Effects of Larval Nutrition on Wolbachia-Based Dengue Virus Interference in Aedes aegypti (Diptera: Culicidae).

In order to assess the broad-scale applicability of field releases of Wolbachia for the biological control of insect-transmitted diseases, we determined the relationship between the larval diet of Aedes aegypti L. mosquitoes infected with Wolbachia strains and their susceptibility to dengue virus (DENV) infection via intrathoracic injection and oral inoculation. Larvae were reared on diets that varied in the quantity of food which had the effect of modifying development time and adult body size. Wolbachia wMel infection was associated with highly significant reductions in dengue serotype 2 (DENV-2) infection rates of between 80 and 97.5% following intrathoracic injection of adults emerging from three diet levels. Reductions were 100% in two diet level treatments following oral inoculation. Similarly, wMelPop infection was associated with highly significant reductions in DENV-2 infection rates of between 95 and 100% for intrathoracic injection and 97.5 and 100% for oral inoculation across diet level treatments. Larval diet level had no significant effect on DENV-2 infection rates in the presence of Wolbachia infection in mosquitoes that were intrathoracically injected with the virus. This indicates that the effectiveness of Wolbachia on vector competence disruption within Ae. aegypti is unlikely to be compromised by variable larval nutrition in field settings.

Role of envelope N-linked glycosylation in Ross River virus virulence and transmission.

With an expanding geographical range and no specific treatments, human arthritogenic alphaviral disease poses a significant problem worldwide. Previous in vitro work with Ross River virus (RRV) demonstrated that alphaviral N-linked glycosylation contributes to type I IFN (IFN-αß) induction in myeloid dendritic cells. This study further evaluated the role of alphaviral N-linked glycans in vivo, assessing the effect of glycosylation on pathogenesis in a mouse model of RRV-induced disease and on viral infection and dissemination in a common mosquito vector, Aedes vigilax. A viral mutant lacking the E1-141 glycosylation site was attenuated for virus-induced disease, with reduced myositis and higher levels of IFN-γ induction at peak disease contributing to improved viral clearance, suggesting that glycosylation of the E1 glycoprotein plays a major role in the pathogenesis of RRV. Interestingly, RRV lacking E2-200 glycan had significantly reduced replication in the mosquito vector A. vigilax, whereas loss of either of the E1 or E2-262 glycans had little effect on the competence of the mosquito vector. Overall, these results indicate that glycosylation of the E1 and E2 glycoproteins of RRV provides important determinants of viral virulence and immunopathology in the mammalian host and replication in the mosquito vector.

Mass spectrometry identification of age-associated proteins from the malaria mosquitoes Anopheles gambiae s.s. and Anopheles stephensi.

This study investigated proteomic changes occurring in Anopheles gambiae and Anopheles stephensi during adult mosquito aging. These changes were evaluated using two-dimensional difference gel electrophoresis (2D-DIGE) and the identities of aging related proteins were determined using capillary high-pressure liquid chromatography (capHPLC) coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometry (MS). Here, we have described the techniques used to determine age associated proteomic changes occurring in heads and thoraces across three age groups; 1, 9 and 17 d old A. gambiae and 4 age groups; 1, 9, 17 and 34 d old A. stephensi. We have provided normalised spot volume raw data for all protein spots that were visible on 2D-DIGE images for both species and processed Orbitrap mass spectrometry data. For public access, mass spectrometry raw data are available via ProteomeXchange with identifier PXD002153. A detailed description of this study has been described elsewhere [1].

Proteomic changes occurring in the malaria mosquitoes Anopheles gambiae and Anopheles stephensi during aging.

The age of mosquitoes is a crucial determinant of their ability to transmit pathogens and their resistance to insecticides. We investigated changes to the abundance of proteins found in heads and thoraces of the malaria mosquitoes Anopheles gambiae and Anopheles stephensi as they aged. Protein expression changes were assessed using two-dimensional difference gel electrophoresis and the identity of differentially expressed proteins was determined by using either matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry or capillary high-pressure liquid chromatography coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometer. Protein biomarkers were validated by semi quantitative Western blot analysis. Nineteen and nine age dependent protein spots were identified for A. stephensi and A. gambiae, respectively. Among the proteins down-regulated with age were homologs of ADF/Cofilin, cytochome c1, heat shock protein-70 and eukaryotic translation initiation factor 5A (eIF5a). Proteins up-regulated with age included probable methylmalonate-semialdehyde dehydrogenase, voltage-dependent anion-selective channel and fructose bisphosphate aldolase. Semi quantitative Western blot analysis confirmed expression patterns observed by 2-D DIGE for eIF5a and ADF/Cofilin. Further work is recommended to determine whether these biomarkers are robust to infection, blood feeding and insecticide resistance. Robust biomarkers could then be incorporated into rapid diagnostic assays for ecological and epidemiological studies. BIOLOGICAL SIGNIFICANCE: In this study, we have identified several proteins with characteristic changes in abundance in both A. gambiae and A. stephensi during their aging process. These changes may highlight underlying mechanisms beneath the relationship between mosquito age and factors affecting Plasmodium transmission and mosquito control. The similarity of changes in protein abundance between these species and the primary dengue vector Aedes aegypti, has revealed conserved patterns of aging-specific protein regulation.

Adult survivorship of the dengue mosquito Aedes aegypti varies seasonally in central Vietnam.

The survival characteristics of the mosquito Aedes aegypti affect transmission rates of dengue because transmission requires infected mosquitoes to survive long enough for the virus to infect the salivary glands. Mosquito survival is assumed to be high in tropical, dengue endemic, countries like Vietnam. However, the survival rates of wild populations of mosquitoes are seldom measured due the difficulty of predicting mosquito age. Hon Mieu Island in central Vietnam is the site of a pilot release of Ae. aegypti infected with a strain of Wolbachia pipientis bacteria (wMelPop) that induces virus interference and mosquito life-shortening. We used the most accurate mosquito age grading approach, transcriptional profiling, to establish the survival patterns of the mosquito population from the population age structure. Furthermore, estimations were validated on mosquitoes released into a large semi-field environment consisting of an enclosed house, garden and yard to incorporate natural environmental variability. Mosquito survival was highest during the dry/cool (January-April) and dry/hot (May-August) seasons, when 92 and 64% of Hon Mieu mosquitoes had survived to an age that they were able to transmit dengue (12 d), respectively. This was reduced to 29% during the wet/cool season from September to December. The presence of Ae. aegypti older than 12 d during each season is likely to facilitate the observed continuity of dengue transmission in the region. We provide season specific Ae. aegypti survival models for improved dengue epidemiology and evaluation of mosquito control strategies that aim to reduce mosquito survival to break the dengue transmission cycle.

Increased container-breeding mosquito risk owing to drought-induced changes in water harvesting and storage in Brisbane, Australia.

BACKGROUND: Extended drought conditions in south-east Queensland during the early 2000s have resulted in a culture of water harvesting and legislated water restrictions. Aedes notoscriptus is a container-breeding mosquito vector of Ross River and Barmah Forest viruses. METHODS: From 2008-2009, the larval habitats and seasonal abundance of domestic container-breeding mosquitoes were recorded from three suburbs of Brisbane. A knowledge, attitudes and practice questionnaire was administered to householders. A low-cost, desktop methodology was used to predict the proportion of shaded premises compared with front-of-property estimates. RESULTS: We highlight changes in the frequency of container categories for A. notoscriptus as a response to human behavioural changes to drought. Garden accoutrements, discarded household items and water storage containers accounted for 66.2% (525/793) of positive containers and 77.5% (73 441/94 731) of all immature mosquitoes. Of all household premises surveyed, 52.6% (550/1046) contained rainwater tanks and 29.4% (308/1046) harvested water in other containers, contrasting with a previous 1995 survey where neither category was observed. Both Premise Condition Index and shade directly correlated with positive premises. CONCLUSIONS: Human response to drought has resulted in new habitats for domestic container-breeding mosquitoes. This recent trend of prolific water storage is similar to earlier years (1904-1943) in Brisbane when Aedes aegypti was present and dengue epidemics occurred.

Proteomic biomarkers for ageing the mosquito Aedes aegypti to determine risk of pathogen transmission.

Biomarkers of the age of mosquitoes are required to determine the risk of transmission of various pathogens as each pathogen undergoes a period of extrinsic incubation in the mosquito host. Using the 2-D Difference Gel Electrophoresis (2-D DIGE) procedure, we investigated the abundance of up to 898 proteins from the Yellow Fever and dengue virus vector, Aedes aegypti, during ageing. By applying a mixed-effects model of protein expression, we identified five common patterns of abundance change during ageing and demonstrated an age-related decrease in variance for four of these. This supported a search for specific proteins with abundance changes that remain tightly associated with ageing for use as ageing biomarkers. Using MALDI-TOF/TOF mass spectrometry we identified ten candidate proteins that satisfied strict biomarker discovery criteria (identified in two out of three multivariate analysis procedures and in two cohorts of mosquitoes). We validated the abundances of the four most suitable candidates (Actin depolymerising factor; ADF, Eukaryotic initiation factor 5A; eIF5A, insect cuticle protein Q17LN8, and Anterior fat body protein; AFP) using semi-quantitative Western analysis of individual mosquitoes of six ages. The redox-response protein Manganese superoxide dismutase (SOD2) and electron shuttling protein Electron transfer oxidoreductase (ETO) were subject to post-translational modifications affecting their charge states with potential effects on function. For the four candidates we show remarkably consistent decreases in abundance during ageing, validating initial selections. In particular, the abundance of AFP is an ideal biomarker candidate for whether a female mosquito has lived long enough to be capable of dengue virus transmission. We have demonstrated proteins to be a suitable class of ageing biomarkers in mosquitoes and have identified candidates for epidemiological studies of dengue and the evaluation of new disease reduction projects targeting mosquito longevity.

Effect of Wolbachia on replication of West Nile virus in a mosquito cell line and adult mosquitoes.

Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that wMelpop and wMel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia-infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia. Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with wMel strain of Wolbachia was not inhibited, whereas wMelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when wMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia-free mosquitoes and were completely inhibited in the presence of Wolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in wMelPop strain of Wolbachia.

Efficacy of residual insecticide Biflex AquaMax applied as barrier treatments for managing mosquito populations in suburban residential properties in southeast Queensland.

The pyrethroid insecticide Biflex AquaMax (bifenthrin) was evaluated for its ability to suppress coastal mosquito populations in a controlled suburban backyard study in Mango Hill, Queensland, Australia. The insecticide was applied to perimeter vegetation, fencing in selected backyards, or both, and mosquito populations were monitored weekly from 3 wk before to 8 wk after treatment (11 wk total) by using CDC miniature light traps and human bait landing rates. In addition, bioassays were conducted on vegetation and wood surfaces to monitor residual activity. The two most common species in light trap collections were the Ross River Virus vectors Aedes vigilax (Skuse) and Culex annulirostris Skuse. After treatment, Ae. vigilax populations in treated properties were significantly lower (75-90%) compared with untreated properties. In contrast, Cx. annulirotris, Coquillettidia xanthogaster (Edwards) and Mansonia uniformis (Theobald) populations were not impacted by the treatment. Bioassays revealed that the product gave better residual mortality when applied to vegetation (94.6 +/- 11.8%) than wooden surfaces (75.9 +/- 29.1%) during the 8-wk posttreatment period.

Low entomological impact of new water supply infrastructure in southern Vietnam, with reference to dengue vectors.

We did a prospective study in southern Vietnam where new water infrastructure was added. New 1,200-L tanks may present potential breeding grounds for Aedes aegypti, particularly when sealed lids were not always supplied. Some householders in these communes received a piped water supply, however there was no reduction in water storage practices. The prevalence of Aedes aegypti immatures in tank and tap households reached 73%, but were non-significantly different from each other and from control households that received no infrastructure. In all three communes, standard jars comprised from 48% to 71% of containers but were associated with > 90% of III-IV instars and pupae on occasions. In contrast, project tanks contributed from 0-21% of the total population. Non-functional or no lids were apparent 4 months after installation in 45-76% of new tanks, but there was no difference between communes with lids and without lids.

Water level flux in household containers in Vietnam--a key determinant of Aedes aegypti population dynamics.

We examined changes in the abundance of immature Aedes aegypti at the household and water storage container level during the dry-season (June-July, 2008) in Tri Nguyen village, central Vietnam. We conducted quantitative immature mosquito surveys of 171 containers in the same 41 households, with replacement of samples, every two days during a 29-day period. We developed multi-level mixed effects regression models to investigate container and household variability in pupal abundance. The percentage of houses that were positive for I/II instars, III/IV instars and pupae during any one survey ranged from 19.5-43.9%, 48.8-75.6% and 17.1-53.7%, respectively. The mean numbers of Ae. aegypti pupae per house ranged between 1.9-12.6 over the study period. Estimates of absolute pupal abundance were highly variable over the 29-day period despite relatively stable weather conditions. Most variability in pupal abundance occurred at the container rather than the household level. A key determinant of Ae. aegypti production was the frequent filling of the containers with water, which caused asynchronous hatching of Ae. aegypti eggs and development of cohorts of immatures. We calculated the probability of the water volume of a large container (>500 L) increasing or decreasing by ≥20% to be 0.05 and 0.07 per day, respectively, and for small containers (<500 L) to be 0.11 and 0.13 per day, respectively. These human water-management behaviors are important determinants of Ae. aegypti production during the dry season. This has implications for choosing a suitable Wolbachia strain for release as it appears that prolonged egg desiccation does not occur in this village.

High household economic burden caused by hospitalization of patients with severe dengue fever cases in Can Tho province, Vietnam.

During 2006-2007, a cohort of 144 confirmed dengue cases in Can Tho Province, Vietnam were compared with a matching set of 144 households that had no dengue cases. Approximately 6-9 months after sickness, there were no significant differences in terms of knowledge of the etiology of dengue, mosquito breeding habitats, and prevention measures in respondents from both sets of households. There was also no difference in the abundance of Aedes aegypti (Linn.) adults but the average numbers of late instar and pupal Ae. aegypti per household were greater in the negative control houses. Thus, the risk seemed to be no higher in case households, although it is conceivable that changes may have occurred in either group over the intervening period. The average cost for a dengue patient was 2,798,000 Vietnamese Dong (VND) (US$167.77), 2,154,000 VND for direct costs, and 644,000 VND for indirect costs. There was a 22% difference in cost for those with and without health insurance. In terms of impact on family economies, 47.2% had to borrow money for treatment, and after 6 months, 71.7% had not begun or had only managed part repayments. Approximately 72.9% indicated that the cost of supporting a dengue patient had impacted on the family economy, with the loss averaging 36% of the annual income in the lowest economic quartile.

Impacts of Wolbachia infection on predator prey relationships: evaluating survival and horizontal transfer between wMelPop infected Aedes aegypti and its predators.

The wMelPop strain of Wolbachia is currently being investigated for its potential use as a biological control agent to reduce the ability of Aedes aegypti (L.) mosquitoes to transmit dengue viruses. The survival of a potential wMelPop infected Ae. aegypti strain for field release is important as a higher susceptibility to predation in the wMelPop strain could result in difficulties in achieving fixation. We investigated immature and adult survival as a function of susceptibility to predation by six naturally occurring predator species; cyclopoid copepods, fish, predatory Toxorhynchites mosquito larvae and a salticid jumping spider. The trials indicated that wMelPop infected and uninfected Ae. aegypti larvae and adults were equally susceptible to predation to all six tested predators. In addition to evaluating any potential fitness costs to the infected host, we were unable to demonstrate horizontal transfer of wMelPop via consumption of infected Ae. aegypti larvae to the above predators. That susceptibility to predation was consistent across mosquito life stage, predator species and experimental venue is strong evidence that despite the neurotrophic and extensive nature of wMelPop infection, behavioral changes are not occurring, or at least not a determining factor in survival when exposed to a predator. Based on our results and the ecology of Wolbachia and mosquito predators, horizontal transfer of wMelPop from Ae. aegypti into naturally occurring predators is not cause for concern.

Community-based control of Aedes aegypti by using Mesocyclops in southern Vietnam.

We previously reported a new community-based mosquito control strategy that resulted in elimination of Aedes aegypti (Linn.) in 40 of 46 communes in northern and central Vietnam, and with annual recurrent total costs (direct and indirect) of only $0.28-$0.89 international dollars per person. This control strategy was extended to four provinces in southern Vietnam in Long An and Hau Giang (2004-2007) and to Long An, Ben Tre, and Vinh Long (2005-2010). In a total of 14 communes with 124,743 residents, the mean ± SD of adult female Ae. aegypti was reduced from 0.93 ± 0.62 to 0.06 ± 0.09, and the reduction of immature Ae. aegypti averaged 98.8%. By the final survey, no adults could be collected in 6 of 14 communes, and one commune, Binh Thanh, also had no immature forms. Although the community-based programs also involved community education and clean-up campaigns, the prevalence of Mesocyclops in large water storage containers > 50 liters increased from 12.77 ± 8.39 to 75.69 ± 9.17% over periods of 15-45 months. At the conclusion of the study, no confirmed dengue cases were detected in four of the five communes for which diagnostic serologic analysis was performed. The rate of progress was faster in communes that were added in stages to the program but the reason for this finding was unclear. At the completion of the formal project, sustainability funds were set up to provide each commune with the financial means to ensure that community-based dengue control activities continued.

Effects of Beauveria bassiana on survival, blood-feeding success, and fecundity of Aedes aegypti in laboratory and semi-field conditions.

The fungus Beauveria bassiana reduces Aedes aegypti longevity in laboratory conditions, but effects on survival, blood-feeding behavior, and fecundity in realistic environmental conditions have not been tested. Adult, female Ae. aegypti infected with B. bassiana (FI-277) were monitored for blood-feeding success and fecundity in the laboratory. Fungal infection reduced mosquito-human contact by 30%. Fecundity was reduced by (mean ± SD) 29.3 ± 8.6 eggs per female per lifetime in the laboratory; egg batch size and viability were unaffected. Mosquito survival, blood-feeding behavior, and fecundity were also tested in 5 meter × 7 meter × 4 meter semi-field cages in northern Queensland, Australia. Fungal infection reduced mosquito survival in semi-field conditions by 59-95% in large cages compared with 61-69% in small cages. One semi-field cage trial demonstrated 80% reduction in blood-feeding; a second trial showed no significant effect. Infection did not affect fecundity in large cages. Beauveria bassiana can kill and may reduce biting of Ae. aegypti in semi-field conditions and in the laboratory. These results further support the use of B. bassiana as a potential biocontrol agent against Ae. aegypti.

Abundance and prevalence of Aedes aegypti immatures and relationships with household water storage in rural areas in southern Viet Nam.

Since 2000, the Government of Viet Nam has committed to provide rural communities with increased access to safe water through a variety of household water supply schemes (wells, ferrocement tanks and jars) and piped water schemes. One possible, unintended consequence of these schemes is the concomitant increase in water containers that may serve as habitats for dengue mosquito immatures, principally Aedes aegypti. To assess these possible impacts we undertook detailed household surveys of Ae. aegypti immatures, water storage containers and various socioeconomic factors in three rural communes in southern Viet Nam. Positive relationships between the numbers of household water storage containers and the prevalence and abundance of Ae. aegypti immatures were found. Overall, water storage containers accounted for 92-97% and 93-96% of the standing crops of III/IV instars and pupae, respectively. Interestingly, households with higher socioeconomic levels had significantly higher numbers of water storage containers and therefore greater risk of Ae. aegypti infestation. Even after provision of piped water to houses, householders continued to store water in containers and there was no observed decrease in water storage container abundance in these houses, compared to those that relied entirely on stored water. These findings highlight the householders' concerns about the limited availability of water and their strong behavoural patterns associated with storage of water. We conclude that household water storage container availability is a major risk factor for infestation with Ae. aegypti immatures, and that recent investment in rural water supply infrastructure are unlikely to mitigate this risk, at least in the short term.

Investigation of environmental influences on a transcriptional assay for the prediction of age of Aedes aegypti (Diptera: Culicidae) mosquitoes.

We examined the effects of environmental regulation of gene transcription on the accuracy of a transcriptional profiling method for determining insect age. In combined temperature/nutrition treatments, Aedes aegypti (L.) mosquitoes were maintained in the laboratory at three different temperatures (20, 26, and 32 degrees C), and larvae were fed on low, medium, and high diet regimens. Adult mosquitoes of distinct size classes were produced. Transcription of three age-responsive genes (Ae-15848, Ae-8505, and Ae-4274) was measured from 1-, 10-, and 19-d-old specimens using a quantitative reverse-transcription polymerase chain reaction method incorporating dual-labeled TaqMan probes. Temperature had a significant effect on transcript abundance for two of the model genes (Ae-15848 and Ae-8505), and transcription of model genes was unaffected by the main effect of larval diet level; however, significant temperature by diet level interactions were observed. Total RNA yield from individual mosquitoes varied according to adult age and temperature, and when combined with wing length, provided a useful predictor variable in age prediction models. More accurate age predictions were achieved from models generated at the same temperature as test mosquitoes; however, whereas significant differences in mean predicted ages were observed between 1- and 10-d-old mosquitoes, differences between 10 and 19 d were nonsignificant. This study highlights the effect of environmental regulation on gene transcription age grading and the need to identify additional gene biomarkers of age to improve the classification of older mosquitoes.

Quantitative relationships between immature and emergent adult Aedes aegypti (Diptera: Culicidae) populations in water storage container habitats.

Although quantitative surveillance data for immature stages of Aedes aegypti are often used to prioritize containers or specific types of containers for control, the relationship between immature and emergent adult populations under field conditions is largely unknown. We examined the relationships between abundance of III/IV instars and pupae, and emerging adult population for a series of water storage containers in southern Vietnam. A large proportion of III/IV instars failed to progress to adulthood, and the relationships between III/IV instars and adults were poor. Collected IV instars appeared to be nutritionally deprived, although their size and nutrient levels were not reliable indicators of emergence success. Conversely, pupal abundance was a good indicator of emerging adult populations, especially over the ensuing 48-h period. Although there were clear advantages of pupal surveillance over surveillance of III/IV instars for the estimation of adult mosquito productivity, there were practical limitations associated with the enumeration of pupae, and their comparatively low densities may preclude the identification of potentially productive containers.

Sustainability and cost of a community-based strategy against Aedes aegypti in northern and central Vietnam.

We previously reported a new community-based mosquito control that resulted in the elimination of Aedes aegypti in 40 of 46 communes in northern and central Vietnam. During 2007 and 2008, we revisited Nam Dinh and Khanh Hoa provinces in northern and central Vietnam, respectively, to evaluate whether or not these programs were still being maintained 7 years and 4.5 years after formal project activities had ceased, respectively. Using a previously published sustainability framework, we compared 13 criteria from Tho Nghiep commune in Nam Dinh where the local community had adopted our community-based project model using Mesocyclops from 2001. These data were compared against a formal project commune, Xuan Phong, where our successful intervention activities had ceased in 2000 and four communes operating under the National Dengue Control Program with data available. In Khanh Hoa province, we compared 2008 data at Ninh Xuan commune with data at project completion in 2003 and benchmarked these, where possible, against an untreated control commune, Ninh Binh, where few control activities had been undertaken. The three communes where the above community-based strategy had been adopted were rated as well-sustained with annual recurrent total costs (direct and indirect) of $0.28-0.89 international dollars per person.

Melanotaenia duboulayi influence oviposition site selection by Culex annulirostris (Diptera: Culicidae) and Aedes notoscriptus (Diptera: Culicidae) but not Culex quinquefasciatus (Diptera: Culicidae).

Some species of mosquito can detect the presence of larvivorous fish and select against ovipositing in pools supporting them. The effect of kairomones released by the crimson-spotted rainbowfish Melanotaenia duboulayi (Castelnau) on the oviposition behavior of the freshwater mosquitoes Culex annulirostris Skuse, Culex quinquefasciatus Say, and Aedes notoscriptus Skuse was evaluated in both laboratory and outdoor artificial pool experiments. In the laboratory, colony-reared Cx. annulirostris selected against ovipositing in water that had contained M. duboulayi at densities of one fish per 5, 30, 180, and 1,080 liters. In contrast, gravid Ae. notoscriptus showed a preference for water that had contained the highest density of M. duboulayi (1 in 5 liters). Gravid Cx. quinquefasciatus were neither repelled nor attracted to water that had previously contained M. duboulayi. In outdoor artificial pool experiments, wild Cx. annulirostris females selected against ovipositing in pools containing caged M. duboulayi stocked at rates of 1, 4, and 10 g per 1,000 liters. When fish were removed from the pools, the repellent effect persisted for at least 24 h, whereas at the lower stocking rate, an avoidance response was not noted until 48 h after fish were placed in pools. This suggests a volatile substance, however, the exact nature of the kairomone/s has not been identified.