from it as a potential low cost bait for outdoor monitoring of the elusive malaria vector Anopheles gambiae s.s. METHODS: This study investigated preferential egg laying of individual An. gambiae s.s. in hay infusion or in tap water treated with volatiles detected in hay infusion headspace compared to tap water alone, using two-choice egg-count bioassays. Infusions were prepared by mixing 90 g of dried Bermuda grass (hay) with 24 L of unchlorinated tap water in a bucket, and leaving it for 3 days at ambient temperature and humidity. The volatiles in the headspace of the hay infusion were sampled with Tenax TA traps for 20 h and analysed using gas chromatography coupled to mass spectrometry. RESULTS: In total, 18 volatiles were detected in the infusion headspace. Nine of the detected compounds and nonanal were selected for bioassays. Eight of the selected compounds have previously been suggested to attract/stimulate egg laying in An. gambiae s.s. Gravid females were significantly (p < 0.05) less likely to lay eggs in hay infusion dilutions of 25, 50 and 100 % and in tap water containing any of six compounds (3-methylbutanol, phenol, 4-methylphenol, nonanal, indole, and 3-methylindole) compared to tap water alone. The oviposition response to 10 % hay infusion or any one of the remaining four volatiles (4-hepten-1-ol, phenylmethanol, 2-phenylethanol, or 4-ethylphenol) did not differ from that in tap water. CONCLUSIONS: Anopheles gambiae s.s. prefers to lay eggs in tap water rather than Bermuda grass hay infusion. This avoidance of the hay infusion appears to be mediated by volatile organic compounds from the infusion. It is, therefore, unlikely that Bermuda grass hay infusion as formulated and used in gravid traps for Culex and Aedes mosquitoes will be suitable baits for monitoring gravid An. gambiae s.s.

Analysing the oviposition behaviour of malaria mosquitoes: design considerations for improving two-choice egg count experiments.

BACKGROUND: Choice egg-count bioassays are a popular tool for analysing oviposition substrate preferences of gravid mosquitoes. This study aimed at improving the design of two-choice experiments for measuring oviposition substrates preferences of the malaria vector Anopheles gambiae senso lato, a mosquito that lays single eggs. METHODS: In order to achieve high egg-laying success of female An. gambiae sensu stricto (s.s.) and Anopheles arabiensis mosquitoes in experiments, four factors were evaluated: (1) the time provided for mating; (2) the impact of cage size, mosquito age and female body size on insemination; (3) the peak oviposition time; and, (4) the host sources of blood meal. Choice bioassays, with one mosquito released in each cage containing two oviposition cups both with the same oviposition substrate (100 ml water), were used to measure and adjust for egg-laying characteristics of the species. Based on these characteristics an improved design for the egg-count bioassay is proposed. RESULTS: High oviposition rates [84%, 95% confidence interval (CI) 77-89%] were achieved when 300 male and 300 blood-fed female An. gambiae s.s. were held together in a cage for 4 days. The chances for oviposition dropped (odds ratio 0.30; 95% CI 0.14-0.66) when human host source of blood meal was substituted with a rabbit but egg numbers per female were not affected. The number of eggs laid by individual mosquitoes was overdispersed (median = 52, eggs, interquartile range 1-214) and the numbers of eggs laid differed widely between replicates, leading to a highly heterogeneous variance between groups and/or rounds of experiments. Moreover, one-third of mosquitoes laid eggs unequally in both cups with similar substrates giving the illusion of choice. Sample size estimations illustrate that it takes 165 individual mosquitoes to power bioassays sufficiently (power = 0.8, p = 0.05) to detect a 15% shift in comparative preferences of two treatments. CONCLUSION: Two-choice egg count bioassays with Anopheles are best done with a two-tier design that (1) implements a parallel series of experiments with mosquitoes given a choice of two identical substrates choices and, (2) uses a single mosquito in each test cage rather than groups of mosquitoes to assess the preference of a test or control solution. This approach, with sufficient replication, lowers the risk detecting pseudopreferences.

Discovery of an oviposition attractant for gravid malaria vectors of the Anopheles gambiae species complex.

BACKGROUND: New strategies are needed to manage malaria vector populations that resist insecticides and bite outdoors. This study describes a breakthrough in developing 'attract and kill' strategies targeting gravid females by identifying and evaluating an oviposition attractant for Anopheles gambiae s.l. METHODS: Previously, the authors found that gravid An. gambiae s.s. females were two times more likely to lay eggs in lake water infused for six days with soil from a natural oviposition site in western Kenya compared to lake water alone or to the same but autoclaved infusion. Here, the volatile chemicals released from these substrates were analysed with a gas-chromatograph coupled to a mass-spectrometer (GC-MS). Furthermore, the behavioural responses of gravid females to one of the compounds identified were evaluated in dual choice egg-count bioassays, in dual-choice semi-field experiments with odour-baited traps and in field bioassays. RESULTS: One of the soil infusion volatiles was readily identified as the sesquiterpene alcohol cedrol. Its widespread presence in natural aquatic habitats in the study area was confirmed by analysing the chemical headspace of 116 water samples collected from different aquatic sites in the field and was therefore selected for evaluation in oviposition bioassays. Twice as many gravid females were attracted to cedrol-treated water than to water alone in two choice cage bioassays (odds ratio (OR) 1.84; 95% confidence interval (CI) 1.16-2.91) and in experiments conducted in large-screened cages with free-flying mosquitoes (OR 1.92; 95% CI 1.63-2.27). When tested in the field, wild malaria vector females were three times more likely to be collected in the traps baited with cedrol than in the traps containing water alone (OR 3.3; 95% CI 1.4-7.9). CONCLUSION: Cedrol is the first compound confirmed as an oviposition attractant for gravid An. gambiae s.l. This finding paves the way for developing new 'attract and kill strategies' for malaria vector control.

Habitat discrimination by gravid Anopheles gambiae sensu lato--a push-pull system.

BACKGROUND: The non-random distribution of anopheline larvae in natural habitats suggests that gravid females discriminate between habitats of different quality. Whilst physical and chemical cues used by Culex and Aedes vector mosquitoes for selecting an oviposition site have been extensively studied, those for Anopheles remain poorly explored. Here the habitat selection by Anopheles gambiae sensu lato (s.l.), the principal African malaria vector, was investigated when presented with a choice of two infusions made from rabbit food pellets, or soil. METHODS: Natural colonization and larval survival was evaluated in artificial ponds filled randomly with either infusion. Dual-choice, egg-count bioassays evaluated the responses of caged gravid females to (1) two- to six-day old infusions versus lake water; (2) autoclaved versus non-autoclaved soil infusions; and assessed (3) the olfactory memory of gravid females conditioned in pellet infusion as larvae. RESULTS: Wild Anopheles exclusively colonized ponds with soil infusion and avoided those with pellet infusion. When the individual infusions were tested in comparison with lake water, caged An. gambiae sensu stricto (s.s.) showed a dose response: females increasingly avoided the pellet infusion with increasing infusion age (six-day versus lake water: odds ratio (OR) 0.22; 95% confidence interval (CI) 0.1-0.5) and showed increasing preference to lay eggs as soil infusion age increased (six-day versus lake water: OR 2.1; 95% CI 1.4-3.3). Larvae survived in soil infusions equally well as in lake water but died in pellet infusions. Anopheles gambiae s.s. preferred to lay eggs in the non-autoclaved soil (OR 2.6; 95% CI 1.8-3.7) compared with autoclaved soil. There was no change in the avoidance of pellet infusion by individuals reared in the infusion compared with those reared in lake water. CONCLUSION: Wild and caged An. gambiae s.l. females discriminate between potential aquatic habitats for oviposition. These choices benefit the survival of the offspring. Although the study was not designed to distinguish between stimuli that acted over a distance or on contact, it could be demonstrated that the choice of habitat is mediated by chemical cues based on both preference and avoidance. These cues, if identified, might be developed for 'push-pull' strategies to improve malaria vector monitoring and control.

Electric nets and sticky materials for analysing oviposition behaviour of gravid malaria vectors.

BACKGROUND: Little is known about how malaria mosquitoes locate oviposition sites in nature. Such knowledge is important to help devise monitoring and control measures that could be used to target gravid females. This study set out to develop a suite of tools that can be used to study the attraction of gravid Anopheles gambiae s.s. towards visual or olfactory cues associated with aquatic habitats. METHODS: Firstly, the study developed and assessed methods for using electrocuting nets to analyse the orientation of gravid females towards an aquatic habitat. Electric nets (1m high × 0.5m wide) were powered by a 12V battery via a spark box. High and low energy settings were compared for mosquito electrocution and a collection device developed to retain electrocuted mosquitoes when falling to the ground. Secondly, a range of sticky materials and a detergent were tested to quantify if and where gravid females land to lay their eggs, by treating the edge of the ponds and the water surface. A randomized complete block design was used for all experiments with 200 mosquitoes released each day. Experiments were conducted in screened semi-field systems using insectary-reared An. gambiae s.s. Data were analysed by generalized estimating equations. RESULTS: An electric net operated at the highest spark box energy of a 400 volt direct current made the net spark, creating a crackling sound, a burst of light and a burning smell. This setting caught 64% less mosquitoes than a net powered by reduced voltage output that could neither be heard nor seen (odds ratio (OR) 0.46; 95% confidence interval (CI) 0.40-0.53, p < 0.001). Three sticky boards (transparent film, glue coated black fly-screen and yellow film) were evaluated as catching devices under electric nets and the transparent and shiny black surfaces were found highly attractive (OR 41.6, 95% CI 19.8 - 87.3, p < 0.001 and OR 28.8, 95% CI 14.5 - 56.8, p < 0.001, respectively) for gravid mosquitoes to land on compared to a yellow sticky film board and therefore unsuitable as collection device under the e-nets. With a square of four e-nets around a pond combined with yellow sticky boards on average 33% (95% CI 28-38%) of mosquitoes released were collected. Sticky materials and detergent in the water worked well in collecting mosquitoes when landing on the edge of the pond or on the water surface. Over 80% of collected females were found on the water surface (mean 103, 95% CI 93-115) as compared to the edge of the artificial pond (mean 24, 95% CI 20-28). CONCLUSION: A square of four e-nets with yellow sticky boards as a collection device can be used for quantifying the numbers of mosquitoes approaching a small oviposition site. Shiny sticky surfaces attract gravid females possibly because they are visually mistaken as aquatic habitats. These materials might be developed further as gravid traps. Anopheles gambiae s.s. primarily land on the water surface for oviposition. This behaviour can be exploited for the development of new trapping and control strategies.

The tsetse fly Glossina fuscipes fuscipes (Diptera: Glossina) harbours a surprising diversity of bacteria other than symbionts.

Three different bacterial species are regularly described from tsetse flies. However, no broad screens have been performed to investigate the existence of other bacteria in this medically and agriculturally important vector insect. Utilising both culture dependent and independent methods we show that Kenyan populations of Glossina fuscipes fuscipes harbour a surprising diversity of bacteria. Bacteria were isolated from 72% of flies with 23 different bacterial species identified. The Firmicutes phylum dominated with 16 species of which seven belong to the genus Bacillus. The tsetse fly primary symbiont, Wigglesworthia glossinidia, was identified by the culture independent pathway. However, neither the secondary symbiont Sodalis nor Wolbachia was detected with either of the methods used. Two other bacterial species were identified with the DNA based method, Bacillus subtilis and Serratia marcescens. Further studies are needed to determine how tsetse flies, which only ever feed on vertebrate blood, pick up bacteria and to investigate the possible impact of these bacteria on Glossina longevity and vector competence.

Field evaluation of two novel sampling devices for collecting wild oviposition site seeking malaria vector mosquitoes: OviART gravid traps and squares of electrocuting nets.

BACKGROUND: New sampling tools are needed for collecting exophilic malaria mosquitoes in sub-Saharan Africa to monitor the impact of vector control interventions. The OviART gravid trap and squares of electrocuting nets (e-nets) were recently developed under semi-field conditions for collecting oviposition site seeking Anopheles gambiae (sensu stricto) (s.s.). This study was designed to evaluate the efficacy of these traps for sampling malaria vectors under field conditions. METHODS: Prior to field testing, two modifications to the prototype OviART gravid trap were evaluated by (i) increasing the surface area and volume of water in the artificial pond which forms part of the trap, and (ii) increasing the strength of the suction fan. Six sampling tools targeting gravid females (Box gravid trap, detergent-treated ponds, e-nets insect glue-treated ponds, sticky boards and sticky floating-acetate sheets) were compared under field conditions to evaluate their relative catching performance and to select a method for comparison with the OviART gravid trap. Finally, the trapping efficacy of the OviART gravid trap and the square of e-nets were compared with a Box gravid trap during the long rainy season in three household clusters in western Kenya. RESULTS: The OviART gravid trap prototype's catch size was doubled by increasing the pond size [rate ratio (RR) 1.9; 95 % confidence interval (CI) 1.1-3.4] but a stronger fan did not improve the catch. The square of e-nets performed better than the other devices, collecting three times more gravid Anopheles spp. than the Box gravid trap (RR 3.3; 95 % CI 1.4-7.6). The OviART gravid trap collections were comparable to those from the e-nets and 3.3 (95 % CI 1.5-7.0) times higher than the number of An. gambiae senso lato (s.l.) collected by the Box gravid trap. CONCLUSION: Both OviART gravid trap and squares of e-nets collected wild gravid Anopheles gambiae (s.l.) where natural habitats were within 200-400 m of the trap. Whilst the e-nets are difficult to handle and might therefore only be useful as a research device, the OviART gravid trap presents a promising new surveillance tool. Further field testing is needed in different eco-epidemiological settings to provide recommendations for its use.

Analysing chemical attraction of gravid Anopheles gambiae sensu stricto with modified BG-Sentinel traps.

BACKGROUND: Cues that guide gravid Anopheles gambiae sensu lato to oviposition sites can be manipulated to create new strategies for monitoring and controlling malaria vectors. However, progress towards identifying such cues is slow in part due to the lack of appropriate tools for investigating long-range attraction to putative oviposition substrates. This study aimed to develop a relatively easy-to-use bioassay system that can effectively analyse chemical attraction of gravid Anopheles gambiae sensu stricto. METHODS: BG-Sentinel™ mosquito traps that use fans to dispense odourants were modified to contain aqueous substrates. Choice tests with two identical traps set in an 80 m(2) screened semi-field system were used to analyse the catch efficacy of the traps and the effectiveness of the bioassay. A different batch of 200 gravid An. gambiae s.s. was released on every experimental night. Choices tested were (1) distilled versus distilled water (baseline) and (2) distilled water versus soil infusion. Further, comparisons were made of distilled water and soil infusions both containing 150 g/l of Sodium Chloride (NaCl). Sodium Chloride is known to affect the release rate of volatiles from organic substrates. RESULTS: When both traps contained distilled water, 45% (95 confidence interval (CI) 33-57%) of all released mosquitoes were trapped. The proportion increased to 84% (95 CI 73-91%) when traps contained soil infusions. In choice tests, a gravid female was twice as likely to be trapped in the test trap with soil infusion as in the trap with distilled water (odds ratio (OR) 1.8, 95% CI 1.3-2.6). Furthermore, the attraction of gravid females towards the test trap with infusion more than tripled (OR 3.4, 95% CI 2.4-4.8) when salt was added to the substrates. CONCLUSION: Minor modifications of the BG-Sentinel™ mosquito trap turned it into a powerful bioassay tool for evaluating the orientation of gravid mosquitoes to putative oviposition substrates using olfaction. This study describes a useful tool for investigating olfactory attraction of gravid An. gambiae s.s. and provides additional evidence that gravid mosquitoes of this species are attracted to and can be baited with attractive substrates such as organic infusions over a distance of several metres.

Evaluation of the influence of electric nets on the behaviour of oviposition site seeking Anopheles gambiae s.s.

BACKGROUND: Electric nets (e-nets) are used to analyse the flight behaviour of insects and have been used extensively to study the host-oriented flight of tsetse flies. Recently we adapted this tool to analyse the oviposition behaviour of gravid malaria vectors, Anopheles gambiae s.s., orienting towards aquatic habitats and traps by surrounding an artificial pond with e-nets and collecting electrocuted mosquitoes on sticky boards on the ground next to the nets. Here we study whether e-nets themselves affect the responses of gravid An. gambiae s.s.. METHODS: Dual-choice experiments were carried out in 80 m2 screened semi-field systems where 200 gravid An. gambiae s.s. were released each night for 12 nights per experiment. The numbers of mosquito landing on or approaching an oviposition site were studied by adding detergent to the water in an artificial pond or surrounding the pond with a square of e-nets. We also assessed whether the supporting framework of the nets or the sticky boards used to retain electrocuted mosquitoes influenced the catch. RESULTS: Two similar detergent treated ponds presented in choice tests caught an equal proportion of the mosquitoes released, whereas a pond surrounded by e-nets caught a higher proportion than an open pond (odds ratio (OR) 1.7, 95% confidence interval (CI) 1.1 - 2.7; p<0.017). The separate evaluation of the impact of the square of electric nets and the yellow boards on the approach of gravid females towards a pond suggests that the tower-like construction of the square of electric nets did not restrict the approach of females but the yellow sticky boards on the ground attract gravid females to a source of water (OR 2.7 95% CI 1.7 - 4.3; p<0.001). CONCLUSION: The trapping efficiency of the electric nets is increased when large yellow sticky boards are placed on the ground next to the e-nets to collect electrocuted mosquitoes, possibly because of increased visual contrast to the aquatic habitat. It is therefore important when comparing two treatments that the same trapping device is used in both. The importance of contrast around artificial habitats might be exploited to improve collections of An. gambiae s.s. in gravid traps.

Development of a gravid trap for collecting live malaria vectors Anopheles gambiae s.l.

BACKGROUND: Effective malaria vector control targeting indoor host-seeking mosquitoes has resulted in fewer vectors entering houses in many areas of sub-Saharan Africa, with the proportion of vectors outdoors becoming more important in the transmission of this disease. This study aimed to develop a gravid trap for the outdoor collection of the malaria vector Anopheles gambiae s.l. based on evaluation and modification of commercially available gravid traps. METHODS: Experiments were implemented in an 80 m(2) semi-field system where 200 gravid Anopheles gambiae s.s. were released nightly. The efficacy of the Box, CDC and Frommer updraft gravid traps was compared. The Box gravid trap was tested to determine if the presence of the trap over water and the trap's sound affected catch size. Mosquitoes approaching the treatment were evaluated using electrocuting nets or detergents added to the water in the trap. Based on the results, a new gravid trap (OviART trap) that provided an open, unobstructed oviposition site was developed and evaluated. RESULTS: Box and CDC gravid traps collected similar numbers (relative rate (RR) 0.8, 95% confidence interval (CI) 0.6-1.2; p = 0.284), whereas the Frommer trap caught 70% fewer mosquitoes (RR 0.3, 95% CI 0.2-0.5; p < 0.001). The number of mosquitoes approaching the Box trap was significantly reduced when the trap was positioned over a water-filled basin compared to an open pond (RR 0.7 95% CI 0.6-0.7; p < 0.001). This effect was not due to the sound of the trap. Catch size increased by 60% (RR 1.6, 1.2-2.2; p = 0.001) with the new OviART trap. CONCLUSION: Gravid An. Gambiae s.s. females were visually deterred by the presence of the trapping device directly over the oviposition medium. Based on these investigations, an effective gravid trap was developed that provides open landing space for egg-laying Anopheles.

Midgut bacterial dynamics in Aedes aegypti.

In vector mosquitoes, the presence of midgut bacteria may affect the ability to transmit pathogens. We have used a laboratory colony of Aedes aegypti as a model for bacterial interspecies competition and show that after a blood meal, the number of species (culturable on Luria-Bertani agar) that coexist in the midgut is low and that about 40% of the females do not harbor any cultivable bacteria. We isolated species belonging to the genera Bacillus, Elizabethkingia, Enterococcus, Klebsiella, Pantoea, Serratia, and Sphingomonas, and we also determined their growth rates, antibiotic resistance, and ex vivo inhibition of each other. To investigate the possible existence of coadaptation between midgut bacteria and their host, we fed Ae. aegypti cohorts with gut bacteria from human, a frog, and two mosquito species and followed the bacterial population growth over time. The dynamics of the different species suggests coadaptation between host and bacteria, and interestingly, we found that Pantoea stewartii isolated from Ae. aegypti survive better in Ae. aegypti as compared to P. stewartii isolated from the malaria mosquito Anopheles gambiae.

Optimizing the colour and fabric of targets for the control of the tsetse fly Glossina fuscipes fuscipes.

BACKGROUND: Most cases of human African trypanosomiasis (HAT) start with a bite from one of the subspecies of Glossina fuscipes. Tsetse use a range of olfactory and visual stimuli to locate their hosts and this response can be exploited to lure tsetse to insecticide-treated targets thereby reducing transmission. To provide a rational basis for cost-effective designs of target, we undertook studies to identify the optimal target colour. METHODOLOGY/PRINCIPAL FINDINGS: On the Chamaunga islands of Lake Victoria , Kenya, studies were made of the numbers of G. fuscipes fuscipes attracted to targets consisting of a panel (25 cm square) of various coloured fabrics flanked by a panel (also 25 cm square) of fine black netting. Both panels were covered with an electrocuting grid to catch tsetse as they contacted the target. The reflectances of the 37 different-coloured cloth panels utilised in the study were measured spectrophotometrically. Catch was positively correlated with percentage reflectance at the blue (460 nm) wavelength and negatively correlated with reflectance at UV (360 nm) and green (520 nm) wavelengths. The best target was subjectively blue, with percentage reflectances of 3%, 29%, and 20% at 360 nm, 460 nm and 520 nm respectively. The worst target was also, subjectively, blue, but with high reflectances at UV (35% reflectance at 360 nm) wavelengths as well as blue (36% reflectance at 460 nm); the best low UV-reflecting blue caught 3× more tsetse than the high UV-reflecting blue. CONCLUSIONS/SIGNIFICANCE: Insecticide-treated targets to control G. f. fuscipes should be blue with low reflectance in both the UV and green bands of the spectrum. Targets that are subjectively blue will perform poorly if they also reflect UV strongly. The selection of fabrics for targets should be guided by spectral analysis of the cloth across both the spectrum visible to humans and the UV region.

Improving the cost-effectiveness of artificial visual baits for controlling the tsetse fly Glossina fuscipes fuscipes.

Tsetse flies, which transmit sleeping sickness to humans and nagana to cattle, are commonly controlled by stationary artificial baits consisting of traps or insecticide-treated screens known as targets. In Kenya the use of electrocuting sampling devices showed that the numbers of Glossina fuscipes fuscipes (Newstead) visiting a biconical trap were nearly double those visiting a black target of 100 cm x 100 cm. However, only 40% of the males and 21% of the females entered the trap, whereas 71% and 34%, respectively, alighted on the target. The greater number visiting the trap appeared to be due to its being largely blue, rather than being three-dimensional or raised above the ground. Through a series of variations of target design we show that a blue-and-black panel of cloth (0.06 m(2)) flanked by a panel (0.06 m(2)) of fine black netting, placed at ground level, would be about ten times more cost-effective than traps or large targets in control campaigns. This finding has important implications for controlling all subspecies of G. fuscipes, which are currently responsible for more than 90% of sleeping sickness cases.

Prospects for developing odour baits to control Glossina fuscipes spp., the major vector of human African trypanosomiasis.

We are attempting to develop cost-effective control methods for the important vector of sleeping sickness, Glossina fuscipes spp. Responses of the tsetse flies Glossina fuscipes fuscipes (in Kenya) and G. f. quanzensis (in Democratic Republic of Congo) to natural host odours are reported. Arrangements of electric nets were used to assess the effect of cattle-, human- and pig-odour on (1) the numbers of tsetse attracted to the odour source and (2) the proportion of flies that landed on a black target (1x1 m). In addition responses to monitor lizard (Varanus niloticus) were assessed in Kenya. The effects of all four odours on the proportion of tsetse that entered a biconical trap were also determined. Sources of natural host odour were produced by placing live hosts in a tent or metal hut (volumes approximately 16 m(3)) from which the air was exhausted at approximately 2000 L/min. Odours from cattle, pigs and humans had no significant effect on attraction of G. f. fuscipes but lizard odour doubled the catch (P<0.05). Similarly, mammalian odours had no significant effect on landing or trap entry whereas lizard odour increased these responses significantly: landing responses increased significantly by 22% for males and 10% for females; the increase in trap efficiency was relatively slight (5-10%) and not always significant. For G. f. quanzensis, only pig odour had a consistent effect, doubling the catch of females attracted to the source and increasing the landing response for females by approximately 15%. Dispensing CO(2) at doses equivalent to natural hosts suggested that the response of G. f. fuscipes to lizard odour was not due to CO(2). For G. f. quanzensis, pig odour and CO(2) attracted similar numbers of tsetse, but CO(2) had no material effect on the landing response. The results suggest that identifying kairomones present in lizard odour for G. f. fuscipes and pig odour for G. f. quanzensis may improve the performance of targets for controlling these species.

Janibacter anophelis sp. nov., isolated from the midgut of Anopheles arabiensis.

A Gram-positive, aerobic, non-motile strain, H2.16BT, isolated from the midgut of the mosquito Anopheles arabiensis was investigated using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity studies, strain H2.16BT was shown to belong to the genus Janibacter, being most closely related to Janibacter melonis (98.3%), Janibacter terrae (98.5%) and Janibacter limosus (98.5%). Chemotaxonomic data (meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall and major fatty acids of iso-C16:0, C17:1omega8c and C17:0)) supported the allocation of the strain to the genus Janibacter. The results of DNA-DNA hybridization and physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain H2.16BT from closely related species. Thus, H2.16BT represents a novel species of the genus Janibacter, for which the name Janibacter anophelis sp. nov. is proposed. The type strain is H2.16BT (=CCUG 49715T=CIP 108728T).

Thorsellia anophelis gen. nov., sp. nov., a new member of the Gammaproteobacteria.

A Gram-negative, rod-shaped organism (CCUG 49520T) was isolated from the midgut of the mosquito Anopheles arabiensis. 16S rRNA gene sequence analysis demonstrated that this isolate is unique, showing <93% similarity to species of the families Enterobacteriaceae and Vibrionaceae. The quinone system consisted exclusively of ubiquinone Q-8; the polar lipid profile consisted of the major compounds phosphatidylethanolamine and phosphatidylglycerol, a moderate to minor amount of two unknown aminophospholipids, an unknown phospholipid and two unknown polar lipids; the polyamine pattern was characterized by the predominant compound 1,3-diaminopropane and showed some significant differences when compared with members of the Enterobacteriaceae and Vibrionaceae. On the basis of 16S rRNA gene sequence analysis in combination with chemotaxonomic data, strain CCUG 49520T is considered to represent a new genus and species, for which the name Thorsellia anophelis gen. nov., sp. nov. is proposed. The type strain is CCUG 49520T (=CIP 108754T).

16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts.

Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the gamma-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito.