Next generation sequencing improves the resolution of detecting mixed host blood meal sources in field collected arboviral mosquito vectors.

Accurate knowledge of blood meal hosts of different mosquito species is critical for identifying potential vectors and establishing the risk of pathogen transmission. We compared the performance of Miseq next generation sequencing approach relative to conventional Sanger sequencing approach in identification of mosquito blood meals using genetic markers targeting the 12S rRNA and cytochrome oxidase I (COI) genes. We analysed the blood meals of three mosquito vector species (Aedes aegypti, Aedes simpsoni s.l. and Culex pipiens s.l.) collected outdoors, and compared the frequency of single- versus multiple-blood feeding. Single host blood meals were mostly recovered for Sanger-based sequencing of the mitochondrial 12S rRNA gene, whereas Miseq sequencing employing this marker and the COI marker detected both single and multiple blood meal hosts in individual mosquitoes. Multiple blood meals (two or more hosts) which mostly included humans were detected in 19%-22.7% of Ae. aegypti samples. Most single host blood meals for this mosquito species were from humans (47.7%-57.1%) and dogs (9.1%-19.0%), with livestock, reptile and rodent hosts collectively accounting for 4.7%-28.9% of single host blood meals. The frequency of two or more host blood meals in Ae. simpsoni s.l. was 26.3%-45.5% mostly including humans, while single host blood meals were predominantly from humans (31.8%-47.4%) with representation of rodent, reptile and livestock blood meals (18.2%-68.2%). Single host blood meals from Cx. pipiens s.l. were mostly from humans (27.0%-39.4%) and cows (11.5%-27.36%). Multiple blood meal hosts that mostly included humans occurred in 21.2%-24.4% of Cx. pipiens s.l. samples. Estimated human blood indices ranged from 53%-76% for Ae. aegypti, 32%-82% for Ae. simpsoni s.l. and 26%-61% for Cx. pipiens s.l. and were consistently lower for Sanger-based sequencing approach compared to Miseq-based sequencing approach. These findings demonstrate that Miseq sequencing approach is superior to Sanger sequencing approach as it can reliably identify mixed host blood meals in a single mosquito, improving our ability to understand the transmission dynamics of mosquito-borne pathogens.

Culex pipiens and Culex restuans larval interactions shape the bacterial communities in container aquatic habitats.

Container aquatic habitats host a community of aquatic insects, primarily mosquito larvae that browse on container surface microbial biofilm and filter-feed on microorganisms in the water column. We examined how the bacterial communities in these habitats respond to feeding by larvae of two container-dwelling mosquito species, Culex pipiens and Cx. restuans. We also investigated how the microbiota of these larvae is impacted by intra- and interspecific interactions. Microbial diversity and richness were significantly higher in water samples when mosquito larvae were present, and in Cx. restuans compared to Cx. pipiens larvae. Microbial communities of water samples clustered based on the presence or absence of mosquito larvae and were distinct from those of mosquito larvae. Culex pipiens and Cx. restuans larvae harbored distinct microbial communities when reared under intraspecific conditions and similar microbial communities when reared under interspecific conditions. These findings demonstrate that mosquito larvae play a major role in structuring the microbial communities in container habitats and that intra- and interspecific interactions in mosquito larvae may shape their microbiota. This has important ecological and public health implications since larvae of the two mosquito species are major occupants of container habitats while the adults are vectors of West Nile virus.

Comparative Transcriptomic Analysis of Insecticide-Resistant Aedes aegypti from Puerto Rico Reveals Insecticide-Specific Patterns of Gene Expression.

Aedes aegypti transmits major arboviruses of public health importance, including dengue, chikungunya, Zika, and yellow fever. The use of insecticides represents the cornerstone of vector control; however, insecticide resistance in Ae. aegypti has become widespread. Understanding the molecular basis of insecticide resistance in this species is crucial to design effective resistance management strategies. Here, we applied Illumina RNA-Seq to study the gene expression patterns associated with resistance to three widely used insecticides (malathion, alphacypermethrin, and lambda-cyhalothrin) in Ae. aegypti populations from two sites (Manatí and Isabela) in Puerto Rico (PR). Cytochrome P450s were the most overexpressed detoxification genes across all resistant phenotypes. Some detoxification genes (CYP6Z7, CYP28A5, CYP9J2, CYP6Z6, CYP6BB2, CYP6M9, and two CYP9F2 orthologs) were commonly overexpressed in mosquitoes that survived exposure to all three insecticides (independent of geographical origin) while others including CYP6BY1 (malathion), GSTD1 (alpha-cypermethrin), CYP4H29 and GSTE6 (lambda-cyhalothrin) were uniquely overexpressed in mosquitoes that survived exposure to specific insecticides. The gene ontology (GO) terms associated with monooxygenase, iron binding, and passive transmembrane transporter activities were significantly enriched in four out of six resistant vs. susceptible comparisons while serine protease activity was elevated in all insecticide-resistant groups relative to the susceptible strain. Interestingly, cuticular-related protein genes (chinase and chitin) were predominantly downregulated, which was also confirmed in the functional enrichment analysis. This RNA-Seq analysis presents a detailed picture of the candidate detoxification genes and other pathways that are potentially associated with pyrethroid and organophosphate resistance in Ae. aegypti populations from PR. These results could inform development of novel molecular tools for detection of resistance-associated gene expression in this important arbovirus vector and guide the design and implementation of resistance management strategies.

Increased Phenoloxidase Activity Constitutes the Main Defense Strategy of Trichoplusia ni Larvae against Fungal Entomopathogenic Infections.

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.

Heterocypris incongruens maintains an egg bank in stormwater habitats and influences the development of larval mosquito, Culex restuans.

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.

Bioactivity of brassica seed meals and its compounds as ecofriendly larvicides against mosquitoes.

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.

Combining TEMPO and methyl undecenoate to produce an effective anti-mosquito compound with convenient spin-labeling.

A general method to spin-label a fatty acid was demonstrated as well as an assay of the effectiveness of methyl 10-undecenoate and the spin-labeled version, against the larvae of Aedes aegypti. The LC50s were 66 and 58 µL/120 mL (55 and 48 ppm) respectively, and the LC90s were 108 and 90 µL/120 mL (113 and 90) ppm. This shows that the spin-label has very little effect on the larvicidal activity of the compound. This opens the possibility of the use of spin-labeling as a tool to determine mechanisms of larvicidal effectiveness, as it can be employed without altering the system under study.

Repellency and toxicity of a CO2-derived cedarwood oil on hard tick species (Ixodidae).

The repellency and toxicity of a CO2-derived cedarwood oil (CWO) was evaluated against actively questing unfed nymphs of four species of hard ticks: Amblyomma americanum (L.), Dermacentor variabilis (Say), Ixodes scapularis Say, and Rhipicephalus sanguineus (Latreille). Using a vertical climb bioassay for repellency, nymphs of these species avoided a CWO-treated filter paper in proportional responses to treatment concentrations. At 60 min of exposure, I. scapularis nymphs were most sensitive with 50% repellency concentration (RC50) of 19.8 µg cm-2, compared with RC50 of 30.8, 83.8 and 89.6 µg cm-2 for R. sanguineus, D. variabilis and A. americanum, respectively. Bioassays determined the lethal concentration for 50% (LC50) and 90% (LC90) mortality of nymphs exposed to CWO in treated vials after 24- and 48-h exposure. After 24 h exposure, the LC50 values were 1.25, 3.45 and 1.42 µg cm-2 and LC90 values were 2.39, 7.59 and 4.14 µg cm-2 for D. variabilis, I. scapularis and R. sanguineus, respectively, but had minimal effect on A. americanum. After 48 h exposure, the LC50 values were 4.14, 0.78, 0.79 and 0.52 µg cm-2, and LC90 values were 8.06, 1.48, 1.54 and 1.22 µg cm-2 for A. americanum, D. variabilis, I. scapularis and R. sanguineus, respectively. The repellency of CWO on tick species decreased with time. The repellency and toxicity bioassays demonstrated concentration-dependent responses of tick nymphs to the oil, indicating the potential of the CO2-derived cedarwood oil be developed as an eco-friendly repellent and/or acaricide.

Multiple mosquito AMPs are needed to potentiate their antifungal effect against entomopathogenic fungi.

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.

Resistance to permethrin alters the gut microbiota of Aedes aegypti.

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.

Insecticide resistance status in Anopheles gambiae (s.l.) in coastal Kenya.

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.

The larval environment strongly influences the bacterial communities of Aedes triseriatus and Aedes japonicus (Diptera: Culicidae).

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.

Blood meal source and mixed blood-feeding influence gut bacterial community composition in Aedes aegypti.

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.

Effect of life stage and pesticide exposure on the gut microbiota of Aedes albopictus and Culex pipiens L.

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.

Microbial communities of container aquatic habitats shift in response to Culex restuans larvae.

We examined how larvae of Culex restuans mosquito influences the bacterial abundance, composition and diversity in simulated container aquatic habitats. The microbiota of Cx. restuans larvae were also characterized and compared to those of their larval habitats. The presence of Cx. restuans larvae altered the bacterial community composition and reduced the bacterial abundance, diversity and richness. Azohydromonas sp., Delftia sp., Pseudomonas sp., Zooglea sp., unclassified Enterobacteriaceae and unclassified Bacteroidales were suppressed while Prosthecobacter sp., Hydrogenaphaga sp., Clostridium sp., unclassified Clostridiaceae and Chryseobacterium sp. were enhanced in the presence of Cx. restuans larvae. Cx. restuans larvae harbored distinct and less diverse bacterial community compared to their larval habitats. These findings demonstrate that Cx. restuans larvae play a key role in structuring the microbial communities in container aquatic habitats and may lower the nutritional quality and alter the decomposition process and food web dynamics in these aquatic systems. The findings also demonstrate that mosquito larvae are highly selective of the bacterial taxa from the larval environment that colonize their bodies. These findings provide new opportunities for more focused studies to identify the specific bacterial taxa that serve as food for mosquito larvae and those that could be harnessed for disease control.

Insecticidal Activity of Commiphora erythraea Essential Oil and Its Emulsions Against Larvae of Three Mosquito Species.

The use of essential oils as ecofriendly tools for vector management is one of the mainstreams for biopesticide research. We evaluated the larvicidal properties of Commiphora erythraea (opoponax) essential oil and its fractions against Culex restuans Theobald, Culex pipiens L., and Aedes aegypti L. 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 was also investigated. Bisabolene was the most abundant chemical constituent in the whole essential oil (33.9%), fraction 2 (62.5%), and fraction 4 (23.8%) while curzerene (32.6%) and α-santalene (30.1%) were the dominant chemical constituents in fractions 1 and 3, respectively. 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. 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 metabolic pathways for 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 the use of Hex-Am as an emulsifier can enhance the insecticidal properties of C. erythraea essential oil.

Leptospermum scoparium essential oil is a promising source of mosquito larvicide and its toxicity is enhanced by a biobased emulsifier.

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.

Amylose Inclusion Complexes as Emulsifiers for Garlic and Asafoetida Essential Oils for Mosquito Control.

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.

Host species and site of collection shape the microbiota of Rift Valley fever vectors in Kenya.

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.