Holobionts are defined as a host and its microbiota, however, only a fraction of the bacteria are inherited vertically and thus coevolve with the host. The "it's the song, not the singer" theory proposes that functional traits, instead of taxonomical microbiota composition, could be preserved across generations if interspecies interaction patterns perpetuate themselves. We tested conservation of functional composition across generations using zooplankton, mosquito, and plant datasets. Then, we tested if there is a change of functional microbiota composition over time within a generation in human datasets. Finally, we simulated microbiota communities to investigate if (pairwise) interactions can lead to multiple stable community compositions. Our results suggest that the vertically transmitted microbiota starts a predictable change of functions performed by the microbiota over time, whose robustness depends on the arrival of diverse migrants. This succession culminates in a stable functional composition state. The results suggest that the host-microbiota interaction and higher order interactions in general have an important contribution to the robustness of the final community. If the proposed mechanism proves to be valid for a diverse array of host species, this would support the concept of holobionts being used as units of selection, including animal breeding, suggesting this has a wider applicability.

Anopheles mosquitoes feed on plant nectars as their main source of sugar. Wang et al. show that Asaia bacteria proliferate in the midgut of mosquitoes that feed on glucose or trehalose. Asaia increases the lumenal pH by downregulating mosquito vacuolar ATPase expression, therefore increasing Plasmodium gametogenesis and vector competence.

BACKGROUND:Pyrethroid insecticides are widely used in many countries for chemical-based control of . Regardless of their efficacy, the constant use of insecticides has induced insecticide resistance mechanisms, such as knockdown resistance () in mosquitoes. Sri Lankan Vector Controlling Entities (VCE) have been using a variety of pyrethroid insecticides as the primary approach for dengue control. However, development of any resistance among the mosquitoes has been limitedly studied in the country. Therefore, the current study was conducted to evaluate the prevalence of F1534C, V1016G, and S989P mutations among mosquito populations in three dengue endemic high-risk regions of Sri Lanka. . Immature (both pupae and larvae) stages of mosquitoes were collected from Colombo, Gampaha, and Kandy districts of Sri Lanka from February 2018 to December 2019. Polymerase Chain Reaction- (PCR-) based assay for molecular genotyping of mutations was performed to identify the prevalence of mutations in collected populations, separately. The frequencies of the resistant and susceptible kdr alleles were determined by using the Hardy-Weinberg equilibrium. RESULTS:The populations from Colombo, Gampaha, and Kandy districts showed 46%, 42%, and 22% of F1534C mutation allele frequencies, along with 15%, 12%, and 6% of V1016G mutation allele frequencies, respectively. The mutation allele frequencies of S989 in Colombo, Gampaha, and Kandy districts were 9.5%, 8.5%, and 4.5%, respectively. The wild-type (PP) genotype remained predominant within all the three districts, whereas the homogenous (QQ) mutation genotype occurred only in minority. The abundance of Q allele frequency in mosquitoes was relatively higher for all the three mutations in Colombo. CONCLUSIONS:The findings clearly indicate that long-term insecticide applications and multiple use of pyrethroids have led to the acquisition of mutations, leading to the development of insecticide resistance among local populations, especially in the Colombo and Gampaha districts. Therefore, evaluation of the prevalence levels of these mutations highlights the necessity for shifting towards novel vector control strategies.

ETHNOPHARMACOLOGICAL RELEVANCE:Dengue virus (DENV) is a re-emerging mosquito-borne flavivirus that has recently engendered large epidemics around the world. Consequently antivirals with effective anti-DENV therapeutic activity are urgently required. In the 18th century, Europeans, as well as native inhabitants of North America, were known to adapt the medicinal property of the common perennial plant Eupatorium perfoliatum L. to treat fever and infections. Previous studies have shown that Eupatorium perfoliatum L. possesses anti-inflammatory, anti-oxidative, anti-plasmodial, anti-bacterial and antiviral activities. However, to the best of our knowledge, no anti-DENV activity of E. perfoliatum L. has been investigated at the molecular level so far. AIM OF STUDY:Here, for the first time we have attempted to study the action of E. perfoliatum extract and its few bioactive components i.e., quercetin, caffeic acid and eupafolin against wild primary clinical isolate of DENV-2 infection in an in vitro model. MATERIALS AND METHODS:The presence of the bioactive components in the E. perfoliatum extract, were analyzed by HPLC- DAD. Then, CC as well as IC values of the extract and its bioactive components were measured against DENV in HepG2 cell line. After that, the antiviral activity was studied by Time of addition assay using qRT-PCR. Further, the downstream signalling action of E. perfoliatum extract, was studied by Human phosphorylation MAPK antibody array, followed by immunofluorescence microscopy. Moreover, a molecular docking analysis was done to study the binding affinity of bioactive components of E. perfoliatum extract with TIM-1 transmembrane receptor protein, which is known for viral internalization. RESULT:We found that E. perfoliatum extract has marked antiviral activity during pre-treatment against DENV infection in HepG2 cell line. The extract also significantly reduced the DENV induced autophagy in HepG2 cell line as detected by LC3 II localization. The presence of different bioactive compounds in E. perfoliatum extract were confirmed by HPLC-DAD. In the bioactive components, in parallel to earlier studies, quercetin showed the most significant preventive action against DENV infection. Further, in molecular docking analysis also, quercetin showed the strongest binding affinity towards DENV membrane receptor TIM-1 protein. CONCLUSION:Our findings suggests that E. perfoliatum extract has significant potential to be an anti-DENV therapeutic agent. Moreover, among the bioactive components, quercetin may have a prophylaxis role in executing the antiviral activity of E. perfoliatum extract against DENV infection.

Nutrition affects multiple aspects of insect physiology such as body size and fecundity, but we lack a detailed understanding of how nutrition influences the reproductive physiology of male insects such as mosquitoes. Given that female mosquitoes are vectors of many deadly diseases and can quickly proliferate, understanding how male nutrition impacts female fecundity could be of critical importance. To uncover the relationship between nutrition in adult male mosquitoes and its impacts on reproductive physiology, we reared larvae of the Northern house mosquito, Culex pipiens, on a standard lab diet and divided adult males among three different dietary treatments: low (3%), moderate (10%), and high (20%) sucrose. We found that although overall body size did not differ among treatments, one-week-old males raised on the 3% sucrose diet had significantly smaller male accessory glands (MAGs) compared to males that consumed the 10% and the 20% sucrose diets. Diet affected whole-body lipid content but did not affect whole-body protein content. Using nuclear magnetic resonance (NMR) spectroscopy, we found that diet altered the metabolic composition of the MAGs, including changes in lactic acid, formic acid, and glucose. We also observed changes in protein and lipid abundance and composition in MAGs. Females who mated with males on the 3% diet were found to produce significantly fewer larvae than females who had mated with males on the 10% diet. Taken together, our results demonstrate that the diet of adult male mosquitoes clearly affects male reproductive physiology and female fecundity.

In vertebrates and invertebrates, the insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) cascade is highly conserved and plays a vital role in many different physiological processes. Among the many tissues that respond to IIS in mosquitoes, the fat body has a central role in metabolism, lifespan, reproduction, and innate immunity. We previously demonstrated that fat body specific expression of active Akt, a key IIS signaling molecule, in adult Anopheles stephensi and Aedes aegypti activated the IIS cascade and extended lifespan. Additionally, we found that transgenic females produced more vitellogenin (Vg) protein than non-transgenic mosquitoes, although this did not translate into increased fecundity. These results prompted us to further examine how IIS impacts immunity, metabolism, growth and development of these transgenic mosquitoes. We observed significant changes in glycogen, trehalose, triglycerides, glucose, and protein in young (3-5 d) transgenic mosquitoes relative to non-transgenic sibling controls, while only triglycerides were significantly changed in older (18 d) transgenic mosquitoes. More importantly, we demonstrated that enhanced fat body IIS decreased both the prevalence and intensity of Plasmodium falciparum infection in transgenic An. stephensi. Additionally, challenging transgenic An. stephensi with Gram-positive and Gram-negative bacteria altered the expression of several antimicrobial peptides (AMPs) and two anti-Plasmodium genes, nitric oxide synthase (NOS) and thioester complement-like protein (TEP1), relative to non-transgenic controls. Increased IIS in the fat body of adult female An. stephensi had little to no impact on body size, growth or development of progeny from transgenic mosquitoes relative to non-transgenic controls. This study both confirms and expands our understanding of the critical roles insulin signaling plays in regulating the diverse functions of the mosquito fat body.

Aedes aegypti is a major vector of Zika, dengue, and other arboviruses. Permethrin adulticidal spraying, which targets the voltage-gated sodium channel (VGSC), is commonly done to reduce local mosquito populations and protect humans from exposure to arbovirus pathogens transmitted by this dangerous pest. Permethrin resistance, however, is a growing problem and understanding its underlying molecular basis may identify avenues to combat it. We identified a single G:C polymorphism in pre-miR-33 that was genetically associated with permethrin resistance; resulting isoforms had structural differences that may affect DICER-1/pre-miRNA processing rates. We then assessed the effects of overexpression of pre-miR-33 isoforms on permethrin toxicological phenotypes, VGSC transcript abundance and protein levels for two genetically related mosquito strains. One strain had its naturally high permethrin resistance levels maintained by periodic treatment, and the other was released from selection. VGSC protein levels were lower in the permethrin resistant strain than in the related permethrin-susceptible strain. Overexpression of the G-pre-miR-33 isoform reduced VGSC expression levels in both strains. To further elucidate changes in gene expression associated with permethrin resistance, exome-capture gDNA deep sequencing, genetic association mapping and subsequent gene set enrichment analysis revealed that transport genes, in particular, were selected in resistant versus susceptible mosquitoes. Collectively, these data indicate that miR-33 regulates VGSC expression as part of a nuanced system of neuronal regulation that contributes to a network of heritable features determining permethrin resistance.

Insecticide resistance among mosquito species is now a pervasive phenomenon that threatens to jeopardize global malaria vector control efforts. Evidence of links between the mosquito microbiota and insecticide resistance is emerging, with significant enrichment of insecticide degrading bacteria and enzymes in resistant populations. Using 16S rRNA amplicon sequencing, we characterized and compared the microbiota of Anopheles coluzzii in relation to their deltamethrin resistance and exposure profiles. Comparisons between 2- and 3-day-old deltamethrin-resistant and -susceptible mosquitoes demonstrated significant differences in microbiota diversity. , , and genera, each of which comprised insecticide-degrading species, were significantly enriched in resistant mosquitoes. Susceptible mosquitoes had a significant reduction in alpha diversity compared to resistant individuals, with and dominating microbial profiles. There was no significant difference in deltamethrin-exposed and -unexposed 5- to 6-day-old individuals, suggesting that insecticide exposure had minimal impact on microbial composition. and were also dominant in 5- to 6-day-old mosquitoes, which had reduced microbial diversity compared to 2- to 3-day-old mosquitoes. Our findings revealed significant alterations of Anopheles coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for insecticide resistance surveillance. qPCR detection of and was consistent with 16S rRNA sequencing, suggesting that population-level field screening of bacterial microbiota may be feasibly integrated into wider resistance monitoring, if reliable and reproducible markers associated with phenotype can be identified. Control of insecticide-resistant vector populations remains a significant challenge to global malaria control and while substantial progress has been made elucidating key target site mutations, overexpressed detoxification enzymes and alternate gene families, the contribution of the mosquito microbiota to phenotypic insecticide resistance has been largely overlooked. We focused on determining the effects of deltamethrin resistance intensity on Anopheles coluzzii microbiota and identifying any microbial taxa associated with phenotype. We demonstrated a significant reduction in microbial diversity between deltamethrin-resistant and -susceptible mosquitoes. Insecticide degrading bacterial species belonging to , , and genera were significantly enriched in resistant mosquitoes, while and dominated microbial profiles of susceptible individuals. Our results revealed significant alterations of Anopheles coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for surveillance and opportunities for designing innovative control techniques to prevent the further evolution and spread of insecticide resistance.

BACKGROUND:Sugar feeding is a fundamental behaviour of many mosquito species. For Aedes albopictus, an important vector of dengue virus and chikungunya virus, little is known about its sugar-feeding behaviour, and no studies have been conducted on this in the southern hemisphere. This knowledge is pivotal for determining the potential of attractive targeted sugar baits (ATSBs) to control this important vector. METHODS:The prevalence of sugar was assessed in 1808 Ae. albopictus from Masig Island, Torres Strait, Australia collected between 13 and 25 March 2020. Fructose presence and content in field-collected Ae. albopictus were quantified using the cold anthrone assay. RESULTS:Significantly more male (35.8%) than female (28.4%) Ae. albopictus were sugar fed. There was a significant interaction between sex and time of day on the probability of capturing sugar-fed Ae. albopictus. For both sexes, fructose prevalence and content were higher in mosquitoes caught in the morning than in the afternoon. Female Ae. albopictus collected in the residential habitat were significantly more likely to be sugar fed than those collected in the woodland habitat. CONCLUSIONS:These findings provide baseline information about the sugar-feeding patterns of Ae. albopictus and provide essential information to enable an assessment of the potential of ATSBs for vector suppression and control on Masig Island, with relevance to other locations where this species occurs.

Efficient reverse genetics approaches are critical for the study of many organisms. The CRISPR/Cas9 gene editing system has led to a plethora of new tools for geneticists. Here, we successfully established a simplified CRISPR/Cas9 system for the malaria model parasite Plasmodium berghei. The homologous directed repair (HDR) template is provided as a linear template with homologous arms of 600-700bp while the CRISPR elements sgRNA and Cas9 are encoded from a single plasmid utilizing the Ribozyme-Guide-Ribozyme (RGR) expression strategy. Our approach eliminates the need for negative selection markers since the plasmid cannot be incorporated into the genome. As a test case we inserted the FLAG encoding sequence into the ACT2 locus using this new approach. We showed that the genetic modification of this locus had no adverse effects on the completion of the P. berghei life cycle, including transmission through the mosquito.

We previously determined that several diets used to rear and other mosquito species support the development of larvae with a gut microbiota but do not support the development of axenic larvae. In contrast, axenic larvae have been shown to develop when fed other diets. To understand the mechanisms underlying this dichotomy, we developed a defined diet that could be manipulated in concert with microbiota composition and environmental conditions. Initial studies showed that axenic larvae could not grow under standard rearing conditions (27 °C, 16-h light: 8-h dark photoperiod) when fed a defined diet but could develop when maintained in darkness. Downstream assays identified riboflavin decay to lumichrome as the key factor that prevented axenic larvae from growing under standard conditions, while gut community members like rescued development by being able to synthesize riboflavin. Earlier results showed that conventional and gnotobiotic but not axenic larvae exhibit midgut hypoxia under standard rearing conditions, which correlated with activation of several pathways with essential growth functions. In this study, axenic larvae in darkness also exhibited midgut hypoxia and activation of growth signaling but rapidly shifted to midgut normoxia and arrested growth in light, which indicated that gut hypoxia was not due to aerobic respiration by the gut microbiota but did depend on riboflavin that only resident microbes could provide under standard conditions. Overall, our results identify riboflavin provisioning as an essential function for the gut microbiota under most conditions larvae experience in the laboratory and field.

Arboviruses are transmitted by specific vectors, and the reasons for this specificity are not fully understood. One contributing factor is the existence of tissue barriers within the vector such as the midgut escape barrier. We used microRNA (miRNA) targeting of Sindbis virus (SINV) to study how replication in midgut cells contributes to overcoming this barrier in the mosquito Aedes aegypti. SINV constructs were designed to be attenuated specifically in midgut cells by inserting binding sites for midgut-specific miRNAs into either the 3' untranslated region (MRE3'miRT) or the structural open reading frame (MRE-ORFmiRT) of the SINV genome. Both miRNA-targeted viruses replicated less efficiently than control viruses in the presence of these miRNAs. When mosquitoes were given infectious blood meals containing miRNA-targeted viruses, only around 20% (MRE3'miRT) or 40% (MRE-ORFmiRT) of mosquitoes developed disseminated infection. In contrast, dissemination occurred in almost all mosquitoes fed control viruses. Deep sequencing of virus populations from individual mosquitoes ruled out selection for mutations in the inserted target sequences as the cause for dissemination in these mosquitoes. In mosquitoes that became infected with miRNA-targeted viruses, titers were equivalent to those of mosquitoes infected with control virus in both the midgut and the carcass, and there was no evidence of a threshold titer necessary for dissemination. Instead, it appeared that if infection was successfully established in the midgut, replication and dissemination were largely normal. Our results support the hypothesis that replication is an important factor in allowing SINV to overcome the midgut escape barrier but hint that other factors are also likely involved. When a mosquito ingests an arbovirus during a blood meal, the arbovirus must escape from the midgut of the vector and infect the salivary glands in order to be transmitted to a new host. We used tissue-specific miRNA targeting to examine the requirement for Sindbis virus (SINV) to replicate in midgut epithelium in order to cause disseminated infection in the mosquito Aedes aegypti. Our results indicate that specifically reducing the ability of SINV to replicate in the mosquito midgut reduces its overall ability to establish infection in the mosquito, but if infection is established, replication and dissemination occur normally. These results are consistent with an importance for replication in the midgut epithelium in aiding arboviruses in crossing the midgut barrier.

In the present work, we established two novel embryonic cell lines from the mosquito Aedes fluviatilis containing or not the naturally occurring symbiont bacteria Wolbachia, which were called wAflu1 and Aflu2, respectively. We also obtained wAflu1 without Wolbachia after tetracycline treatment, named wAflu1.tet. Morphofunctional characterization was performed to help elucidate the symbiont-host interaction in the context of energy metabolism regulation and molecular mechanisms of the immune responses involved. The presence of Wolbachia pipientis improves energy performance in A. fluviatilis cells; it affects the regulation of key energy sources such as lipids, proteins, and carbohydrates, making the distribution of actin more peripheral and with extensions that come into contact with neighboring cells. Additionally, innate immunity mechanisms were activated, showing that the wAflu1 and wAflu1.tet cells are responsive after the stimulus using Gram negative bacteria. Therefore, this work confirms the natural, mutually co-regulating symbiotic relationship between W. pipientis and A. fluviatilis, modulating the host metabolism and immune pathway activation. The results presented here add important resources to the current knowledge of Wolbachia-arthropod interactions.

A number of recent papers report that standing genetic variation in natural populations includes ubiquitous polymorphisms within target sites for Cas9-based gene drive (CGD) and that these "drive resistant alleles" (DRA) preclude the successful application of CGD for managing these populations. Here we report the results of a survey of 1280 genomes of the mosquitoes Anopheles gambiae, An. coluzzii, and Aedes aegypti in which we determine that ~90% of all protein-encoding CGD target genes in natural populations include at least one target site with no DRAs at a frequency of ≥1.0%. We conclude that the abundance of conserved target sites in mosquito genomes and the inherent flexibility in CGD design obviates the concern that DRAs present in the standing genetic variation of mosquito populations will be detrimental to the deployment of this technology for population modification strategies.

BACKGROUND: mosquitoes infected with the Mel strain of are less susceptible than wild-type to dengue virus infection. METHODS:We conducted a cluster-randomized trial involving releases of Mel-infected mosquitoes for the control of dengue in Yogyakarta, Indonesia. We randomly assigned 12 geographic clusters to receive deployments of Mel-infected (intervention clusters) and 12 clusters to receive no deployments (control clusters). All clusters practiced local mosquito-control measures as usual. A test-negative design was used to assess the efficacy of the intervention. Patients with acute undifferentiated fever who presented to local primary care clinics and were 3 to 45 years of age were recruited. Laboratory testing was used to identify participants who had virologically confirmed dengue (VCD) and those who were test-negative controls. The primary end point was symptomatic VCD of any severity caused by any dengue virus serotype. RESULTS:After successful introgression of Mel into the intervention clusters, 8144 participants were enrolled; 3721 lived in intervention clusters, and 4423 lived in control clusters. In the intention-to-treat analysis, VCD occurred in 67 of 2905 participants (2.3%) in the intervention clusters and in 318 of 3401 (9.4%) in the control clusters (aggregate odds ratio for VCD, 0.23; 95% confidence interval [CI], 0.15 to 0.35; P = 0.004). The protective efficacy of the intervention was 77.1% (95% CI, 65.3 to 84.9) and was similar against the four dengue virus serotypes. The incidence of hospitalization for VCD was lower among participants who lived in intervention clusters (13 of 2905 participants [0.4%]) than among those who lived in control clusters (102 of 3401 [3.0%]) (protective efficacy, 86.2%; 95% CI, 66.2 to 94.3). CONCLUSIONS:Introgression of Mel into populations was effective in reducing the incidence of symptomatic dengue and resulted in fewer hospitalizations for dengue among the participants. (Funded by the Tahija Foundation and others; AWED ClinicalTrials.gov number, NCT03055585; Indonesia Registry number, INA-A7OB6TW.).

As vectors of disease, mosquitoes are a global threat to human health. The Anopheles mosquito is the deadliest mosquito species as the insect vector of the malaria-causing parasite, which kills hundreds of thousands every year. These mosquitoes are reliant on their sense of smell (olfaction) to guide most of their behaviors, and a better understanding of Anopheles olfaction identifies opportunities for reducing the spread of malaria. This review takes a detailed look at Anopheles olfaction. We explore a range of topics from chemosensory receptors, olfactory neurons, and sensory appendages to behaviors guided by olfaction (including host-seeking, foraging, oviposition, and mating), to vector management strategies that target mosquito olfaction. We identify many research areas that remain to be addressed.

West Nile virus (WNV) is the most prevalent arbovirus found throughout the United States. Surveillance of surface breeding Culex vectors involved in WNV transmission is primarily conducted using CDC Gravid traps. However, anecdotal claims from mosquito abatement districts in Louisiana assert that other trap types may be more suited to WNV surveillance. To test the validity of these assertions, we conducted a series of trapping trials and WNV surveillance over 3 yr to compare the efficacy of multiple trap types. First, we compared the CDC Gravid trap, CO2-baited New Standard Miniature Blacklight traps, and CO2-baited CDC light traps with either an incandescent light, a red light, or no light. We found that the CDC Gravid trap and CO2-baited no-light CDC Light trap collected the most mosquitoes. Second, we conducted additional, long-term trapping and WNV surveillance to compare these two trap types. We found that CO2-baited no-light CDC traps collected more of the local WNV vector, Culex quinquefasciatus (Say, Diptera, Culicidae), and detected WNV with greater sensitivity. Finally, we conducted trapping to compare the physiological states of Cx. quinquefasciatus and diversity of collected mosquitoes. CO2-baited no-light CDC light traps collected more unfed Cx. quinquefasciatus while Gravid traps collected more blooded Cx. quinquefasciatus; both traps collected the same number of gravid Cx. quinquefasciatus. Additionally, we found that CO2-baited no-light CDC light traps collected a larger diversity of mosquito species than Gravid traps.

Dengue virus infection, transmitted via mosquito bites, poses a substantial risk to global public health. Studies suggest that the mosquito's microbial community can profoundly influence vector-borne pathogen transmissions, including dengue virus. Ascogregarina culicis (Ross) of the phylum Apicomplexa is among the most common parasites of Aedes aegypti (Linnaeus), the principal vector of dengue. Despite a high prevalence worldwide, including in the areas where dengue is endemic, the impact of A. culicis on Ae. aegypti vector competence for dengue virus is unknown. This study aimed to investigate the effects of A. culicis infection on mosquito size and fitness, as measured by wing length, and the susceptibility to dengue virus infection in Ae. aegypti. Our results showed that there was no statistically significant difference in wing lengths between Ae. aegypti infected and not infected with A. culicis. Furthermore, A. culicis infection did not significantly affect dengue virus infection or disseminated infection rate. However, there was a significant association between shorter wings and higher dengue virus infection rate, whereby a 0.1-mm increase in wing length decreased the odds of the mosquito being infected by 32%. Thus, based on our result, A. culicis infection does not influence the body size and dengue virus infection in Ae. aegypti. This study helps to shed light on a common but neglected eukaryotic mosquito parasite.

BACKGROUND:Transmission of pathogens by vector mosquitoes is intrinsically linked with mosquito's reproductive strategy because anautogenous mosquitoes require vertebrate blood to develop a batch of eggs. Each cycle of egg maturation is tightly linked with the intake of a fresh blood meal for most species. Mosquitoes that acquire pathogens during the first blood feeding can transmit the pathogens to susceptible hosts during subsequent blood feeding and also vertically to the next generation via infected eggs. Large-scale gene-expression changes occur following each blood meal in various tissues, including ovaries. Here we analyzed mosquito ovary transcriptome following a blood meal at three different time points to investigate blood-meal induced changes in gene expression in mosquito ovaries. RESULTS:We collected ovaries from Aedes aegypti that received a sugar meal or a blood meal on days 3, 10 and 20 post blood meal for transcriptome analysis. Over 4000 genes responded differentially following ingestion of a blood meal on day 3, and 660 and 780 genes on days 10 and 20, respectively. Proteins encoded by differentially expressed genes (DEGs) on day 3 include odorant binding proteins (OBPs), defense-specific proteins, and cytochrome P450 detoxification enzymes. In addition, we identified 580 long non-coding RNAs that are differentially expressed at three time points. Gene ontology analysis indicated that genes involved in peptidase activity, oxidoreductase activity, extracellular space, and hydrolase activity, among others were enriched on day 3. Although most of the DEGs returned to the nonsignificant level compared to the sugar-fed mosquito ovaries following oviposition on days 10 and 20, there remained differences in the gene expression pattern in sugar-fed and blood-fed mosquitoes. CONCLUSIONS:Enrichment of OBPs following blood meal ingestion suggests that these genes may have other functions besides being part of the olfactory system. The enrichment of immune-specific genes and cytochrome P450 genes indicates that ovaries become well prepared to protect their germ line from any pathogens that may accompany the blood meal or from environmental contamination during oviposition, and to deal with the detrimental effects of toxic metabolites.

BACKGROUND:The bacterial communities associated with mosquito eggs are an essential component of the mosquito microbiota, yet there are few studies characterizing and comparing the microbiota of mosquito eggs to other host tissues. METHODS:We sampled gravid female Culex pipiens L. and Culex restuans Theobald from the field, allowed them to oviposit in the laboratory, and characterized the bacterial communities associated with their egg rafts and midguts for comparison through MiSeq sequencing of the 16S rRNA gene. RESULTS:Bacterial richness was higher in egg rafts than in midguts for both species, and higher in Cx pipiens than Cx. restuans. The midgut samples of Cx. pipiens and Cx. restuans were dominated by Providencia. Culex pipiens and Cx. restuans egg rafts samples were dominated by Ralstonia and Novosphingobium, respectively. NMDS ordination based on Bray-Curtis distance matrix revealed that egg-raft samples, or midgut tissues harbored similar bacterial communities regardless of the mosquito species. Within each mosquito species, there was a distinct clustering of bacterial communities between egg raft and midgut tissues. CONCLUSION:These findings expand the list of described bacterial communities associated with Cx. pipiens and Cx. restuans and the additional characterization of the egg raft bacterial communities facilitates comparative analysis of mosquito host tissues, providing a basis for future studies seeking to understand any functional role of the bacterial communities in mosquito biology.

BACKGROUND:The direct toxicological impact of insecticides on vector mosquitoes has been well emphasized; however, behavioural responses such as excito-repellency and physical avoidance as a result of insecticide exposure have not been much studied. We have demonstrated the excito-repellency and behavioural avoidance in certain vector mosquito species on exposure to a slow-release insecticidal paint (SRIP) formulation in addition to direct toxicity. METHODS:A SRIP formulation developed by the Defence Research and Development Establishment, Gwalior, contains chlorpyriphos, deltamethrin and pyriproxyfen as active insecticides. Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti mosquitoes were used to study the excito-repellency response of the formulation. The experiments were performed in a specially designed dual-choice exposure and escape chamber made of transparent polymethyl methacrylate. For the experiments, the SRIP formulation was applied undiluted at a rate of 8 m per kg on 15 cm metallic surfaces. Mosquitoes were introduced into the exposure chamber, and observations of the movement of mosquitoes into the escape chamber through the exit portal were taken at 1-min intervals for up to 30 min. RESULTS:The evaluated formulation displayed strong excito-repellency against all three tested vector mosquito species. Results showed that the ET (escape time 50%) for Ae. aegypti, An. stephensi and Cx. quinquefasciatus was 20.9 min, 14.5 min and 17.9 min for contact exposure (CE) respectively. Altogether in CE, the escape rates were stronger in An. stephensi mosquitoes at different time intervals compared to Ae. aegypti and Cx. quinquefasciatus mosquitoes. The probit analysis revealed that the determined ET did not deviate from linearity for both non-contact exposure (NCE) and placebo exposure (PE) (χ ≤ 7.9; p = 1.0) for Ae. aegypti mosquitoes and for NCE (χ = 8.3; p = 1.0) and PE (χ = 1.7; p = 1.0) treatments in Cx. quinquefasciatus. Mortality (24 h) was found to be statistically higher (F = 6.4; p = 0.02) in An. stephensi for CE but did not vary for NCE (p ≥ 0.3) and PE (p = 0.6) treatments among the tested mosquito species. Survival probability response suggested that all the three tested species displayed similar survival responses for similar exposures (χ ≤ 2.3; p ≥ 0.1). CONCLUSION:The study demonstrates the toxicity and strong behavioural avoidance in known vector mosquito species on exposure to an insecticide-based paint formulation. The combination of insecticides in the present formulation will broaden the overall impact spectrum for protecting users from mosquito bites. The efficacy data generated in the study provide crucial information on the effectiveness of the tested formulation and could be useful in reducing the transmission intensity and disease risk in endemic countries.

Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts, and they are central to RNA interference (RNAi). RNAi is critical for regulation of gene expression and antiviral defense in Aedes aegypti mosquitoes, which transmit Zika, chikungunya, dengue, and yellow fever viruses. In mosquitoes, AGO1 mediates miRNA interactions, while AGO2 mediates siRNA interactions. We applied AGO-crosslinking immunoprecipitation (AGO-CLIP) for both AGO1 and AGO2, and we developed a universal software package for CLIP analysis (CLIPflexR), identifying 230 small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map in mosquitoes. RNAi network maps predicted expression levels of small RNA targets in specific tissues. Additionally, this resource identified unexpected, context-dependent AGO2 target preferences, including endogenous viral elements and 3'UTRs. Finally, contrary to current thinking, mosquito AGO2 repressed imperfect targets. These findings expand our understanding of small RNA networks and have broad implications for the study of antiviral RNAi.

Studies capturing the high efficiency of green-synthesized metal nanoparticles (NPs) in targeting mosquito vectors of the world's main infectious diseases suggest the NPs' possible utilization as bio-insecticides. However, it is necessary to confirm that these potential bio-insecticides are not harmful to non-target organisms that are often sympatric and natural enemies of the vectors of these diseases. In this systematic review, we comprehensively analyse the content of 56 publications focused on the potentially deleterious effects of NPs on these non-target organisms. Current research on biosynthesised NPs, characterization, and impact on mosquito vectors and non-target larvivorous organisms is reviewed and critically discussed. Finally, we pinpoint some major challenges that merit future investigation. Plants (87.5%) were mainly used for synthesizing NPs in the studies. NPs were found to be spherical or mainly spherical in shape with a large distribution size. In most of the included studies, NPs showed interesting mosquitocidal activity (LC < 50 ppm). Some plant families (e.g., Meliaceae, Poaceae, Lamiaceae) have produced NPs with a particularly high larvicidal and pupicidal activity (LC < 10 ppm). Regarding non-target organisms, most of the studies concluded that NPs were safe to them, with boosted predatory activity in NP-treated milieu. In contrast, some studies reported NP-elicited adverse effects (i.e., genotoxic, nuclear, and enzymatic effects) on these non-target organisms. This review outlines the promising mosquitocidal effects of biosynthesized NPs, recognizing that NPs' potential usage is currently limited by the harm NPs are thought pose to non-target organism. It is of utmost importance to investigate green NPs to determine whether laboratory findings have applications in the real world.

Plant-nectar-derived sugar is the major energy source for mosquitoes, but its influence on vector competence for malaria parasites remains unclear. Here, we show that Plasmodium berghei infection of Anopheles stephensi results in global metabolome changes, with the most significant impact on glucose metabolism. Feeding on glucose or trehalose (the main hemolymph sugars) renders the mosquito more susceptible to Plasmodium infection by alkalizing the mosquito midgut. The glucose/trehalose diets promote proliferation of a commensal bacterium, Asaia bogorensis, that remodels glucose metabolism in a way that increases midgut pH, thereby promoting Plasmodium gametogenesis. We also demonstrate that the sugar composition from different natural plant nectars influences A. bogorensis growth, resulting in a greater permissiveness to Plasmodium. Altogether, our results demonstrate that dietary glucose is an important determinant of mosquito vector competency for Plasmodium, further highlighting a key role for mosquito-microbiota interactions in regulating the development of the malaria parasite.

Vector control and personal protection against anthropophilic mosquitoes mainly rely on the use of insecticides and repellents. The search for mosquito-attractive semiochemicals has been the subject of intense studies for decades, and new compounds or odor blends are regularly proposed as lures for odor-baited traps. We present a comprehensive and up-to-date review of all the studies that have evaluated the attractiveness of volatiles to mosquitoes, including individual chemical compounds, synthetic blends of compounds, or natural host or plant odors. A total of 388 studies were analysed, and our survey highlights the existence of 105 attractants (77 volatile compounds, 17 organism odors, and 11 synthetic blends) that have been proved effective in attracting one or several mosquito species. The exhaustive list of these attractants is presented in various tables, while the most common mosquito attractants - for which effective attractiveness has been demonstrated in numerous studies - are discussed throughout the text. The increasing knowledge on compounds attractive to mosquitoes may now serve as the basis for complementary vector control strategies, such as those involving lure-and-kill traps, or the development of mass trapping. This review also points out the necessity of further improving the search for new volatile attractants, such as new compound blends in specific ratios, considering that mosquito attraction to odors may vary over the life of the mosquito or among species. Finally, the use of mosquito attractants will undoubtedly have an increasingly important role to play in future integrated vector management programs.

Mosquitoes are the main arthropod vectors of human pathogens. The current methods for mosquito identification include morphological and molecular methods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), now routinely used for bacterial identification, has recently emerged in the field of entomology. The aim of this study was to use MALDI-TOF MS to identify mosquito colonies from French Polynesia. Five hundred specimens from French Polynesia belonging to three species, Aedes aegypti, Aedes polynesiensis, and Culex quinquefasciatus, were included in the study. Testing the legs of these mosquitoes by MALDI-TOF MS revealed a 100% correct identification of all specimens at the species level. The MALDI-TOF MS profiles obtained allowed differentiation of male from female mosquitoes and the specific identification of female mosquito colonies of the same species but different geographic origin.

The majority of mosquito-borne illness is spread by a few mosquito species that have evolved to specialize in biting humans, yet the precise causes of this behavioral shift are poorly understood. We address this gap in the arboviral vector Aedes aegypti. We first collect and characterize the behavior of mosquitoes from 27 sites scattered across the species' ancestral range in sub-Saharan Africa, revealing previously unrecognized variation in preference for human versus animal odor. We then use modeling to show that over 80% of this variation can be predicted by two ecological factors-dry season intensity and human population density. Finally, we integrate this information with whole-genome sequence data from 375 individual mosquitoes to identify a single underlying ancestry component linked to human preference. Genetic changes associated with human specialist ancestry were concentrated in a few chromosomal regions. Our findings suggest that human-biting in this important disease vector originally evolved as a by-product of breeding in human-stored water in areas where doing so provided the only means to survive the long, hot dry season. Our model also predicts that the rapid urbanization currently taking place in Africa will drive further mosquito evolution, causing a shift toward human-biting in many large cities by 2050.

Chikungunya virus (CHIKV) that causes chikungunya fever, is an alphavirus that belongs to the Togaviridae family containing a single-stranded RNA genome. Mosquitoes of the Aedes species act as the vectors for this virus and can be found in the blood, which can be passed from an infected person to a mosquito through mosquito bites. CHIKV has drawn much attention recently because of its potential of causing an epidemic. As the detailed mechanism of its pathogenesis inside the host system is still lacking, in this in silico research we have hypothesized that CHIKV might create miRNAs, which would target the genes associated with host cellular regulatory pathways, thereby providing the virus with prolonged refuge. Using bioinformatics approaches we found several putative miRNAs produced by CHIKV. Then we predicted the genes of the host targeted by these miRNAs. Functional enrichment analysis of these targeted genes shows the involvement of several biological pathways regulating antiviral immune stimulation, cellular proliferation, and cell cycle, thereby provide themselves with prolonged refuge and facilitate their pathogenesis, which in turn may lead to disease conditions. Finally, we analyzed a publicly available microarray dataset (GSE49985) to determine the altered expression levels of the targeted genes and found genes associated with pathways such as cell differentiation, phagocytosis, T-cell activation, response to cytokine, autophagy, Toll-like receptor signaling, RIG-I like receptor signaling and apoptosis. Our finding presents novel miRNAs and their targeted genes, which upon experimental validation could facilitate in developing new therapeutics to combat CHIKV infection and minimize CHIKV mediated diseases.

Dengue virus (DENV) is a mosquito-borne virus that poses an incomparable public health problem, particularly in tropical and subtropical areas. Vaccination remains the most rational measure for controlling DENV infection. In this study, an ultraviolet irradiation (UV)-inactivated DENV-2 carried by -trimethyl chitosan nanoparticles (UV-inactivated DENV2 TMC NPs) was investigated as a potential non-replicating dengue vaccine candidate. Using a human model, the human monocyte-derived dendritic cells (MoDCs), we showed that TMC served as both a vaccine vehicle and a potent adjuvant. TMC NPs not only efficiently enhanced UV-inactivated DENV2 internalization into MoDCs but also greatly increased the breadth of UV-inactivated DENV2 immunogenicity to drive the maturation of MoDCs. Moreover, UV-inactivated DENV2 TMC NPs were highly immunogenic in mice, inducing greater levels of antibodies (total IgG, IgG1, IgG2a and neutralizing antibodies) and T cells (activated CD4⁺ and CD8⁺ T cells) against DENV-2 compared to soluble DENV-2 immunogens. Notably, the neutralizing activity of sera from mice immunized with UV-inactivated DENV2 TMC NPs was significantly augmented in the presence of complement activation, leading to the strong elimination of both DENV-2 particles and infected cells. We further showed that the immunogenicity of an inactivated dengue-based vaccine was significantly improved in a concentration-dependent manner. These positive results warrant further investigations of this platform of vaccine delivery for tetravalent vaccines or monovalent vaccines in sequential immunizations.

Plasmodium species, the causative agent of malaria, have a complex life cycle involving two hosts. The sporozoite life stage is characterized by an extended phase in the mosquito salivary glands followed by free movement and rapid invasion of hepatocytes in the human host. This transmission stage has been the subject of many transcriptomics and proteomics studies and is also targeted by the most advanced malaria vaccine. We applied Bayesian data integration to determine which proteins are not only present in sporozoites but are also specific to that stage. Transcriptomic and proteomic Plasmodium data sets from 26 studies were weighted for how representative they are for sporozoites, based on a carefully assembled gold standard for Plasmodium falciparum (Pf) proteins known to be present or absent during the sporozoite life stage. Of 5418 Pf genes for which expression data were available at the RNA level or at the protein level, 975 were identified as enriched in sporozoites and 90 specific to them. We show that Pf sporozoites are enriched for proteins involved in type II fatty acid synthesis in the apicoplast and GPI anchor synthesis, but otherwise appear metabolically relatively inactive in the salivary glands of mosquitos. Newly annotated hypothetical sporozoite-specific and sporozoite-enriched proteins highlight sporozoite-specific functions. They include PF3D7_0104100 that we identified to be homologous to the prominin family, which in human has been related to a quiescent state of cancer cells. We document high levels of genetic variability for sporozoite proteins, specifically for sporozoite-specific proteins that elicit antibodies in the human host. Nevertheless, we can identify nine relatively well-conserved sporozoite proteins that elicit antibodies and that together can serve as markers for previous exposure. Our understanding of sporozoite biology benefits from identifying key pathways that are enriched during this life stage. This work can guide studies of molecular mechanisms underlying sporozoite biology and potential well-conserved targets for marker and drug development.

In many developing countries, there are certain health problems faced by the public, one among them is Malaria. This tropical disease is mainly caused by Plasmodium falciparum. It is categorized as a disaster to public health, which increases both mortality and morbidity. Numerous drugs are in practice to control this disease and their vectors. Eco-friendly control tools are required to battle against vector of this significant disease. Nanotechnology plays a major role in fighting against malaria. The present paper synthesized Zinc oxide nanoparticles (ZnO NPs) using zinc nitrate via simple green routes with the help of aqueous peel extract of Lagenaria siceraria (L. siceraria). The synthesized ZnO NPs were characterized by various biophysical methods. Moreover, the extract of L. siceraria and their mediated ZnO NPs was experimented against III instar larvae of An. stephensi. The impact of the treatment based on ZnO NPs concerning histology and morphology of mosquito larval was further observed. In the normal laboratory environment, the efficiency of predation of Poeciliareticulata (P. reticulata) against An. Stephensi larvae was found to be 44%, whereas in aqueous L. siceraria extract and its mediated ZnO NPs contaminated environment, P. reticulate showed predation efficiency of about 45.8% and 61.13% against An. Stephensi larva. L. siceraria synthesized ZnO NPs were examined against the Plasmodium falciparum CQ-sensitive strains. The L. siceraria extract and its mediated ZnO NPs showed the cytotoxic effects against HeLa cell lines with an IC value of 62.5 µg/mL. This study concludes that L. siceraria peel extract and L. siceraria synthesized ZnO NPs represent a valuable green option to fight against malarial vectors and parasites.

Getah virus (GETV), a mosquito-borne virus belonging to the Alphavirus genus of family Togaviridae, has become increasingly problematic, which poses a huge threat to the safety of animals and public health. In order to detect GETV quickly and accurately, we have developed a SYBR Green I real-time quantitative reverse transcription PCR (RT-qPCR) assay for GETV with the detection limit of 66 copies/μL, excellent correlation coefficient (R) of 0.9975, and amplification efficiency (E) of 98.90%, the target selected was the non-structural protein 3 of GETV. The sensitivity of it was higher than that of ordinary RT-PCR by 1000 folds, and the inter-assay and intra-assay CV values were all less than 0.99%. The newly developed RT-qPCR assay exhibited good sensitivity and reproducibility, which will provide technical support for the reliable and specific rapid diagnosis, and quantitative analysis of GETV infection.

BACKGROUND:A study conducted at the International Airport of Cluj-Napoca, Romania, with the aim of investigating the presence/absence of invasive Aedes mosquito species resulted in finding Aedes japonicus japonicus (Theobald 1901) eggs in one of the ovitraps placed on site. METHODS:The study was carried out between 30 June and 29 September 2020. On 24 August, 26 eggs were collected and later hatched at the University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca's insectary. On 15 October another adult female Ae. japonicus was caught entering a building in the center of the city, about 7 km from the first sampling spot. RESULTS:The mosquitoes were identified morphologically and confirmed by molecular analysis, based on the genetic analysis of the mitochondrial gene cytochrome c oxidase subunit 1 (COI). CONCLUSION:This is the first report of the species in Romania, highlighting the need for surveillance and implemented control methods. However, in Romania to our knowledge only Aedes albopictus has been established; further studies are required to learn about this new invasive species' status in Romania.

BACKGROUND:Dengue virus (DENV) is a mosquito-borne arbovirus transmitted by Aedes mosquitoes, but is not endemic in all areas where this vector is found. For example, the relatively sparse distribution of cases in West Africa is generally attributed to the refractory nature of West African Aedes aegypti (Ae. aegypti) to DENV infection, and particularly the forest-dwelling Ae. aegypti formosus. However, recent studies have shown these mosquitoes to be competent vectors within some West African countries that have suffered outbreaks in the past, such as Senegal. There is however little information on the vector competence of the Ae. aegypti in West African countries such as Ghana with no reported outbreaks. METHODS:This study examined the vector competence of 4 Ae. aegypti colonies from urban, semi-urban, and two rural locations in Ghana in transmitting DENV serotypes 1 and 2, using a single colony from Vietnam as control. Midgut infection and virus dissemination were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), while the presence and concentration of DENV in the saliva of infectious mosquitoes was determined by the focus forming assay. RESULTS:There were significant differences in the colonies' susceptibility to virus infection, dissemination, and transmission. All examined Ghanaian mosquitoes were refractory to infection by DENV serotype 2, while some colonies exhibited potential to transmit DENV serotype 1. None of the tested colonies were as competent as the control group colony. CONCLUSIONS:These findings give insight into the possible risk of outbreaks, particularly in the urban areas in the south of Ghana, and highlight the need for continuous surveillance to determine the transmission status and outbreak risk. This study also highlights the need to prevent importation of different DENV strains and potential invasion of new highly vector-competent Ae. aegypti strains, particularly around the ports of entry.

Recognition of Zika virus (ZIKV) sexual transmission (ST) among humans challenges our understanding of the maintenance of mosquito-borne viruses in nature. Here we dissected the relative contributions of the components of male reproductive system (MRS) during early male-to-female ZIKV transmission by utilizing mice with altered antiviral responses, in which ZIKV is provided an equal opportunity to be seeded in the MRS tissues. Using microRNA-targeted ZIKV clones engineered to abolish viral infectivity to different parts of the MRS or a library of ZIKV genomes with unique molecular identifiers, we pinpoint epithelial cells of the epididymis (rather than cells of the testis, vas deferens, prostate, or seminal vesicles) as a most likely source of the sexually transmitted ZIKV genomes during the early (most productive) phase of ZIKV shedding into the semen. Incorporation of this mechanistic knowledge into the development of a live-attenuated ZIKV vaccine restricts its ST potential.

Microbes or parasites spread vector-borne diseases by mosquitoes without being affected themselves. Insecticides used in vector control produce a substantial problem for human health. This study synthesized zinc oxide nanoparticles (ZnO NPs) using Lawsonia inermis L. and were characterized by UV-vis, FT-IR, SEM with EDX, and XRD analysis. Green synthesized ZnO NPs were highly toxic against Anopheles stephensi, whose lethal concentrations values ranged from 5.494 ppm (I instar), 6.801 ppm (II instar), 9.336 ppm (III instar), 10.736 ppm (IV instar), and 12.710 ppm (pupae) in contrast to L. inermis treatment. The predation efficiency of the teleost fish Gambusia affinis and the copepod Mesocyclops aspericornis against A. stephensi was not affected by exposure at sublethal doses of ZnO NPs. The predatory potency for G. affinis was 45 (I) and 25.83% (IV), copepod M. aspericornis was 40.66 (I) and 10.8% (IV) while in an ZnO NPs contaminated environment, the predation by the fish G. affinis was boosted to 71.33 and 34.25%, and predation of the copepod M. aspericornis was 60.35 and 16.75%, respectively. ZnO NPs inhibited the growth of several microbial pathogens including the bacteria (Escherichia coli and Bacillus subtilis) and the fungi (Alternaria alternate and Aspergillus flavus), respectively. ZnO NPs decreased the cell viability of Hep-G2 with IC value of 21.63 µg/mL (R = 0.942; P < 0.001) while the concentration increased from 1.88 to 30 µg/mL. These outcomes support the use of L. inermis mediated ZnO NPs for mosquito control and drug development.

To combat health challenges associated with mosquito-borne diseases, the larvicidal activity of metallic nanoparticles, food-grade polymeric nano-capsules and insecticides was investigated against larvae of Aedes albopictus as an effective alternate control approach. The Ae. albopictus was identified using sequencing and phylogenetic analyses of COXI, CYTB and ITS2 genes. The characterization of synthesized nanostructures was performed through Zetasizer, UV-VIS spectroscopy, atomic force microscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy. The mosquito larvae were exposed to varying concentration of nanostructures and insecticides, and their percentage mortality was evaluated at different time intervals of 24 h and 48 h exposure. The highest efficacy was observed in zinc oxide nanoparticles (ZnO-NPs) and polymeric nanocapsules FG-Cur E-III (LC50 = 0.24 mg/L, LC90 = 0.6 mg/L) and (LC50 = 3.8 mg/L, LC90 = 9.33 mg/L), respectively, after 24 h; while (LC50 = 0.18 mg/L, LC90 = 0.43 mg/L) and (LC50 = 1.95 mg/L, LC90 = 6.46 mg/L), respectively, after 48 h against fourth instar larvae of Ae. albopictus. Ag, CuO, NiTiO3 and CoTiO3 nanoparticles evaluated in this study also showed promising larvicidal activity. Although ZnO-NPs proved to be effective larvicides, their possible toxicity (producing ROS species) can limit their use. The curcumin nanostructures (FG-Cur E-III) stabilized by food-grade materials are thought to exert their larvicidal activity by binding to sterol carrier protein-2, and depriving the larvae from the essential dietary cholesterol, and bears effective larvicidal potential as safe alternative for chemical larvicides, due to their environment friendly, food-grade and easy biodegradability.