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Abstract Strategic, sustainable, and ecofriendly alternatives to chemical pesticides are needed to effectively control mosquitoes and reduce the incidence of their vectored diseases. We evaluated several Brassicaceae (mustard family) seed meals as sources of plant derived isothiocyanates produced from the enzymatic hydrolysis of biologically inactive glucosinolates for the control of Aedes aegypti (L., 1762). Five defatted seed meals (Brassica juncea (L) Czern., 1859, Lepidium sativum L., 1753, Sinapis alba L., 1753, Thlaspi arvense L., 1753, and Thlaspi arvense—heat inactivated and three major chemical products of enzymatic degradation (allyl isothiocyanate, benzyl isothiocyanate and 4-hydroxybenzyl isothiocyanate) were assayed to determine toxicity (LC50) to Ae. aegypti larvae. All seed meals except the heat inactivated T. arvense were toxic to mosquito larvae. L. sativum seed meal was the most toxic treatment to larvae (LC50 = 0.04 g/120 mL dH2O) at the 24-h exposure. At the 72-h evaluation, the LC50 values for B. juncea, S. alba and T. arvense seed meals were 0.05, 0.08 and 0.1 g/120 mL dH2O, respectively. Synthetic benzyl isothiocyanate was more toxic to larvae 24-h post treatment (LC50 = 5.29 ppm) compared with allyl isothiocyanate (LC50 = 19.35 ppm) and 4-hydroxybenzyl isothiocyanate (LC50 = 55.41 ppm). These results were consistent with the higher performance of the benzyl isothiocyanate producing L. sativum seed meal. Isothiocyanates produced from seed meals were more effective than the pure chemical compounds, based on calculated LC50 rates. Using seed meal may provide an effective method of delivery for mosquito control. This is the first report evaluating the efficacy of five Brassicaceae seed meals and their major chemical constituent against mosquito larvae and demonstrates how natural compounds from Brassicaceae seed meals can serve as a promising ecofriendly larvicides to control mosquitoes.

Abstract Dormant propagules can provide a rapid colonization source for temporary aquatic habitats and set the trajectory for community dynamics, yet the egg banks of stormwater management systems have received little attention. We asked which species hatched from the sediment of drainage ditches in Champaign County, IL, and found bdelloid rotifers and ostracods (Heterocypris incongruens) to be the most common taxa. These sites also are colonized by mosquitoes, and we established laboratory experiments to examine interspecific interactions between common co‐occurring taxa. Culex restuans larvae were reared in the presence or absence of H. incongruens at two intra‐ and interspecific densities (20 or 40 total individuals) and their survivorship to adulthood, development time to adulthood, adult body size, and sex ratio were determined. Survival for Cx. restuans was significantly lower at high larval density than at low larval density in both treatments. Culex restuans larvae reared in the presence of H. incongruens had a shorter development time to adulthood and emerged as larger adults compared to those reared in the absence of H. incongruens. The sex ratios in the H. incongruens treatments were female‐biased whereas those in the Culex‐only treatments were male‐biased. These differences may have epidemiological implications, as only female mosquitoes serve as disease vectors. Our results emphasize the importance of understanding interspecific interactions in influencing larval mosquito development traits.

Mosquito resistance to microbial infections, including fungal entomopathogens that are selected for mosquito control, depend on a range of antimicrobial effectors, among them antimicrobial peptides (AMPs). These short peptides, along the antimicrobial effector lysozyme, act by disrupting the microbial cell membrane or by interfering with microbial physiological processes. While the induction of AMPs and lysozyme during fungal entomopathogenic infections have been reported, their contribution to the mosquito antifungal response has not been evaluated. In this study, we assessed the induction of Ae. aegypti AMPs and lysozyme genes at two points of infection and against distinct entomopathogenic fungi. Our results indicate that fungal infection elicits the expression of cecropin, defensin, diptericin, holotricin, and lysozyme, but do not affect those of attacin or gambicin. We further evaluated the role of these antimicrobial effectors via RNAi-based depletion of select AMPs during challenges with two entomopathogenic fungi. Our results reveal that AMPs and lysozyme are critical to the antifungal response, acting in concert, rather than individually, to potentiate their antimicrobial effect against entomopathogenic fungi. This study further contributes to a better understanding of the mechanisms that confer resistance to entomopathogenic fungi in an important mosquito vector.

The cabbage looper Trichoplusia ni is an important agricultural pest worldwide and is frequently used as a model organism for assessing entomopathogenic fungi virulence, though few studies have measured the host response repertoire to fungal biocontrol agents. Here, we quantified the immune response of T. ni larvae following exposure to two entomopathogenic fungal species: Beauveria bassiana and Cordyceps javanica. Results from our study demonstrate that T. ni larvae exposed to fungal entomopathogens had higher total phenoloxidase activity compared to controls, indicating that the melanization cascade is one of the main immune components driving defense against fungal infection and contrasting observations from other insect–fungi interaction studies. We also observed differences in host response depending on the species of entomopathogenic fungi, with significantly higher induction observed during infections with B. bassiana than with C. javanica. Larvae exposed to B. bassiana had an increased expression of genes involved in prophenoloxidase response and the Imd, JNK, and Jak/STAT immune signaling pathways. Our results indicate a notable absence of Toll pathway-related responses, further contrasting results to other insect–fungi pathosystems. Important differences were also observed in the induction of antimicrobial effectors, with B. bassiana infections eliciting three antimicrobial effectors (lysozyme, gloverin, and cecropin), while C. javanica only induced cecropin expression. These results provide insight into the host response strategies employed by T. ni for protection against entomopathogenic fungi and increase our understanding of insect–fungal entomopathogen interactions, aiding in the design of more effective microbial control strategies for this important agricultural pest.

Abstract Insecticide resistance has emerged as a persistent threat to the fight against vector-borne diseases. We compared the gut microbiota of permethrin-selected (PS) strain of Aedes aegypti relative to the parent (KW) strain from Key West, Florida. Bacterial richness but not diversity was significantly higher in PS strain compared to KW strain. The two mosquito strains also differed in their gut microbial composition. Cutibacterium spp., Corynebacterium spp., Citricoccus spp., Leucobacter spp., Acinetobacter spp., Dietzia spp., and Anaerococcus spp. were more abundant in PS strain than in KW strain. In contrast, Sphingomonas spp., Aquabacterium spp., Methylobacterium spp., Flavobacterium spp., Lactobacillus spp., unclassified Burkholderiaceae and unclassified Nostocaceae were more abundant in KW strain compared to PS strain. PS strain was enriched with propionate metabolizers, selenate reducers, and xylan, chitin, and chlorophenol degraders while KW strain was enriched with sulfur oxidizers, sulfur metabolizers, sulfate reducers and naphthalene and aromatic hydrocarbons degraders. These findings demonstrate an association between the gut microbiota and insecticide resistance in an important vector species and sets the foundation for future studies to investigate the contribution of gut microbiota to evolution of insecticide resistance in disease vectors.

Abstract 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.

Abstract Mosquito bacterial communities are essential in mosquito biology, and knowing the factors shaping these bacterial communities is critical to their application in mosquito-borne disease control. This study investigated how the larval environment influences the bacterial communities of larval stages of two container-dwelling mosquito species, Aedes triseriatus, and Aedes japonicus. Larval and water samples were collected from tree holes and used tires at two study sites, and their bacteria characterized through MiSeq sequencing of the 16S rRNA gene. Bacterial richness was highest in Ae. japonicus, intermediate in Ae. triseriatus, and lowest in water samples. Dysgonomonas was the dominant bacterial taxa in Ae. triseriatus larvae; the unclassified Comamonadaceae was dominant in water samples from waste tires, while Mycobacterium and Carnobacterium, dominated Ae. japonicus. The two mosquito species harbored distinct bacterial communities that were different from those of the water samples. The bacterial communities also clustered by habitat type (used tires vs. tree holes) and study site. These findings demonstrate that host species, and the larval sampling environment are important determinants of a significant component of bacterial community composition and diversity in mosquito larvae and that the mosquito body may select for microbes that are generally rare in the larval environment.

Abstract Background The guts of blood-sucking insects host a community of bacteria that can shift dramatically in response to biotic and abiotic factors. Identifying the key factors structuring these microbial communities has important ecological and epidemiological implications. Methods We used the yellow fever mosquito, Aedes aegypti, to investigate the impact of mixed blood meals on gut microbiota of vector mosquitoes. Adult females were experimentally fed on sugar or blood from chicken, rabbit or a mixture of chicken and rabbit blood, and their gut microbiota were characterized using 16S rRNA gene amplification and MiSeq sequencing. Results The gut bacterial communities of mosquitoes fed on the three blood meal treatments clustered separately, suggesting that host species identity and mixed blood-feeding are key determinants of gut bacterial community composition in mosquitoes. Mixed blood meal had a synergistic effect on both operational taxonomic unit (OTU) richness and the Shannon diversity index, suggesting that mixed blood-feeding can offset the nutritional deficit of blood meals from certain host species. The microbial communities observed in this study were distinct from those identified from similarly fed Ae. aegypti from our previous study. Conclusions These findings demonstrate that vector host-feeding preferences can influence gut microbial composition and diversity, which could potentially impact pathogen acquisition and transmission by the vector. The results also demonstrate that different microenvironmental conditions within the laboratory may play an important role in structuring the microbial communities of independently reared mosquito colonies.

Fungal entomopathogens are potential tools for the control of mosquito vectors that transmit infectious agents that cause disease in humans and animals. During the infection process, effective recognition of the invading fungi by the mosquito, is a crucial step in mounting an appropriate anti-fungal response. In this study, we investigated the role of peptidoglycan recognition receptors (PGRPs) in host resistance to fungal entomopathogens at the early stages of infection. Our study identified the induction of PGRP-LA, -LB, -LD, -LE, and -S1 during infection with two different fungal entomopathogenic strains. Furthermore, our data shows temporal differences in PGRP elicitation, with most PGRPs displaying significant upregulation at 60 h post-infection. Depletion of certain PGRPs via RNAi silencing resulted in a significant increase in fungal proliferation and a reduction in mosquito survival that was fungal strain-specific. Our data indicates that PGRPs play an important role in the antifungal response and expands our understanding of the factors that determine host susceptibility to fungal entomopathogens.

Abstract Background Microbial communities that inhabit the mosquito body play an import role in host biology and may have potential for mosquito control. However, the forces that shape these microbial communities are poorly understood. Methods To gain a better understanding of how host location influences the composition and diversity of mosquito microbiota, we performed a survey of microbial communities in mosquito samples collected from six USA states using HiSeq sequencing of the 16S rRNA gene. Results A total of 284 bacterial operational taxonomic units (OTUs) belonging to 14 phyla were detected in nine mosquito species, with Proteobacteria, Firmicutes and Actinobacteria accounting for 95% of total sequences. OTU richness varied markedly within and between mosquito species. The microbial composition and diversity was heavily influenced by the site of mosquito collection, suggesting that host location plays an important role in shaping the mosquito microbiota. Conclusions Variation in microbial composition and diversity between mosquitoes from different locations may have important implications on vector competence and transmission dynamics of mosquito-borne pathogens. Future studies should investigate the environmental factors responsible for these variations and the role of key bacteria characterized in this study on mosquito biology and their potential application in symbiotic control of mosquito-borne diseases.

Abstract Background Understanding the major predictors of disease vectors such as mosquitoes can guide the development of effective and timely strategies for mitigating vector-borne disease outbreaks. This study examined the influence of selected environmental, weather and sociodemographic factors on the spatial and temporal distribution of the southern house mosquito Culex quinquefasciatus Say in New Orleans, Louisiana, USA. Methods Adult mosquitoes were collected over a 4-year period (2006, 2008, 2009 and 2010) using CDC gravid traps. Socio-demographic predictors were obtained from the United States Census Bureau, 2005–2009 American Community Survey and the City of New Orleans Department of Code Enforcement. Linear mixed effects models and ERDAS image processing software were used for statistical analysis and image processing. Results Only two of the 22 predictors examined were significant predictors of Cx. quinquefasciatus abundance. Mean temperature during the week of mosquito collection was positively associated with Cx. quinquefasciatus abundance while developed high intensity areas were negatively associated with Cx. quinquefasciatus abundance. Conclusion The findings of this study illustrate the power and utility of integrating biophysical and sociodemographic data using GIS analysis to identify the biophysical and sociodemographic processes that increase the risk of vector mosquito abundance. This knowledge can inform development of accurate predictive models that ensure timely implementation of mosquito control interventions.

La Crosse encephalitis is a viral disease that has emerged in new locations across the Appalachian region of the United States. Conventional wisdom suggests that ongoing emergence of La Crosse virus (LACV) could stem from the invasive Asian tiger (Aedes albopictus) mosquito. Efforts to prove this, however, are complicated by the numerous transmission routes and species interactions involved in LACV dynamics. To analyze LACV transmission by Asian tiger mosquitoes, we constructed epidemiologic models. These models accurately predict empirical infection rates. They do not, however, support the hypothesis that Asian tiger mosquitoes are responsible for the recent emergence of LACV at new foci. Consequently, we conclude that other factors, including different invasive mosquitoes, changes in climate variables, or changes in wildlife densities, should be considered as alternative explanations for recent increases in La Crosse encephalitis.

Abstract Background Exotic invasive plants alter the structure and function of native ecosystems and may influence the distribution and abundance of arthropod disease vectors by modifying habitat quality. This study investigated how invasive plants alter the ecology of Culex pipiens, an important vector of West Nile virus (WNV) in northeastern and midwestern regions of the United States. Methods Field and laboratory experiments were conducted to test the hypothesis that three native leaf species (Rubus allegheniensis, blackberry; Sambucus canadensis, elderberry; and Amelanchier laevis, serviceberry), and three exotic invasive leaf species (Lonicera maackii, Amur honeysuckle; Elaeagnus umbellata, autumn olive; and Rosa multiflora, multiflora rose) alter Cx. pipiens oviposition site selection, emergence rates, development time, and adult body size. The relative abundance of seven bacterial phyla in infusions of the six leaf species also was determined using quantitative real-time polymerase chain reaction to test the hypothesis that variation in emergence, development, and oviposition site selection is correlated to differences in the diversity and abundance of bacteria associated with different leaf species, important determinants of nutrient quality and availability for mosquito larvae. Results Leaf detritus from invasive honeysuckle and autumn olive yielded significantly higher adult emergence rates compared to detritus from the remaining leaf species and honeysuckle alleviated the negative effects of intraspecific competition on adult emergence. Conversely, leaves of native blackberry acted as an ecological trap, generating high oviposition but low emergence rates. Variation in bacterial flora associated with different leaf species may explain this asymmetrical production of mosquitoes: emergence rates and oviposition rates were positively correlated to bacterial abundance and diversity, respectively. Conclusions We conclude that the displacement of native understory plant species by certain invasive shrubs may increase production of Cx. pipiens with potential negative repercussions for human and wildlife health. These findings may be relevant to mosquito control and invasive plant management practices in the geographic range of Cx. pipiens. Further, our discovery of a previously unknown ecological trap for an important vector of WNV has the potential to lead to novel alternatives to conventional insecticides in mosquito control by exploiting the apparent “attract-kill” properties of this native plant species.

Although the insecticidal properties of some plant essential oils are well-documented, their use in integrated pest and vector management is complicated by their high volatility, low thermal stability, high sensitivity to oxidation, and low solubility in water. We investigated the use of bio-based N-1-hexadecylammonium chloride and sodium palmitate amylose inclusion complexes as emulsifiers for two essential oils, garlic and asafoetida, known to be highly toxic to mosquito larvae. Four emulsions of each essential oil based on amylose hexadecylammonium chloride and amylose sodium palmitate inclusion complexes were evaluated for their toxicity against Aedes aegypti L. larvae relative to bulk essential oils. All emulsions were significantly more toxic than the bulk essential oil with the lethal dosage ratios ranging from 1.09−1.30 relative to bulk essential oil. Droplet numbers ranged from 1.11 × 109 to 9.55 × 109 per mL and did not change significantly after a 6-month storage period. These findings demonstrated that amylose inclusion complexes enhanced the toxicity of essential oils and could be used to develop new essential oil based larvicides for use in integrated vector management.

The growing awareness that microbial symbionts residing in mosquito midguts can interrupt transmission of vector-borne diseases has stimulated interest in understanding their potential role in mosquito biology. Fluorescent proteins are powerful molecular markers that provide for detailed analysis of the function and behavior of specific midgut bacterial isolates without disturbing the normal gut microbiota. The aim of this study was to label bacterial isolates from the midgut of Ochlerotatus triseriatus, the primary vector of La Crosse virus, with green, yellow, and red fluorescent proteins (GFP, YFP, RFP) via electroporation. We also assessed the stability of GFP-, YFP-, and RFP-bearing plasmids and their effect on bacterial growth. Seven of eleven bacterial species could not be labeled despite several attempts. Labeling of Escherichia coli and Enterobacter cloacae was successfully achieved with all three fluorescent proteins. In contrast, labeling of Aerococcus viridans was achieved with GFP only and labeling of Aeromonas hydrophila was achieved with GFP and YFP only. The stability of GFP plasmid varied among bacterial species with A. hydrophila followed by E. cloacae having the most stable GFP label. In contrast, YFP and RFP plasmids were very stable in all bacterial species possessing these labels. GFP plasmid reduced the growth of labeled strains relative to wild type but this effect was not evident in YFP and RFP plasmids. These findings suggest that some mosquito midgut bacterial isolates can effectively be labeled with GFP, YFP and RFP plasmids allowing non-destructive studies on their functions within the vector.

Background & objectives: 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.Methods: 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. Results: 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. Interpretation & conclusion: 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.

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

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

Background & objectives: An entomological survey was conducted to determine the relationshipbetween malaria and lymphatic filariasis transmission by Anopheles gambiae s.l. and An. funestus intwo inland villages along the Kenyan coast.Methods: Mosquitoes were sampled inside houses by pyrethrum spray sheet collection (PSC). In thelaboratory, the mosquitoes were sorted to species, dissected for examination of filarial infection andthe anophelines later tested for Plasmodium falciparum circumsporozoite proteins by an enzymelinkedimmunosorbent assay (ELISA).Results: From a total of 2,032 female mosquitoes collected indoors, An. gambiae s.l constituted 94.4%while the remaining 5.6% comprised of An. funestus and Culex quinquefasciatus. None of the Cx.quinquefasciatus was positive for filarial worms. P. falciparum sporozoite rate for An. gambiae s.l.from both villages was significantly higher than Wuchereria bancrofti infectivity rate. Similarly, theentomological inoculation rate for An. gambiae s.l. was significantly higher than the corresponding W.bancrofti infective biting rate and transmission potential for both the villages. Mass treatment of peoplewith filaricidal drugs in Shakahola in the ongoing global elimination of lymphatic filariasis campaignseemed to have reduced the indices of filariasis transmission but had no effect on malaria transmission.Interpretation & conclusion: These results indicate the intensity of malaria transmission by anophelinesto be much higher than that of lymphatic filariasis in areas where both diseases co-exist and re-emphasisethe need to integrate the control of the two diseases in such areas.

Synthetic pesticides are the cornerstone of vector-borne disease control, but alternatives are urgently needed to tackle the growing problem of insecticide resistance and concerns over environmental safety. Leptospermum scoparium J.R. Forst and G. Forst (manuka) essential oil and its four fractions were analyzed for chemical composition and toxicity against Aedes aegypti larvae. The use of bio-based amylose-N-1-hexadecylammonium chloride inclusion complexes (Hex-Am) as an emulsifier for L. scoparium essential oil was also investigated. Fraction 1 was inactive, fractions 2 (LC50 = 12.24 ppm) and 3 (LC50 = 20.58 ppm) were more toxic than the whole essential oil (LC50 = 47.97 ppm), and fraction 4 (LC50 = 35.87 ppm) had similar toxicity as the whole essential oil. Twenty-one chemical constituents were detected in L. scoparium essential oil compared to 16, 5, 19 and 25 chemical constituents in fractions, 1, 2, 3 and 4 respectively. The two most dominant chemical constituents were calamenene (17.78%) and leptospermone (11.86%) for L. scoparium essential oil, calamenene (37.73%) and ledene (10.37%) for fraction 1, leptospermone (56.6%) and isoleptospermone (19.73) for fraction 2, cubenol (24.30%) and caryophyllene oxide (12.38%) for fraction 3, and γ-gurjunene (21.62%) and isoleptospermone (7.88%) for fraction 4. Alpha-pinene, ledene, and aromandendrene were 2-7 times less toxic than the whole essential suggesting that the toxicity of L. scoparium essential oil was either due to other chemical constituents that were not tested or due synergist interactions among chemical constituents. Leptospermum scoparium essential oil-Hex-Am emulsion (LC50 = 29.62) was more toxic than the whole essential oil. These findings suggest that L. scoparium essential oil is a promising source of mosquito larvicide and that Hex-Am is an excellent emulsifier for L. scoparium essential oil for use as a larvicide.

The composition and structure of microbial communities associated with mosquitoes remain poorly understood despite their important role in host biology and potential to be harnessed as novel strategies for mosquito-borne disease control. We employed MiSeq sequencing of the 16S rRNA gene amplicons to characterize the bacterial flora of field-collected populations of Aedes mcintoshi and Aedes ochraceus, the primary vectors of Rift Valley fever (RVF) virus in Kenya. Proteobacteria (53.5%), Firmicutes (22.0%) and Actinobacteria (10.0%) were the most abundant bacterial phyla accounting for 85.5% of the total sequences. Non-metric multi-dimensional scaling plots based on Bray-Curtis dissimilarities revealed a clear grouping of the samples by mosquito species, indicating that the two mosquito species harbored distinct microbial communities. Microbial diversity, richness and composition was strongly influenced by the site of mosquito collection and overall, Ae. ochraceus had significantly higher microbial diversity and richness than Ae. mcintoshi. Our findings suggest that host species and site of collection are important determinants of bacterial community composition and diversity in RVF virus vectors and these differences likely contribute to the spatio-temporal transmission dynamics of RVF virus.

BACKGROUND:The transmission of malaria is highly variable and depends on a range of climatic and anthropogenic factors. This study investigates the combined, i.e. direct and indirect, impacts of climate change on the dynamics of malaria through modifications in: (i) the sporogonic cycle of Plasmodium induced by air temperature increase, and (ii) the life cycle of Anopheles vector triggered by changes in natural breeding habitat arising from the altered moisture dynamics resulting from acclimation responses of vegetation under climate change. The study is performed for a rural region in Kilifi county, Kenya. METHODS AND FINDINGS:We use a stochastic lattice-based malaria (SLIM) model to make predictions of changes in Anopheles vector abundance, the life cycle of Plasmodium parasites, and thus malaria transmission under projected climate change in the study region. SLIM incorporates a nonlinear temperature-dependence of malaria parasite development to estimate the extrinsic incubation period of Plasmodium. It is also linked with a spatially distributed eco-hydrologic modeling framework to capture the impacts of climate change on soil moisture dynamics, which served as a key determinant for the formation and persistence of mosquito larval habitats on the land surface. Malaria incidence data collected from 2008 to 2013 is used for SLIM model validation. Projections of climate change and human population for the region are used to run the models for prediction scenarios. Under elevated atmospheric CO2 concentration ([CO2]) only, modeled results reveal wetter soil moisture in the root zone due to the suppression of transpiration from vegetation acclimation, which increases the abundance of Anopheles vectors and the risk of malaria. When air temperature increases are also considered along with elevated [CO2], the life cycle of Anopheles vector and the extrinsic incubation period of Plasmodium parasites are shortened nonlinearly. However, the reduction of soil moisture resulting from higher evapotranspiration due to air temperature increase also reduces the larval habitats of the vector. Our findings show the complicated role of vegetation acclimation under elevated [CO2] on malaria dynamics and indicate an indirect but ignored impact of air temperature increase on malaria transmission through reduction in larval habitats and vector density. CONCLUSIONS:Vegetation acclimation triggered by elevated [CO2] under climate change increases the risk of malaria. In addition, air temperature increase under climate change has opposing effects on mosquito larval habitats and the life cycles of both Anopheles vectors and Plasmodium parasites. The indirect impacts of temperature change on soil moisture dynamics are significant and should be weighed together with the direct effects of temperature change on the life cycles of mosquitoes and parasites for future malaria prediction and control.

Alternative methods of mosquito control are needed to tackle the rising burden of mosquito-borne diseases while minimizing the use of synthetic insecticides, which are threatened by the rapid increase in insecticide resistance in mosquito populations. Fungal biopesticides show great promise as potential alternatives because of their ecofriendly nature and ability to infect mosquitoes on contact. Here we describe the temporospatial interactions between the mosquito Aedes aegypti and several entomopathogenic fungi. Fungal infection assays followed by the molecular assessment of infection-responsive genes revealed an intricate interaction between the mosquito immune system and entomopathogenic fungi. We observed contrasting tissue and time-specific differences in the activation of immune signaling pathways and antimicrobial peptide expression. In addition, these antifungal responses appear to vary according to the fungal entomopathogen used in the infection. Enzyme activity-based assays coupled with gene expression analysis of prophenoloxidase genes revealed a reduction in phenoloxidase (PO) activity in mosquitoes infected with the most virulent fungal strains at 3 and 6d post-fungal infection. Moreover, fungal infection led to an increase in midgut microbiota that appear to be attributed in part to reduced midgut reactive oxygen species (ROS) activity. This indicates that the fungal infection has far reaching effects on other microbes naturally associated with mosquitoes. This study also revealed that despite fungal recognition and immune elicitation by the mosquito, it is unable to successfully eliminate the entomopathogenic fungal infection. Our study provides new insights into this intricate multipartite interaction and contributes to a better understanding of mosquito antifungal immunity.

Abstract Background Mosquitoes transmit a number of arboviruses associated with disease outbreaks in humans and other animals. The majority of medically important arboviruses belong to three families: Togaviridae, Flaviviridae and Bunyaviridae. Several members of these families have overlapping distributions and share common vectors, increasing the potential for arboviral coinfections. This study examined how two model viruses: Sindbis virus (SINV, Togaviridae: Alphavirus) and dengue-4 virus (DENV-4, Flaviviridae: Flavivirus) may interact in C6/36 Aedes albopictus cells and in the mosquito vector Ae. albopictus. Methods C6/36 cells were coinfected, superinfected, or singly infected with SINV and DENV-4 and the two viruses quantified at different time points. Four to seven day old adult females of Ae. albopictus were also fed blood containing one or both viruses and viral infection and dissemination rates determined. Results Sindbis virus suppressed replication of DENV-4 in C6/36 Ae. albopictus cells with greater inhibition occurring when the two arboviruses were inoculated simultaneously compared to sequentially. In addition, Ae. albopictus simultaneously exposed to both arboviruses had significantly lower DENV-4 infection and population dissemination rates compared to those exposed to DENV-4 alone. Conclusion These results suggest that certain Alphaviruses may interfere with DENV-4 transmission by suppressing its replication and increasing vector refractoriness. The findings provide important insights into the potential contribution of mixed arboviral infections to DENV transmission dynamics.

BACKGROUND:The composition and structure of microbial communities that inhabit the mosquito midguts are poorly understood despite their well-documented potential to impede pathogen transmission. METHODOLOGY/PRINCIPAL FINDINGS:We used MiSeq sequencing of the 16S rRNA gene to characterize the bacterial communities of field-collected populations of 12 mosquito species. After quality filtering and rarefaction, the remaining sequences were assigned to 181 operational taxonomic units (OTUs). Approximately 58% of these OTUs occurred in at least two mosquito species but only three OTUs: Gluconobacter (OTU 1), Propionibacterium (OTU 9), and Staphylococcus (OTU 31) occurred in all 12 mosquito species. Individuals of different mosquito species shared similar gut microbiota and it was common for individuals of the same species from the same study site and collection date to harbor different gut microbiota. On average, the microbiota of Aedes albopictus was the least diverse and significantly less even compared to Anopheles crucians, An. quadrimaculatus, Ae. triseriatus, Ae. vexans, Ae. japonicus, Culex restuans, and Culiseta inornata. The microbial community of Cx. pipiens and Ae. albopictus differed significantly from all other mosquitoes species and was primarily driven by the dominance of Wolbachia. CONCLUSION AND SIGNIFICANCE:These findings expand the range of mosquito species whose gut microbiota has been characterized and sets the foundation for further studies to determine the influence of these microbiota on vector susceptibility to pathogens.

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.

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.