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The use essential oils as ecofriendly tools for pest and vector management has become one of the mainstreams for biopesticide research. Here, Commiphora erythraea (opoponax) essential oil and its fractions were profiled for their chemical composition and larvicidal activity against Culex restuans, Cx. pipiens, and Aedes aegypti mosquitoes. The effect of C. erythraea essential oil on expression of three cytochrome P450 (CYP450s) and three Glutathione-S-transferases (GSTs) genes and the use of bio-based amylose-N-1-hexadecylammonium chloride inclusion complex (Hex-Am) and amylose-sodium palmitate inclusion complex (Na-Palm) as emulsifiers for C. erythraea essential oil were also investigated. The most abundant chemical constituents were curzerene (32.6%) in fraction 1, spathulenol (62.5%) in fraction 2, and bicyclosesquiphellandrene in the whole essential oil (33.9%) and factions 3 (30.1%) and 4 (23.8%). LC50 values for the whole essential oil were 19.05 ppm for Cx. restuans, 22.61 ppm for Cx. pipiens and 29.83 ppm for Ae. aegypti and differed significantly from each other. None of the four C. erythraea essential oil fractions were active against mosquito larvae. Two CYP450 genes (CYP6M11 and CYP6N12) and one GST gene (GST-2) were significantly upregulated in Ae. aegypti larvae exposed to C. erythraea essential oil suggesting their potential involvement in pathways of metabolic activation and detoxification of C. erythraea essential oil. Essential oil emulsions produced with Hex-Am were more toxic than the whole essential oil while those produced with Na-Palm had similar toxicity as the whole essential oil. These findings demonstrate that C. erythraea essential oil is a promising source of mosquito larvicide and that its larvicidal activity is likely due to synergistic interactions among the major and minor constituents. The findings also demonstrate that the use of Hex-Am as an emulsifier can enhance the insecticidal properties of C. erythraea essential oil.

Background Some plant essential oils have insecticidal properties against mosquitoes and can be harnessed as ecofriendly tools for mosquito control. We conducted bioassays to determine the toxicity of Italian honeysuckle (Lonicera caprifolium) essential oil and its fractions against larvae of the yellow fever mosquito, Aedes aegypti. Results Sixteen constituents were identified in honeysuckle essential oil compared to 15, 15, 15, and 11 constituents in fractions A, B, C, and E, respectively. The chemical constituents for fraction D were not determined due to lack of enough fraction sample. The two major constituents identified were patchouli alcohol (29.3%) and 6-acetyl-1,1,2,4,4,7-hexamethyltetralin (20.6%) in whole essential oil, alpha-bulnesene (27.6%) and 6-acetyl-1,1,2,4,4,7-hexamethyltetralin (23.2%) in fraction A, unknown chemical (47.3%) and diethyl phthalate (19.5%) in fraction B, unknown chemical (38.3%) and diethyl phthalate (23.2%) in fraction C, and patchouli alcohol (58.7%) and diethyl phthalate (20.5%) in fraction E. The LC50 for whole essential oil was 34.4 ppm and significantly higher than 20.6, 19.7, 18.6, and 17.7 ppm for fractions B, C, D, and E, respectively. In contrast, fraction A was inactive. At 50 ppm, all individual constituents tested were less toxic than the whole essential oil with exception of patchouli alcohol, which caused 100% mortality. Conclusion These findings suggest that patchouli alcohol is one of the chemical constituents responsible for bioactivity of honeysuckle essential oil and some of its fractions. The findings also demonstrate that honey suckle essential oil and its fractions can be exploited as a source of ecofriendly larvicides for mosquito control. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Background The rapid and widespread evolution of insecticide resistance has emerged as one of the major challenges facing malaria control programs in sub-Saharan Africa. Understanding the insecticide resistance status of mosquito populations and the underlying mechanisms of insecticide resistance can inform the development of effective and site-specific strategies for resistance prevention and management. The aim of this study was to investigate the insecticide resistance status of Anopheles gambiae (s.l.) mosquitoes from coastal Kenya. Methods Anopheles gambiae (s.l.) larvae sampled from eight study sites were reared to adulthood in the insectary, and 3- to 5-day-old non-blood-fed females were tested for susceptibility to permethrin, deltamethrin, dichlorodiphenyltrichloroethane (DDT), fenitrothion and bendiocarb using the standard World Health Organization protocol. PCR amplification of rDNA intergenic spacers was used to identify sibling species of the An. gambiae complex. The An. gambiae (s.l.) females were further genotyped for the presence of the L1014S and L1014F knockdown resistance (kdr) mutations by real-time PCR. Results Anopheles arabiensis was the dominant species, accounting for 95.2% of the total collection, followed by An. gambiae (s.s.), accounting for 4.8%. Anopheles gambiae (s.l.) mosquitoes were resistant to deltamethrin, permethrin and fenitrothion but not to bendiocarb and DDT. The L1014S kdr point mutation was detected only in An. gambiae (s.s.), at a low allelic frequency of 3.33%, and the 1014F kdr mutation was not detected in either An. gambiae (s.s.) or An. arabiensis. Conclusion The findings of this study demonstrate phenotypic resistance to pyrethroids and organophosphates and a low level of the L1014S kdr point mutation that may partly be responsible for resistance to pyrethroids. This knowledge may inform the development of insecticide resistance management strategies along the Kenyan Coast. Graphical Abstract

Pesticides commonly contaminate the aquatic environments inhabited by mosquito juveniles. However, their role in shaping the mosquito microbiota is not well understood. We hypothesized that environmentally relevant concentrations of atrazine, permethrin and malathion will mediate a shift in the mosquito gut bacterial community structure due to their toxic effect on the aquatic bacterial communities, and reduce mosquito gut bacterial diversity by enriching pesticide-degrading bacterial communities over susceptible taxa. Illumina MiSeq sequencing of the V3-V4 hypervariable regions of the 16 S rRNA gene was used to characterize the microbial communities of larval and adult stages of the two mosquito species and the water samples from microcosms treated with each of the pesticides, separately. Bacterial community composition differed by sample type (larval stage vs. adult stage) and water sampling date (day 3 vs. day 7), but not by pesticide treatment. In larval stages, bacterial OTU richness was highest in samples exposed to malathion, intermediate in permethrin, and lowest in controls. Bacterial richness was significantly higher in larval stages compared to adult stages for all treatments. This study provides a primer for future studies evaluating mosquito microbial responses to exposures to chemical pesticides and the possible implications for mosquito ecology.

The potential for gut microbiota to impede or enhance pathogen transmission is well-documented but the factors that shape this microbiota in mosquito vectors are poorly understood. We characterized and compared the gut microbiota of adult females of Culex restuans (Theobald; Diptera: Culicidae) from different parents. Cx. restuans larvae from nine field-collected egg rafts were reared on a common diet and gut microbiota of newly emerged adult females characterized by MiSeq sequencing of the V4 hypervariable region of the 16S rRNA gene. Bacterial diversity and evenness in individuals from one egg raft were significantly lower compared to those of three of the other eight egg rafts. The gut microbiota of adult females reared from seven of the nine egg rafts clustered together suggesting that individuals from most egg rafts had similar profiles of gut microbiota. These findings suggest that the microbiota of adult females from the same parents do not differ appreciably from the microbiota of adult females from different parents. However, additional studies using mosquitoes separated by geographic distances greater than those studied here and estimating the genetic distances between populations from different egg rafts are needed to provide further insights into the influence of host genetics on gut microbiota. Also worthwhile are studies evaluating how individuals from different egg rafts and harboring different gut microbiota compare in relation to vector competence for different pathogens.

Essential oils are potential alternatives to synthetic insecticides because they have low mammalian toxicity, degrade rapidly in the environment, and possess complex mixtures of bioactive constituents with multi-modal activity against the target insect populations. Twenty-one essential oils were initially screened for their toxicity against Aedes aegypti (L.) larvae and three out of the seven most toxic essential oils (Manuka, oregano, and clove bud essential oils) were examined for their chemical composition and combined toxicity against Ae. aegypti larvae. Manuka essential oil interacted synergistically with oregano essential oil and antagonistically with clove bud essential oil. GC-MS analysis revealed the presence of 21 components in Manuka essential oil and three components each in oregano and clove bud essential oils. Eugenol (84.9%) and eugenol acetate (9.6%) were the principal constituents in clove bud essential oil while carvacrol (75.8%) and m-isopropyltoluene (15.5%) were the major constituents in oregano essential oil. The major constituents in Manuka essential oil were calamenene (20%) and 3-dodecyl-furandione (11.4%). Manuka essential oil interacted synergistically with eugenol acetate and antagonistically with eugenol, suggesting that eugenol was a major contributor to the antagonistic interaction between Manuka and clove bud essential oils. In addition, Manuka interacted synergistically with carvacrol suggesting its contribution to the synergistic interaction between Manuka and oregano essential oils. These findings provide novel insights that can be used to develop new and safer alternatives to synthetic insecticides.

We examined the growth characteristics of Tolypocladium cylindrosporum IBT 41712 and its potential to infect eggs of Aedes aegypti and Ae. albopictus at a low temperature (15°C). When grown on Sabouraud dextrose agar supplemented with yeast extract, the IBT 41712 formed white colonies turning to a slightly darker, off-white color when mature. The mycelia bore swollen conidiophores producing smooth-walled, oblong to cylindrical conidia with varying sizes, ranging from 1.5 to 3.5 μm long. To determine the optimum temperature for the fungus, we cultured the fungus at eight temperatures (4°C, 12°C, 15°C, 21°C, 28°C, 33°C, 37°C, and 40°C) and measured the diametric growth. The optimum temperature for growth was 28°C since it had the highest diametric growth rate (2.1 ± 0.05 mm/day) and the fastest sporulation period (within 8-10 days of incubation). There was no fungal growth at the 3 highest temperatures (33°C, 37°C, and 40°C) but plates incubated at 33°C, when shifted to optimal temperature (28°C), showed visible growth indicating that following incubation at 33°C, the fungus remained viable. The IBT 41712 successfully infected mosquito eggs at 15°C. Fungal treatment induced egg hatch on moist seed-germination paper and this effect was more pronounced in Ae. aegypti compared to Ae. albopictus. When treated eggs were immersed in dH2O 21 days posttreatment, larval hatch of both Ae. aegypti (control = 91%, 1 × 107 conidia/ml, fungal treatment = 0%) and Ae. albopictus (control = 85%, fungal treatment = 28%) was significantly lower in fungal treatment compared to the controls. The ability of the strain to grow in a wide temperature range, and effectively infect mosquito eggs and induce egg hatch at a low temperature warrants further investigation for its potential as a mosquito control agent targeting eggs that overwinter or undergo long diapause.

Invasive alien plants wreak havoc on native ecosystems and using them as a source of biopesticides could improve their management. We examined the toxicity of essential oil of wild carrot (also known as 'Queen Anne's Lace', Daucus carota Linnaeus), an aggressive invader throughout the United States, against Aedes aegypti L., Culex pipiens L., and Culex restuans Theobald larvae. Comparisons were made between essential oil extracted from umbels of local populations of wild carrot versus a commercial brand. Methyl isoeugenol (60.7%) was by far the most abundant constituent in commercial brand oil, whereas α-pinene (33.0%) and β-pinene (25.8%) were the dominant constituents in essential oil extracted from local wild carrot populations. The commercial brand essential oil was significantly more toxic to Cx. restuans larvae (LC50 = 44.4 ppm) compared with Cx. pipiens (LC50 = 51.0 ppm) and Ae. aegypti (LC50 = 54.5 ppm). Essential oil from local populations of wild carrot was significantly more toxic to both Cx. pipiens (LC50 = 42.9) and Cx. restuans (LC50 = 40.3) larvae compared with Ae. aegypti (LC50 = 64.6 ppm) larvae. Three of the nine tested chemical constituents of wild carrot essential oil (terpinolene, para cymene, and γ-terpinene) were consistently more toxic to larvae of the three mosquito species than the whole essential oil. These findings suggest that exploiting wild carrot essential oil and its chemical constituents as a biopesticide for mosquito control could be used as part of multifaceted approaches for controlling this invasive alien plant species.

Ecological traps occur due to a mismatch between a habitat's attractiveness and quality, wherein organisms show preference for low-quality habitats over other available high-quality habitats. Our previous research identified leaf litter from common blackberry (Rubus allegheniensis) as a natural ecological trap for an important vector for West Nile virus (Culex pipiens), attracting mosquitoes to oviposit in habitats deleterious to the survival of their larvae. Here we demonstrate that manipulation of leaf litter in stormwater catch basins, an important source of disease vector mosquitoes in urban environments, can increaseCx. pipiensoviposition but reduce survival. In a series of experiments designed to elucidate the mechanisms that explain the attractive and lethal properties of this native plant, behavioural bioassays suggest that oviposition site selection byCx. pipiensis mediated primarily by chemical cues as leaves decompose. However, we also show that juvenile mosquito survival is mainly related to the suitability of the bacterial community in the aquatic habitat for mosquito nutritional needs, which does not appear to create a cue that influences oviposition choice. This mismatch between oviposition cues and drivers of larval habitat quality may account for the ecological trap phenomenon detected in this study. Our findings provide new insights into potential mechanistic pathways by which ecological traps may occur in nature and proof-of-concept for a new ‘attract-and-kill’ tool for mosquito control.

Aedes aegypti L. and Aedes albopictus Skuse co-occur in a variety of water-filled containers where they compete for resources. Larvae of Ae. albopictus Skuse often outcompete those of Ae. aegypti L., but variation in biotic and abiotic parameters can modify the outcome of this interspecific competition. We tested whether container size can alter the magnitude and direction of intra- and interspecific competition by rearing three Ae. aegypti and Ae. albopictus larval combinations (100:0, 50:50 and 0:100) in three container sizes (small, medium, and large). For both mosquito species, individuals raised in small- and medium-sized containers had shorter development time to adulthood, higher survival to adulthood, and larger adult body size compared to individuals from large containers. For Ae. aegypti but not Ae. albopictus, survival to adulthood was significantly influenced by a two-way interaction between container size and larval competition. The negative effect of interspecific competition was stronger in the small and medium containers and the negative effect of intraspecific competition was stronger in large containers. Our results show that container size can affect the outcome of intra- and interspecific competition between Ae. aegypti and Ae. albopictus and may help account for the observed patterns of both competitive exclusion and coexistence documented in the field for these two medically important mosquito species.

We examined the chemical composition of garlic and asafoetida essential oils and their individual and combined toxicity against larvae of Culex pipiens Linnaeus and Culex restuans Theobald (Diptera: Culicidae). The effect of the two essential oils on egg hatch was also examined. Ten and 12 compounds, respectively, were identified in garlic and asafoetida essential oils. Allyl disulfide (49.13%) and diallyl trisulfide (31.08%) were the most abundant compounds in garlic essential oil accounting for 80.2% of the total oil. In contrast, (E)-sec-butyl propenyl disulfide (30.03%), (Z)-sec-butyl propenyl disulfide (24.32%), and disulfide, methyl 1-(methylthio)propyl (21.87%) were the most abundant compounds in asafoetida essential oil. Allyl disulfide accounted for 7.38% of the total oil in asafoetida essential oil and was one of only three compounds found in both oils. For both mosquito species, garlic essential oil was more toxic than asafoetida essential oil with Cx. restuans (LC50: garlic = 2.7 ppm; asafoetida = 10.1 ppm) being more sensitive than Cx. pipiens (LC50: garlic = 7.5 ppm; asafoetida = 13.5 ppm). When combined, the two essential oils had antagonistic effects. The majority of Culex egg rafts exposed to garlic (73.1%) or asafoetida (55.8%) essential oils failed to hatch and larvae of the few that did hatch mostly died as first instars. Allyl disulfide exhibited strong ovicidal and larvicidal activity suggesting its important contribution to the overall toxicity of the two essential oils. Thus, garlic and asafoetida essential oils are potent mosquito ovicides and larvicides but if used jointly, they could undermine vector control programs.

Ochlerotatus triseriatus (Say), the primary vector of La Crosse virus (LAC), develops in a variety of natural and artificial aquatic containers where it often co-occurs with larvae of other mosquito species. We conducted a field study at two woodlots (South Farms and Trelease Woods) in Urbana, IL, to examine how container type influences vector abundance, body size, and susceptibility to LAC. Mosquito pupae were collected from tree holes, plastic bins, and waste tires, and eclosing adults were identified to species morphologically. Oc. triseriatus and Ochlerotatus japonicus (Theobald) females were orally challenged with LAC and midgut infection rate, disseminated infection rate, and body titer were determined by reverse-transcriptase real-time PCR. Oc. triseriatus was the dominant species collected in tree holes while Oc. japonicus and Culex restuans (Theobald) were mostly dominant in artificial containers. Female Oc. triseriatus and Oc. japonicus collected from plastic bins were significantly larger than those collected from tree holes or waste tires. Oc. japonicus females from South Farms were also significantly larger than those from Trelease Woods. Oc. triseriatus females collected from plastic bins and waste tires were significantly more susceptible to LAC infection relative to females collected from tree holes. In addition, Oc. triseriatus females from waste tires had significantly higher LAC titer relative to Oc. triseriatus from tree holes. For each container type and study site, wing length was not correlated to infection or dissemination rates. These findings suggest that the container type in which Oc.triseriatus develop may contribute to the spatial and temporal dynamics of LAC transmission.

Mosquito-microbe interactions tend to influence larval nutrition, immunity, and development, as well as fitness and vectorial capacity of adults. Understanding the role of different bacterial species not only improves our knowledge of the physiological and ecological consequences of these interactions, but also provides the basis for developing novel strategies for controlling mosquito-borne diseases. We used culture-dependent and culture-independent techniques to characterize the bacterial composition and abundance in water and midgut samples of larval and adult females of Aedes japonicus (Theobald), Aedes triseriatus (Say), and Culex restuans (Theobald) collected from waste tires at two wooded study sites in Urbana, IL. The phylum-specific real-time quantitative polymerase chain reaction assay revealed a higher proportion of Actinobacteria and a lower proportion of gamma-Proteobacteria and Bacteroidetes in water samples and larval midguts compared to adult female midguts. Only 15 of the 57 bacterial species isolated in this study occurred in both study sites. The number of bacterial species was highest in water samples (28 species from Trelease Woods; 25 species from South Farms), intermediate in larval midguts (13 species from Ae. japonicus; 12 species from Ae. triseriatus; 8 species from Cx. restuans), and lowest in adult female midguts (2 species from Ae. japonicus; 3 species from Ae. triseriatus). These findings suggest that the composition and richness of bacterial communities varies both between habitats and among mosquito species and that the reduction in bacteria diversity during metamorphosis is more evident among bacteria detected using the culture-dependent method.

Container aquatic habitats support a specialized community of macroinvertebrates (e.g. mosquitoes) that feed on microbial communities associated with decaying organic matter. These aquatic habitats are often embedded within and around agricultural lands and are frequently exposed to pesticides. We used a microcosm approach to examine the single and combined effects of two herbicides (atrazine, glyphosate), and three insecticides (malathion, carbaryl, permethrin) on microbial communities of container aquatic habitats. MiSeq sequencing of the V4 region of both bacterial and archaeal 16S rRNA gene was used to characterize the microbial communities of indoor microcosms that were either exposed to each pesticide alone, a mix of herbicides, a mix of insecticides, or a mix of all five insecticides. Individual insecticides but not herbicides reduced the microbial diversity and richness and two insecticides, carbaryl and permethrin, also altered the microbial community structure. A mixture of herbicides had no effect on microbial diversity or structure but a mixture of insecticides or all five pesticides reduced microbial diversity and altered the community structure. These findings suggest that exposure of aquatic ecosystems to individual pesticides or their mixtures can disrupt aquatic microbial communities and there is need to decipher how these changes affect resident macroinvertebrate communities.

Although exposure of mosquito larvae to agricultural chemicals such as herbicides is common and widespread, our understanding of how these chemicals affect mosquito ecology and behavior is limited. This study investigated how an environmentally relevant concentration of two herbicides, atrazine and glyphosate, affects mosquito life history traits. One hundred and fifty (150) first instar Aedes (Stegomyia) aegypti (L.) or Aedes (Stegomyia) albopictus (Skuse) larvae were reared in 1.6 L of live oak leaf (Quercus virginiana) infusion in the presence (5 mg/L) or absence (0 mg/L) of atrazine or glyphosate. The containers were monitored daily to determine the emergence rates, sex ratio, male and female emergence times, and female body size. Emergence rates of A. aegypti from atrazine treatment were significantly higher relative to either glyphosate or control treatments (A. aegypti: atrazine = 93 ± 6 % (±95 % CI), glyphosate = 82 ± 5 %, control = 78 ± 5 %), while emergence rates of A. albopictus in atrazine treatments were significantly higher than in glyphosate treatments but not in controls (A. albopictus: atrazine = 84 ± 5 %, glyphosate = 76 ± 4 %, control = 78 ± 4 %). For both mosquito species, a sex ratio distortion with male bias was observed in control and glyphosate treatments, but not in atrazine treatments (A. aegypti: atrazine = 0.90 ± 0.17 (±SE), glyphosate = 1.63 ± 0.21, control = 1.69 ± 0.26; A. albopictus: atrazine = 1.09 ± 0.08, glyphosate = 1.88 ± 0.12, control = 1.37 ± 0.11). Emergence times for both sexes of the two mosquito species were significantly longer in atrazine treatments compared to glyphosate or control treatments (A. aegypti: females: atrazine = 11.20 ± 0.50 (days ± 95 % CI), glyphosate = 9.71 ± 0.23, control = 9.87 ± 0.21; males: atrazine = 9.46 ± 0.27, glyphosate = 8.80 ± 0.25, control = 8.85 ± 0.24; A. albopictus: females: atrazine = 17.40 ± 1.70, glyphosate = 12.4 ± 0.40, control = 12.5 ± 0.30; males: atrazine = 12.96 ± 0.41, glyphosate = 10.48 ± 0.24, control = 10.64 ± 0.37). For A. albopictus but not A. aegypti, adult females from atrazine treatment had significantly longer wing lengths compared to those from glyphosate or control treatments (A. albopictus: atrazine = 3.06 ± 0.07 (mm ± 95 % CI), glyphosate = 2.80 ± 0.07, control = 2.83 ± 0.06). These results demonstrate the potential for atrazine, a widely used herbicide, to influence epidemiologically relevant life history traits of mosquitoes.

Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse) larvae rely on oxidases to reduce toxicity of water soluble toxins from some senescent tree leaf infusions. The mortality of third instar Ae. aegypti larvae in live oak and pin oak leaf infusions increased significantly in the presence of piperonyl butoxide (PBO), a broad inhibitor of cytochrome P450s (CYPs). In contrast, PBO treatment did not increase mortality in water controls or infusions of northern red oak or sugar maple leaf infusions for Ae. aegypti larvae. A similar pattern was observed for Ae. albopictus larvae, that is, an increase in mortality when CYPs were inhibited in live oak leaf infusions and no increase in sugar maple leaf infusions or water controls. However, the fresh live oak leaf infusion (5 d old) was the most toxic infusion to Ae. aegypti, but appeared less toxic to Ae. albopictus than the older infusions. A direct comparison of survival between the two Aedes species revealed Ae. aegypti exhibited a greater mortality than Ae. albopictus in PBO-treated live oak leaf infusions. These findings suggest that toxic components of some leaf litter in larval habitats may impose cryptic energy costs (detoxification).

Contamination of aquatic habitats with anthropogenic nutrients has been associated with an increase in mosquito larval populations but the underlying mechanisms remain poorly understood. We examined the individual and combined effects of two synthetic fertilizers (ammonium sulfate and potassium chloride) on Aedes albopictus survival, development time, and sex ratio. The bacterial and fungal communities of water samples from different fertilizer treatments were also characterized by MiSeq sequencing of the 16S rRNA gene (bacteria) and internal transcribed spacer 1 (fungi) and their relationship with mosquito survival and development determined. Mosquitoes from ammonium sulfate treatment had significantly lower survival rates and longer development times compared to those from control, potassium chloride or a mixture of the two fertilizers. Fertilizer treatment had no significant effects on Ae. albopictus sex ratio although ammonium sulfate treatment tended to be more biased towards males relative to the other treatments. There were no significant effects of fertilizer treatment on fungal communities. However, potassium chloride treatments had lower bacterial diversity compared to the other treatments and the bacterial community structure of control and potassium chloride treatments differed significantly from that of ammonium sulfate and a mixture of the two fertilizers. Microbial composition but not diversity was significantly associated with mosquito survival and development. These findings suggest that anthropogenic nutrients can have a profound impact on mosquito survival and development. In addition to any potential direct effects on mosquito physiology, our results suggest that fertilizers can act indirectly by disrupting the microbial communities that provide a critical food resource for mosquito larvae.

The mechanisms by which natural populations of vector mosquitoes cope with daily and seasonal fluctuations in temperature are poorly understood. We examined the effect of water temperature on expression of stress and immune-related genes in Aedes aegypti larvae. Aedes aegypti 3rd instars were exposed for 24 h to one of 7 constant temperatures (10 degrees C, 15 degrees C, 20 degrees C, 25 degrees C [control], 32 degrees C, 36 degrees C, or 40 degrees C) and expression of antimicrobial peptides (cecropin, defensin), transferrin, and heat shock proteins (HSP70 and HSP83) quantified by real-time reverse-transcriptase polymerase chain reaction. Cecropin, defensin, and transferrin were overexpressed at 36 degrees C and underexpressed at 15 degrees C and 32 degrees C. HSP83 was overexpressed at 10 degrees C and 40 degrees C and underexpressed at 20 degrees C, while HSP70 was underexpressed at 15 degrees C, 32 degrees C, and 36 degrees C. These findings suggest that antimicrobial peptides can serve as biomarkers of thermal stress and that HSP83 may buffer mosquito larvae against extreme temperatures.

Mosquito larvae experience multiple environmental stressors that may modify how subsequent adults interact with pathogens. We evaluated the effect of larval rearing temperature and intraspecific larval competition on adult mosquito immunity and vector competence for Sindbis virus (SINV). Aedes aegypti larvae were reared at two intraspecific densities (150 and 300 larvae) at 20° C and 30° C and the adults were fed artificially on citrated bovine blood containing 10(5) plaque forming units of SINV. Expression of cecropin, defensin, and transferrin was also evaluated in one- and five-day-old female adults. There was a direct relationship between larval density and SINV infection and dissemination rates at low temperature (20° C) and an inverse relationship between larval density and SINV infection rate at high temperature (30° C). Cecropin was only expressed in five-day-old adults that were raised at high temperature as larvae and was 20-fold over-expressed at low compared to high density treatments. Defensin and transferrin were under-expressed in one-day-old adults and over-expressed in five-day-old adults in all competition-temperature combinations relative to low density treatments at 20° C. These findings suggest that interaction between biotic and abiotic conditions of the larval environment may alter adult mosquito immunity resulting in enhanced vector competence for arboviruses.

The role of toxic component of leaf litter in mediating the outcome of mosquito species interactions is not well documented. To examine the effect of leaf litter toxins on mosquito performance and interspecific interactions, we reared monospecific and heterospecific cultures of Aedes aegypti L. and Aedes albopictus Skuse larvae in microcosms with one of five leaf species and measured the expression of five cytochrome P450 genes and life history traits of the two mosquito species. For both mosquito species, survival to adulthood was significantly higher in black alder, black walnut, and cypress infusion compared to sugar maple and eastern white pine infusion. In pine but not in other leaf treatments, the presence of A. albopictus had significant positive effects on A. aegypti wing length and development time to adulthood. A. albopictus from heterospecific cultures were larger than those from monospecific cultures and were smaller and took longer to develop in pine and sugar maple infusions than in the other infusions. Up regulation of CYP6Z6 and CYP9M9 in A. aegypti and A. albopictus respectively appeared to be closely associated with the deleterious effects of sugar maple infusion on mosquito performance as was the down regulation of CYP6N12 (in A. aegypti) and lack of induction of CYP6Z6 and CYP9M9 (in A. aegypti and A. albopictus respectively) in pine infusion. Results suggest that metabolic capabilities that enable the two species to tolerate natural xenobiotics are associated with a fitness cost.

1. It was determined if the predatory midge Corethrella appendiculata Grabham imposes a fitness cost in a native mosquito, Ochlerotatus triseriatus Say, and an invasive mosquito, Aedes albopictus Skuse. The hypothesis that decreased activity of immature prey in the presence of predator cues is associated with life history costs through all life cycle stages was tested. 2. In experiment 1, individual larvae of O. triseriatus or A. albopictus were raised in the presence or absence of predation cues at two resource levels. Prey were video recorded to detect behavioural responses and to measure development time, size at emergence, and adult longevity. In experiment 2, prey populations were reared in similar environments and the frequency of predator cue additions was varied. 3. Only O. triseriatus reduced its activity in the presence of predation cues. Predation cues were associated with longer immature development times and shorter adult life spans in O. triseriatus, whereas in A. albopictus, the cues were associated with a larger size of emerging adults. 4. In the present study, it was found that behavioural modifications during the larval stage can affect mosquitoes through multiple stages of their complex life cycle. The species-specific behavioural differences are probably attributable to the longer evolutionary history O. triseriatus has with predators, relative to the invasive A. albopictus.

Temperature is a key factor influencing mosquito growth and development and is also known to affect insecticide efficacy. We evaluated the effects of larval rearing temperature and exposure to insecticides on adult mosquito fitness and competence for arboviral infection using Sindbis virus (SINV). We exposed newly hatched larvae of Aedes aegypti to an environmentally realistic level of insecticide malathion at 20°C and 30°C and allowed the resulting adults to feed on SINV-infected blood meal. Exposure to malathion significantly reduced survival to adulthood. Statistically significant interactions between temperature and malathion were observed for body size, estimated population growth, and SINV infection and dissemination. Malathion-exposed Ae. aegypti cohorts had significantly higher population growth at 20°C than at 30°C. Body size decreased with higher temperature and malathion-exposed females were larger than unexposed females at 20°C but not at 30°C. Viral infection and dissemination increased with larval rearing temperature and were higher in malathion-exposed than unexposed females at 30°C but not at 20°C. These results show that environmental factors, including those factors used in controlling mosquitoes, experienced by immature stages have latent effects that continue to adulthood and alter vector competence to arboviruses.

Density-dependent processes such as larval competition may be important regulatory factors among some mosquito species. The application of pesticides used for control may alter these density-dependent interactions with consequences for the number of survivors and associated sublethal and chronic effects on these individuals. We examined how intraspecific competition among larvae and low concentrations of malathion alter Aedes aegypti L. and Aedes albopictus Skuse adult life history traits and competence for arboviruses using Sindbis virus as a model system. Larvae were reared at densities of 150 and 300 larvae per container and in the absence or presence of 0.04 parts per million of malathion, before surviving females were exposed to an infectious blood meal containing 10(5) plaque-forming units/ml Sindbis virus. For both species, competition and the presence of malathion reduced survival to adulthood. The presence of malathion eliminated the negative effects of competition that resulted in lengthened development time and smaller-sized adults. For Ae. aegypti, but not Ae. albopictus, high competition conditions and the presence of malathion independently and not interactively led to an increase in virus dissemination from the midgut. Our results suggest that larval competition and chemical contaminants may influence disease transmission directly by altering adult mosquito fitness and indirectly by altering vector interactions with arboviruses.

The chronic effects of exposure of Culex restuans (Theobald) and Aedes albopictus (Skuse) (Diptera: Culicidae) to low concentrations of malathion were examined by exposing larvae of the two species to four malathion doses at 20, 25, and 30 degrees C and maintaining the resulting adults at 25 degrees C. For both species, a significant temperature by malathion interaction on survival was found. Greater temperatures at the highest malathion dosage had significantly lower survivorship than in other treatments, but this effect was not observed at 20 degrees C. These results suggest that low temperature may buffer against some of the negative effects of malathion. For both species, temperature but not malathion had significant effects on female developmental time to adulthood and adult longevity. Temperature also affected adult female size for Ae. albopictus but not Cx. restuans. Ae. albopictus females developed faster as temperature increased, lived longer when larvae were maintained at 30 degrees C than at 20 degrees C, and were larger when larvae were maintained at 25 degrees C than at 20 or 30 degrees C. Cx. restuans females developed faster at 25 and 30 degrees C than at 20 degrees C and lived longer at 25 degrees C than at 20 or 30 degrees C. The estimated finite rate of increase (lambda') for Cx. restuans was significantly lower at 20 degrees C than at 25 and 30 degrees C, whereas that of Ae. albopictus was significantly influenced by an interaction between temperature and malathion with significantly lower lambda' at 20 degrees C than at 25 and 30 degrees C for all malathion treatments except 0.014 mg liter(-1). Understanding how pesticides interact with abiotic environmental conditions will contribute to management decisions about vector control practices.

Decaying leaves provide a major carbon source for mosquito larvae and leaf litter of different plant species vary in their ability to support mosquito growth, survival, and development. Thus analyzing the effects of leaf species treatment on development time of Aedes albopictus has the potential to discover a plant-based strategy for mosquito control. Here, we employ a statistical model named copula that provides a convenient methodology for modeling multivariate dependence to determine the association between leaf litter identify and mosquito performance. A copula that best fits the association of leaf litters on mosquito performance is selected, and statistical tests are performed to check the adequacy of the copula chosen. By computer-based Monte Carlo methods, a large number of simulated development times are generated under the copula chosen. From the simulated development times, we calculate the percentiles to determine expected development time of female Aedes albopictus under the five different leaf species treatments, and compared the results to those when all the effects of leaf infusion are combined.

Interaction of aquatic stages of coexisting mosquito species may have significant influence on resulting adult mosquito populations. We used two coexisting species, Anopheles gambiae s.s. and Culex quinquefasciatus to investigate whether third instars of one species consumed first instars of the other. First instars of one species were readily consumed by a third instar of the other species irrespective food quantity. DNA of Cx. quinquefasciatus was detected in the eight An. gambiae s.s. third instars presumed to have consumed at least one Cx. quinquefasciatus first instar. Likewise, DNA of An. gambiae s.s. was detected in five of eight Cx. quinquefasciatus third instars presumed to have consumed at least one An. gambiae s.s. first instar. A small number of dead first instars was found in the controls indicating that some larvae in the treatment group may have been consumed after they had died. These findings suggest that intraguild predation between the two species may be common in nature and that it is a facultative process that is not induced by food shortage. The findings further suggest that polymerase chain reaction could be a useful technique in the study of this phenomenon in natural habitats.

An ecological study was conducted at three study sites in Mwea Rice Scheme, Kenya to identify the diverse aquatic habitats in which culicine mosquitoes thrived and to explore the best strategies for mosquito control in the area. During the 11-month study period, ten habitat categories and 11 culicine species mainly dominated by Culex quinquefasciatus (72.0%) and Culex annulioris (17.9%) were identified from pupae and late instars larval samples. Two of the 11 culicine species, Ficalbia (Mimomyia) plumosa and Uranotaenia spp., have not been reported previously in the study area. Rurumi had more habitat types than either of the other study sites but the least number of mosquito species. In contrast, Karima had the least number of habitat types but significantly higher density of early instars than the other study sites. The relative abundance of late instars and pupae did not vary significantly among study sites. The contribution of different habitat types to larval production varied markedly between seasons and among study sites. Paddies and canals were perennial contributors of culicine mosquito larvae while the other habitat types were important mainly during the wet season. Some habitat types such as ditches, seeps, marshes, and fishpond were absent in some study sites but of great significance in other study sites. C. quinquefasciatus was positively associated with turbidity at all study sites and also negatively associated with emergent vegetation and distance to the nearest homestead in Karima, emergent vegetation in Kiuria, and other aquatic invertebrates in Rurumi. C. annulioris was positively associated with emergent vegetation at all study sites and also with depth in Kiuria. These findings indicate that besides rice fields and associated habitats, a diversity of other aquatic habitats contribute to culicine adult mosquito production in the study area and that environmental factors that determine the occurrence of a particular mosquito species may vary significantly even among areas of similar land use. This information is critical when designing and implementing mosquito larval control programs.

Distribution of mosquito larvae in inundated rice fields is poorly known despite its profound implications in implementation of vector control programs. Based on oviposition behavior of gravid females and biotic and abiotic conditions of the rice field, distribution of mosquito larvae within the paddy may vary greatly. As a guide to implementation of mosquito vector control program targeting the aquatic stages in the rice fields in Mwea, studies were conducted to determine the distribution of mosquito larvae within the paddy. Twenty-eight cages measuring 50 cm3 were distributed randomly within the paddy during the transplanting stage of the rice growth cycle, and were examined twice per week up to the flowering stage to determine mosquito oviposition pattern. A total of 17,218 mosquito larvae were collected at the periphery and a further 17,570 at the center of the paddy. These comprised 7,461 larvae from the genus Anopheles and 27,327 from genus Culex. The number of pupae collected at the periphery was 1,004 and 1.5 times greater than the number collected at the center. Significantly higher counts of Anopheles larvae were collected at the center (1.00 +/- 0.11) than at the periphery (0.55 +/- 0.05) of the paddy during transplanting stage, but the difference was not significant during the tillering stage. In contrast, significantly higher numbers of Culex larvae were collected from the periphery (3.09 +/- 0.39) than at the center (2.81 +/- 0.24) of the paddy. More pupae were also collected at the center than at the periphery of the paddy. These findings indicate the distribution of Anopheles and Culex larvae in rice fields to be nonrandom; however, for successful achievement of an integrated vector control program targeting the diverse mosquito fauna occurring in rice fields, there is need to target the whole paddy for larvicidal application.

We determined changes in species composition and densities of immature stages of Anopheles arabiensis mosquitoes in relation to rice growth cycle in order to generate data for developing larval control strategies in rice ecosystems. Experimental rice paddies (6.3m x 3.15m) exposed to natural colonization of mosquitoes were sampled weekly for two rice growing cycles between February 2004 and March 2005. Overall, 21,325 Anopheles larvae were collected, of which 91.9% were 1 st and 2 nd instars and 8.1% were 3 rd and 4 th instars. An. arabiensis was the predominant species (84.1%) with other species, An. pharoensis (13.5%), An. funestus (2.1%), An. coustani (0.3%), and An. maculipalpis (0.1%) accounting for only a small proportion of the anophelines collected. Culex quinquefasciatus (65.7%) was the predominant species among the non-anopheline species. Others species collected included: C. annulioris (9.9%), C. poicilipes (7.3%), C. tigripes (7.2%), C. duttoni (0.6%), Aedes aegypti (5.3%), Ae. cumminsii (3.5%), and Ae. vittatus (0.7%). The densities of the major anopheline species were closely related to rice stage and condition of the rice field. An. arabiensis, the predominant species, was most abundant over a three-week period after transplanting. Low densities of larvae were collected during the late vegetative, reproductive, and ripening phases of rice. An increase in larval density ten days post-transplanting was found to correlate with the application of fertilizer (sulphate of ammonia). Culicine and aedine species densities were significantly higher during the post-harvesting period. Our results suggest that the transplanting stage is favorable for the growth of immature stages of An. arabiensis and provides a narrow window for targeted larval intervention in rice. Journal of Vector Ecology 31 (2): 245-251. 2006.

Despite the growing awareness that larval competition can influence adult mosquito life history traits including susceptibility to pathogens, the net effect of larval competition on human risk of exposure to mosquito-borne pathogens remains poorly understood. We examined how intraspecific larval competition affects dengue-2 virus (DENV-2) extrinsic incubation period and vectorial capacity of its natural vector Aedes albopictus. Adult Ae. albopictus from low and high-larval density conditions were orally challenged with DENV-2 and then assayed for virus infection and dissemination rates following a 6, 9, or 12-day incubation period using real-time quantitative reverse transcription PCR. We then modeled the effect of larval competition on vectorial capacity using parameter estimates obtained from peer-reviewed field and laboratory studies. Larval competition resulted in significantly longer development times, lower emergence rates, and smaller adults, but did not significantly affect the extrinsic incubation period of DENV-2 in Ae. albopictus. Our vectorial capacity models suggest that the effect of larval competition on adult mosquito longevity likely has a greater influence on vectorial capacity relative to any competition-induced changes in vector competence. Furthermore, we found that large increases in the viral dissemination rate may be necessary to compensate for small competition-induced reductions in daily survivorship. Our results indicate that mosquito populations that experience stress from larval competition are likely to have a reduced vectorial capacity, even when susceptibility to pathogens is enhanced.

Competitive interactions between mosquitoes Aedes aegypti (L.) and Aedes albopictus (Skuse) may depend on environmental conditions. Pesticides may alleviate density-dependent competition for limited food, and a differential species response to sublethal concentrations may modify interspecific competition. We tested the hypothesis that exposure to malathion alters interspecific resource competition between these two species. In the absence of malathion, Ae. aegypti survivorship and per capita rate of population change were negatively affected by increasing densities of Ae. albopictus. However, the asymmetrical negative effect ofAe. albopictus on Ae. aegypti was eliminated in the presence of malathion. In addition, the presence of malathion resulted in shorter development time compared with the controls. The relative importance of pesticide-mediated coexistence in nature has not been evaluated, so its role in mediating coexistence is unclear; however, these findings underscore the potential of environmental concentrations of malathion, and perhaps other pesticides to facilitate coexistence between species.

Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse) larvae rely on oxidases to reduce toxicity of water soluble toxins from some senescent tree leaf infusions. The mortality of third instar Ae. aegypti larvae in live oak and pin oak leaf infusions increased significantly in the presence of piperonyl butoxide (PBO), a broad inhibitor of cytochrome P450s (CYPs). In contrast, PBO treatment did not increase mortality in water controls or infusions of northern red oak or sugar maple leaf infusions for Ae. aegypti larvae. A similar pattern was observed for Ae. albopictus larvae, that is, an increase in mortality when CYPs were inhibited in live oak leaf infusions and no increase in sugar maple leaf infusions or water controls. However, the fresh live oak leaf infusion (5 d old) was the most toxic infusion to Ae. aegypti, but appeared less toxic to Ae. albopictus than the older infusions. A direct comparison of survival between the two Aedes species revealed Ae. aegypti exhibited a greater mortality than Ae. albopictus in PBO-treated live oak leaf infusions. These findings suggest that toxic components of some leaf litter in larval habitats may impose cryptic energy costs (detoxification).

Stressful environmental conditions during mosquito larval development may enhance susceptibility of adult mosquitoes to viral pathogens. Although anti-viral defense system in mosquitoes remains uncertain, stress-related enhancement of mosquito susceptibility to viral pathogens may be due to alteration of signaling pathways such as the Toll and immune deficiency (IMD) pathways. To test the influence of larval density and Sindbis virus (SINV) infection on mosquito Toll/Imd pathways, 100 or 200 Aedes aegypti larvae were reared in 2L of live oak (Quercus virginiana) leaf infusion and the adults were fed on SINV-infected (treatments) or non-infected (controls) bovine blood. SINV infection status and expression of genes encoding three antimicrobial peptides (cecropin, defensin, diptericin), an iron-binding protein (transferrin), and four regulators of Toll/Imd pathways (caspar, cactus, Rel1A, Rel2) were quantified by RT-qPCR at 7 and 14days post blood meals. Irrespective of larval density, females incubated for 14days after an infectious blood meal had significantly higher SINV titers compared to females from low density treatments that were incubated for 7days. For both larval densities and time intervals, there was significant down-regulation of the Toll/Imd regulator genes in SINV-infected mosquitoes compared to controls. At day 7 post-infection, there was significant down-regulation of cecropin, defensin, diptericin and transferrin in SINV-infected mosquitoes at low larval density but this effect was only observed for diptericin at high larval density. These genes remained suppressed on day 14, except cecropin which was significantly up-regulated at both larval densities, and transferrin which was similar to controls at low larval density. We conclude that SINV infection suppresses Toll/Imd pathways, but high larval density enables SINV to attain maximum titers in Ae. aegypti much earlier compared to low density treatments despite the up-regulation of cecropin.

The effects of anthropogenic chemical contaminants on aquatic organisms are largely influenced by underlying environmental conditions. This study evaluated how larval rearing temperature influences the impact of malathion on the fitness of the yellow fever mosquito Aedes aegypti. Larvae were exposed to water control, and low (0.03mg/L) or high (0.05mg/L) malathion dose at 20°C, 25°C and 30°C and emergence rate, time to emergence, female fecundity and expression of genes encoding two antimicrobial peptides (defensin, cecropin) and an iron-binding protein (transferrin) quantified. High malathion dose at 25°C and 30°C resulted in significantly lower emergence rates compared to control and low malathion dose but this effect was not observed at 20°C. Female time to emergence was inversely proportional to temperature and was significantly shorter in high malathion dose than in control and low malathion dose at 25°C and 30°C but not at 20°C. Regardless of temperature treatment, females from high malathion dose were significantly larger and laid more eggs than their counterparts in control and low malathion dose. Relative to the controls, two immune genes were significantly over-expressed in adult females from malathion-exposed treatments at 20°C (defensin and cecropin) and 25°C (defensin and transferrin) and one gene (defensin) was significantly under-expressed at 30°C. These findings suggest that larval rearing temperature can modify the effect of malathion on fitness traits in mosquitoes.

Mosquito larvae face numerous biotic and abiotic challenges that affect their development and survivorship, as well as adult fitness. We conducted two experiments under semi-natural conditions to evaluate the effects of intraspecific competition, nutrient limitation and sub-lethal doses of malathion on individual life history traits in adult Culex pipiens (Diptera: Culicidae). In the first experiment, larvae of Cx. pipiens were reared at different intraspecific densities and exposed to sub-lethal doses of malathion. In the second experiment, different intraspecific densities of Cx. pipiens larvae were reared under conditions of low or high larval nutrients, and subsequent adults were fed on either water or 10% sucrose solution. Malathion treatment had relatively minor effects compared with density, which had significant negative effects on development rate, survivorship to adulthood, body size (wing length) and longevity. As larval density increased, a sex ratio distortion in survivorship to adulthood emerged, in which a bias towards males was apparent. Nutrient-rich larval environments alleviated, in part, the effects of increasing density and extended the lifespan of mosquitoes fed on water and 10% sucrose. Density-dependent alterations in adult longevity attributable to the larval environment are complex and show contrasting results depending on interactions with other environmental factors. This study suggests that larval resource availability and competition influence Cx. pipiens population growth correlates and have lasting effects on traits that relate to a mosquito's ability to vector pathogens.

The introduction of Aedes albopictus (Skuse) in the United States has been associated with declines in abundance of resident mosquito species, presumably because of resource competition, as larvae of Ae. albopictus have been illustrated as superior competitors under certain resource conditions. We evaluated the hypothesis that varying the type and ratio of two food resources (Foxtail grass: American elm) alters the competitive outcome of Ae. albopictus and Culex pipiens (L.). We measured survivorship, development time, size, and adult longevity, and estimated the population growth index (A') of populations raised both alone and in equal number with the interspecific competitor, across five ratios of the two food resources. Competition was asymmetric with Ae. albopictus, the superior competitor across all resource treatments; however, the competitive advantage Ae. albopictus had over Cx. pipiens was reduced as grass became the predominant resource. With elm as the predominant resource, the population growth index (A') for both Ae. albopictus and Cx. pipiens was lower in intraspecific and interspecific competition treatments, respectively. The treatments also impacted adult life history, as life spans of both Ae. albopictus and Cx. pipiens varied when they emerged from larval conditions with different resource and competition treatments. We discuss the possible differences in the two species' efficiencies in exploiting the two resource types. Despite some resource conditions alleviating the competitive effects of Ae. albopictus on Cx. pipiens, competition remained asymmetric; thus, additional mechanisms are likely operating under field conditions when the two species coexist.

Background Pre-adult stages of malaria vectors in semi-arid areas are confronted with highly variable and challenging climatic conditions. The objective of this study was to determine which larval habitat types are most productive in terms of larval densities in the dry and wet seasons within semi-arid environments, and how vector species productivity is partitioned over time. Methods Larval habitats were mapped and larvae sampled longitudinally using standard dipping techniques. Larvae were identified to species level morphologically using taxonomic keys and to sub-species by polymerase chain reaction (PCR) methods. Physical characteristics of larval habitats, including water depth, turbidity, and presence of floating and emergent vegetation were recorded. Water depth was measured using a metal ruler. Turbidity, pH, conductivity, dissolved oxygen, temperatures salinity and total dissolved solids (TDS) were measured in the field using the hand-held water chemistry meters. Results Mean larval densities were higher in the dry season than during the wet season but the differences in density were not statistically significant (F = 0.04, df = 1, p = 0.8501). Significantly higher densities of larvae were collected in habitats that were shaded and holding turbid, temporary and still water. Presence of emergent or floating vegetation, habitat depth, habitat size and habitat distance to the nearest house did not significantly affect larval density in both villages. There was a weakly positive relationship between larval density and salinity (r = 0.19, p < 0.05), conductivity (r = 0.05, p = 0.45) and total dissolved solids (r = 0.17, p < 0.05). However, the relationship between water temperature and larval density was weakly negative (r = 0.15, p = 0.35). All statistical tests were significant at alpha = 0.05. Conclusion Breeding of malaria vector mosquitoes in Baringo is driven by predominantly human-made and permanent breeding sites in which Anopheles arabiensis and Anopheles funestus breed at a low level throughout the year. Permanent water sources available during the dry season serve as inocula by providing "larval seed" to freshly formed rain-fed habitats during the rainy season. The highly localized and focal nature of breeding sites in these semi-desert environments provides a good opportunity for targeted larval control since the habitats are few, well-defined and easily traceable.

Background The diversity and abundance of Anopheles larvae has significant influence on the resulting adult mosquito population and hence the dynamics of malaria transmission. Studies were conducted to examine larval habitat dynamics and ecological factors affecting survivorship of aquatic stages of malaria vectors in three agro-ecological settings in Mwea, Kenya. Methods Three villages were selected based on rice husbandry and water management practices. Aquatic habitats in the 3 villages representing planned rice cultivation (Mbui Njeru), unplanned rice cultivation (Kiamachiri) and non-irrigated (Murinduko) agro-ecosystems were sampled every 2 weeks to generate stage-specific estimates of mosquito larval densities, relative abundance and diversity. Records of distance to the nearest homestead, vegetation coverage, surface debris, turbidity, habitat stability, habitat type, rice growth stage, number of rice tillers and percent Azolla cover were taken for each habitat. Results Captures of early, late instars and pupae accounted for 78.2%, 10.9% and 10.8% of the total Anopheles immatures sampled (n = 29,252), respectively. There were significant differences in larval abundance between 3 agro-ecosystems. The village with 'planned' rice cultivation had relatively lower Anopheles larval densities compared to the villages where 'unplanned' or non-irrigated. Similarly, species composition and richness was higher in the two villages with either 'unplanned' or limited rice cultivation, an indication of the importance of land use patterns on diversity of larval habitat types. Rice fields and associated canals were the most productive habitat types while water pools and puddles were important for short periods during the rainy season. Multiple logistic regression analysis showed that presence of other invertebrates, percentage Azolla cover, distance to nearest homestead, depth and water turbidity were the best predictors for Anopheles mosquito larval abundance. Conclusion These results suggest that agricultural practices have significant influence on mosquito species diversity and abundance and that certain habitat characteristics favor production of malaria vectors. These factors should be considered when implementing larval control strategies which should be targeted based on habitat productivity and water management.

Mosquito larval development occurs in aquatic habitats that are directly or indirectly exposed to chemical contaminants. Little is known about how interaction of these chemicals with other biotic and abiotic stressors impact mosquito populations. We used two levels of nutrient (low and high) and four larval densities (10, 20, 30, 40) to examine the effects of low concentrations of insecticide malathion, on Culex pipiens L. mosquitoes experiencing stress from larval competition. Addition of malathion at the high nutrient condition enhanced survival with increasing larval densities, but this effect was not observed at low nutrient condition. Males exposed to malathion were significantly larger than those from control treatments while the effect of malathion on size of females varied with larval density and the level of nutrients. Larval exposure to malathion and low nutrient resulted in significantly larger females with increases in larval densities compared with other treatments. The effect of malathion on male longevity varied with larval density and amount of nutrients. At higher densities, male longevity was consistently higher in low nutrient than in high nutrient conditions and addition of malathion in high nutrient treatment increased male longevity at the highest density. These effects are most likely attributable to release from competition among survivors after mortality from malathion and density-dependent effects. We conclude that biotic conditions of the larval habitat can alter the impact of low concentrations of chemical contaminants on mosquito populations in ways that may influence the pattern of disease transmission and the outcome of vector control efforts.

Changes in weather patterns especially rainfall affects the distribution and densities of mosquitoes. The objective of this study was to describe mosquito aquatic habitats, to determine larval abundance, species composition, and habitat types found in Kasayani village of Kibwezi division.A cross-sectional survey of mosquito larval habitats was conducted in Kasayani village in Kibwezi division to determine species composition, larval abundance, and habitat types found in this village. This survey was conducted during the rainy season in November and December 2006 and during the dry season in February and March 2007. Larvae were collected using the standard dipping technique and a total of 24 habitats were sampled. The primary habitats identified were water reservoir tanks, puddles, temporary pools, and tyre tracks.A total of 2660 mosquito larvae were collected of which 2140 (80.45%) were culicines, 503 (18.91%) were Anopheles and 17 (0.64%) were pupae. For culicines, 1787 (83.5%) were categorized as early instars and 353 (16.5%) were as late instars while in the Anopheles, 425 (84.49%) were classified as early instars and 78 (15.51%) were late instars. Morphological identification of the III and IV instar larvae by use of microscopy yielded 16.24% (n = 70) Anopheles gambiae complex, 1.16% (n=5) An. funestus, 0.70% (n=3) An. coustani, 42.46% (n=183) Culex quinquefasciatus, 6.26% (n = 27) Cx. duttoni, and 33.18% (n=143) Ae. aegypti. Puddles, tyre tracks and pools had highly turbid water while water reservoir tanks had clear water. Anopheles gambiae and Cx. quinquefasciatus were found in all habitat categories while Ae. aegypti were found only in water storage tanks.The mosquito larval densities indicate that the inhabitants of this village are at risk of mosquito-borne diseases including malaria, which is one of the greatest causes of morbidity and mortality in this area. Furthermore, mosquito control measures targeting both the mosquito immatures and adults should be enhanced especially during the rainy season to ensure maximum protection of the inhabitants.

Studies were conducted to determine the contribution of diverse larval habitats to adult Anopheles arabiensis Patton and Culex quinquefasciatus Say production in a rice land agro-ecosystem in Mwea, Kenya. Two sizes of cages were placed in different habitat types to investigate the influence of non-mosquito invertebrates on larval mortalities and the contribution of each habitat type to mosquito productivities, respectively. These emergence traps had fine netting material covers to prevent adult mosquitoes from ovipositing in the area covered by the trap and immature mosquitoes from entering the cages. The emergence of Anopheles arabiensis in seeps, tire tracks, temporary pools, and paddies was 10.53%, 17.31%, 12.50%, and 2.14%, respectively, while the corresponding values for Cx. quinquefasciatus were 16.85% in tire tracks, 8.39% in temporary pools, and 5.65% in the paddies from 0.125 m3 cages during the study. Cages measuring 1 m3 were placed in different habitat types which included paddy, swamp, marsh, ditch, pool, and seep to determine larval habitat productivity. An. arabiensis was the predominant anopheline species (98.0%, n = 232), although a few Anopheles coustani Laveran (2.0%, n = 5) emerged from the habitats. The productivity for An. arabiensis larvae was 6.0 mosquitoes per m2 for the temporary pools, 5.5 for paddy, 5.4 for marsh, 2.7 for ditch, and 0.6 for seep. The Cx. quinquefasciatus larval habitat productivity was 47.8 mosquitoes per m2 for paddies, 35.7 for ditches, 11.1 for marshes, 4.2 for seeps, 2.4 for swamps, and 1.0 for temporary pools. Pools, paddy, and marsh habitat types were the most productive larval habitats for An. arabiensis while paddy, ditch, and marsh were the most productive larval habitats for Cx. quinquefasciatus. The most common non-mosquito invertebrate composition in the cages included Dytiscidae, Notonectidae, Belostomatidae, and Ephemerellidae, and their presence negatively affected the number of emergent mosquitoes from the cages. In conclusion, freshly formed habitats are the most productive aquatic habitats, while old and more permanent habitats are the least productive due to natural regulation of mosquito immatures.

Water quality of aquatic habitats is an important determinant of female mosquito oviposition and successful larval development. This study examined the influence of environmental covariates on Anopheles arabiensis mosquito abundance in the Mwea Irrigation Scheme, Central Province of Kenya, prior to implementation of a malaria vector control program. Experimental rice plots were used to examine the environmental covariates responsible for regulating abundance and diversity of the aquatic stages of malaria vectors. Mosquito larval sampling and water quality analysis were done weekly from the flooding stage to the rice maturation stage. Sampling for mosquito larvae was conducted using standard dipping technique. During each larval collection, environmental covariates such as pH, temperature, conductivity, salinity, dissolved oxygen, water depth, and rice stage were measured. Anopheles arabiensis larval density was highest between 1 wk before transplanting and 4 wk after transplanting with peaks at weeks 0, 3, and 8. The fluctuation in values of the various environmental covariates showed characteristic patterns in different rice growth phases depending on the changes taking place due to the agronomic practices. Using a backward linear regression model, the factors that were found to be associated with abundance of An. arabiensis larvae at any of the rice growing phases included the following: dissolved oxygen, pH, turbidity, water depth, rice height, number of rice tillers, salinity, conductivity, and temperature. The environmental covariates associated with abundance of An. arabiensis were associated with early vegetative stage of the rice growth. For effective control of developmental stages of mosquito larvae, the application of larvicides should be done at the vegetative stage and the larvicides should persist until the beginning of the reproductive stage of the rice.

Studies were conducted to determine the effect of ammonium sulfate (AM) and muriate of potash (MOP) fertilizers on survival and development of immature stages of Culex quinquefasciatus Say, a major vector of Bancroftian filariasis in Africa.Twenty I instar larvae each were added in four doses of each fertilizer dissolved in one litre of deionised water and in one litre of deionised water as a control in replicates of five. The larvae were monitored every morning throughout their life and the numbers of each instar surviving were recorded. The experiments were discontinued when all the larvae had died or emerged into adults.An analysis of variance test and Tukey's HSD test revealed a significant impact of fertilizers on survival and development of aquatic stages of Cx. quinquefasciatus. Ammonium sulfate accounted for up to 40% mortality rate, and one week delay in development time and this effect was both instar and dose dependent. None of the MOP dosages had significant impact on survival of immatures of Cx. quinquefasciatus and only the higher dosages showed significant impact on development time but in significantly lower magnitudes compared with similar dosages of ammonium sulfate.These findings demonstrate the toxic effect of fertilizers on immature stages of Cx. quinquefasciatus contrary to field observations.

The succession of mosquito species and abiotic factors affecting their distribution and abundance in rice (Oryza spp.) fields was investigated over a 16-wk rice growing cycle covering the period between January and May 2006. Fifteen experimental rice plots were sampled for mosquito larvae and characterized based on rice height, number of tillers, floating vegetation cover, water depth, water temperature, turbidity, salinity, pH, dissolved oxygen, total dissolved solids, and conductivity. Microscopic identification of 3,025 larvae yielded nine mosquito species predominated by Anopheles arabiensis Patton (45.0%), Culex quinquefasciatus Say (35.8%), Anopheles pharoensis Theobald (9.0%) and Ficalbia splendens Theobald (7.1%). Other species, including Anopheles rufipes Gough, Anopheles coustani Laveran, Anonopheles maculipalpis Giles, Culex annulioris Theobald, and Culex poicilipes Theobald made up 3.1% of the total collection. Anopheles gambiae s.l., Cx. quinquefasciatus, and An. pharoensis occurred throughout the cycle, but they were more abundant up to 4 wk posttransplanting with peaks after fertilizer application. As rice plants became established, three groups of mosquitoes were recognized: the first groups included An. rufipes, Fl. splendens, and Cx. annulioris, which occurred throughout much of the second half of the rice cycle, whereas the second group included Cx. poicilipes, which was found in the middle of the rice cycle. An. coustani and An. maculipalpis formed the third group occurring toward the end of the cycle. Dissolved oxygen, number of tillers, and rice height were negatively associated with the abundance ofAn. arabiensis and Cx. quinquefasciatus larvae. In addition, Cx. quinquefasciatus also was associated with water depth (-ve) and turbidity (+ve). Abundance of An. pharoensis larvae was significantly associated with water temperature (+ve), the number of tillers (-ve), and rice height (-ve), whereas Fl. splendens was significantly associated with the number of tillers (+ve). The results demonstrate a complex nature of the interactions between some of the factors in the ecosystem and mosquito species abundance and calls for time-dependent and species-specific mosquito control operations.

Background & objectives: A study was conducted to characterise larval habitats and to determine spatialheterogeneity of the Anopheles mosquito larvae. The study was conducted from May to June 1999 innine villages along the Kenyan coast.Methods: Aquatic habitats were sampled by use of standard dipping technique. The habitats werecharacterised based on size, pH, distance to the nearest house, coverage of canopy, surface debris, algaeand emergent plants, turbidity, substrate, and habitat type.Results: A total of 110 aquatic habitats like stream pools (n = 10); puddles (n = 65); tire tracks (n =5); ponds (n = 5) and swamps (n = 25) were sampled in nine villages located in three districts of theKenyan coast. A total of 7,263 Anopheles mosquito larvae were collected, 63.9% were early instarsand 36.1% were late instars. Morphological identification of the III and IV instar larvae by use ofmicroscopy yielded 90.66% (n = 2,377) Anopheles gambiae Complex, 0.88% (n = 23) An. funestus,An. coustani 7.63% (n = 200), An. rivulorum 0.42% (n = 11), An. pharoensis 0.19% (n = 5), An.swahilicus 0.08% (n = 2), An. wilsoni 0.04% (n = 1) and 0.11% (n = 3) were unidentified. A subset ofthe An. gambiae Complex larvae identified morphologically, was further analysed using rDNA-PCRtechnique resulting in 68.22% (n = 1,290) An. gambiae s.s., 7.93% (n = 150) An. arabiensis and 23.85%(n = 451) An. merus. Multiple logistic regression model showed that emergent plants (p = 0.019), andfloating debris (p = 0.038) were the best predictors of An. gambiae larval abundance in these habitats.Interpretation & conclusion: Habitat type, floating debris and emergent plants were found to be thekey factors determining the presence of Anopheles larvae in the habitats. For effective larval control,the type of habitat should be considered and most productive habitat type be given a priority in themosquito abatement programme

Introduction of irrigation projects in developing nations has often been blamed for aggravating the problem of mosquito-borne diseases by creating ideal larval habitats for vector mosquitoes. However, whereas several studies have demonstrated the relationship between malaria vectors and irrigation, little work has been done on culicine mosquitoes despite their potential in transmission of filariasis and arboviruses and their significant biting nuisance in these areas. This study examined the diversity of Culex mosquito fauna and their larval habitats at two sites (Murinduko and Kiamachiri) in Mwea, Kenya over a 12-month period. The habitat types present at each site within a 200-meter radius around the study village, including randomly selected paddies and canals, were sampled every two weeks to examine the relationship between vegetation cover, water depth, turbidity, and Culex larval counts. Ten culicine species belonging to four genera were identified, with 73.1% of the total collection comprising of Culex duttoni and Cx. quinquefasciatus. Other species collected included Cx. annulioris, Cx. poicilipes, Cx. cinereus, Cx. tigripes, Cx. trifilatus, Aedes spp., Coquilettidia fuscopennata, and Ficalbia splendens. Murinduko was more diverse than Kiamachiri in terms of species richness (10 versus 7 species) and larval habitat diversity (11 versus 8 habitat types). Paddies, canals, and rain pools were the most diverse habitats in terms of species richness, and ditches, rock pools, and tree holes were the least diverse. Principal component and correlation analyses showed a strong association between three Culex species and the measured habitat characteristics. Culex poicilipes was strongly associated with floating vegetation, Cx. annulioris with clean water containing emergent vegetation, and Cx. quinquefasciatus was associated with turbid water. Seasonal changes in larval counts in water reservoirs and pool and ditch habitats were closely associated with rainfall. These findings provide important information on larval habitat preference for different Culex species, which will be useful in designing and implementation of larval control operations.

Background The survivorship and distribution of Anopheles arabiensis larvae and pupae was examined in a rice agro-ecosystem in Mwea Irrigation Scheme, central Kenya, from August 2005 to April 2006, prior to implementation of larval control programme. Methods Horizontal life tables were constructed for immatures in semi-field condition. The time spent in the various immature stages was determined and survival established. Vertical life tables were obtained from five paddies sampled by standard dipping technique. Results Pre-adult developmental time for An. arabiensis in the trays in the experimental set up in the screen house was 11.85 days from eclosion to emergence. The mean duration of each instar stage was estimated to be 1.40 days for first instars, 2.90 days for second instars, 1.85 days for third instars, 3.80 days for fourth instars and 1.90 days for pupae. A total of 590 individuals emerged into adults, giving an overall survivorship from L1 to adult emergence of 69.4%. A total of 4,956 An. arabiensis immatures were collected in 1,400 dips throughout the sampling period. Of these, 55.9% were collected during the tillering stage, 42.5% during the transplanting period and 1.6% during the land preparation stage. There was a significant difference in the An. arabiensis larval densities among the five stages. Also there was significant variation in immature stage composition for each day's collection in each paddy. These results indicate that the survival of the immatures was higher in some paddies than others. The mortality rate during the transplanting was 99.9% and at tillering was 96.6%, while the overall mortality was 98.3%. Conclusion The survival of An. arabiensis immatures was better during the tillering stage of rice growth. Further the survival of immatures in rice fields is influenced by the rice agronomic activities including addition of nitrogenous fertilizers and pesticides. For effective integrated vector management, the application of larvicides should target An. arabiensis larvae at the tillering stage (early vegetative stage of rice) when their survival in the aquatic habitats is high to significantly reduce them and the larvicides should be long-lasting to have a significant impact on the malaria vector productivity on the habitats.

Background For remote identification of mosquito habitats the first step is often to construct a discrete tessellation of the region. In applications where complex geometries do not need to be represented such as urban habitats, regular orthogonal grids are constructed in GIS and overlaid on satellite images. However, rice land vector mosquito aquatic habitats are rarely uniform in space or character. An orthogonal grid overlaid on satellite data of rice-land areas may fail to capture physical or man-made structures, i.e paddies, canals, berms at these habitats. Unlike an orthogonal grid, digitizing each habitat converts a polygon into a grid cell, which may conform to rice-land habitat boundaries. This research illustrates the application of a random sampling methodology, comparing an orthogonal and a digitized grid for assessment of rice land habitats. Methods A land cover map was generated in Erdas Imagine V8.7® using QuickBird data acquired July 2005, for three villages within the Mwea Rice Scheme, Kenya. An orthogonal grid was overlaid on the images. In the digitized dataset, each habitat was traced in Arc Info 9.1®. All habitats in each study site were stratified based on levels of rice stage Results The orthogonal grid did not identify any habitat while the digitized grid identified every habitat by strata and study site. An analysis of variance test indicated the relative abundance of An. arabiensis at the three study sites to be significantly higher during the post-transplanting stage of the rice cycle. Conclusion Regions of higher Anopheles abundance, based on digitized grid cell information probably reflect underlying differences in abundance of mosquito habitats in a rice land environment, which is where limited control resources could be concentrated to reduce vector abundance.

Lentic freshwater systems including those inhabited by aquatic stages of mosquitoes derive most of their carbon inputs from terrestrial organic matter mainly leaf litter. The leaf litter is colonized by microbial communities that provide the resource base for mosquito larvae. While the microbial biomass associated with different leaf species in container aquatic habitats is well documented, the taxonomic composition of these microbes and their response to common environmental stressors is poorly understood. We used indoor aquatic microcosms to determine the abundances of major taxonomic groups of bacteria in leaf litters from seven plant species and their responses to low concentrations of four pesticides with different modes of action on the target organisms; permethrin, malathion, atrazine and glyphosate. We tested the hypotheses that leaf species support different quantities of major taxonomic groups of bacteria and that exposure to pesticides at environmentally relevant concentrations alters bacterial abundance and community structure in mosquito larval habitats. We found support for both hypotheses suggesting that leaf litter identity and chemical contamination may alter the quality and quantity of mosquito food base (microbial communities) in larval habitats. The effect of pesticides on microbial communities varied significantly among leaf types, suggesting that the impact of pesticides on natural microbial communities may be highly complex and difficult to predict. Collectively, these findings demonstrate the potential for detritus composition within mosquito larval habitats and exposure to pesticides to influence the quality of mosquito larval habitats.

Background We examined algorithms for malaria mapping using the impact of reflectance calibration uncertainties on the accuracies of three vegetation indices (VI)'s derived from QuickBird data in three rice agro-village complexes Mwea, Kenya. We also generated inferential statistics from field sampled vegetation covariates for identifying riceland Anopheles arabiensis during the crop season. All aquatic habitats in the study sites were stratified based on levels of rice stages; flooded, land preparation, post-transplanting, tillering, flowering/maturation and post-harvest/fallow. A set of uncertainty propagation equations were designed to model the propagation of calibration uncertainties using the red channel (band 3: 0.63 to 0.69 μm) and the near infra-red (NIR) channel (band 4: 0.76 to 0.90 μm) to generate the Normalized Difference Vegetation Index (NDVI) and the Soil Adjusted Vegetation Index (SAVI). The Atmospheric Resistant Vegetation Index (ARVI) was also evaluated incorporating the QuickBird blue band (Band 1: 0.45 to 0.52 μm) to normalize atmospheric effects. In order to determine local clustering of riceland habitats Gi*(d) statistics were generated from the ground-based and remotely-sensed ecological databases. Additionally, all riceland habitats were visually examined using the spectral reflectance of vegetation land cover for identification of highly productive riceland Anopheles oviposition sites. Results The resultant VI uncertainties did not vary from surface reflectance or atmospheric conditions. Logistic regression analyses of all field sampled covariates revealed emergent vegetation was negatively associated with mosquito larvae at the three study sites. In addition, floating vegetation (-ve) was significantly associated with immature mosquitoes in Rurumi and Kiuria (-ve); while, turbidity was also important in Kiuria. All spatial models exhibit positive autocorrelation; similar numbers of log-counts tend to cluster in geographic space. The spectral reflectance from riceland habitats, examined using the remote and field stratification, revealed post-transplanting and tillering rice stages were most frequently associated with high larval abundance and distribution. Conclusion NDVI, SAVI and ARVI generated from QuickBird data and field sampled vegetation covariates modeled cannot identify highly productive riceland An. arabiensis aquatic habitats. However, combining spectral reflectance of riceland habitats from QuickBird and field sampled data can develop and implement an Integrated Vector Management (IVM) program based on larval productivity.