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Genetic toolkit is now extended to disease vectors. For genetic control methods such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-based editing and gene drives, promoters with robust and tissue-specific expression are needed. The availability of genomes and machine-learning technologies provides an opportunity to identify promoters in non-model vectors. Vector-borne diseases (VBDs) impose devastating effects on human health and a heavy financial burden. Malaria, Lyme disease, and dengue fever are just a few examples of VBDs that cause severe illnesses. The current strategies to control VBDs consist mainly of environmental modification and chemical use, and to a small extent, genetic approaches. The genetic approaches, including transgenesis/genome modification and gene-drive technologies, provide the basis for developing new tools for VBD prevention by suppressing vector populations or reducing their capacity to transmit pathogens. The regulatory elements such as promoters are required for a robust sex-, tissue-, and stage-specific transgene expression. As discussed in this review, information on the regulatory elements is available for mosquito vectors but is scant for other vectors. [ABSTRACT FROM AUTHOR]

Background: Tick-borne flaviviruses (TBFs) cause thousands of human cases of encephalitis worldwide each year, with some TBF infections progressing to hemorrhagic fever. TBFs are of medical and veterinary importance and strategies to reduce flavivirus transmission by the tick vector may have significant application. Analyses of the proteome of ISE6 cells derived from the black legged tick, Ixodes scapularis infected with the TBF, Langat virus (LGTV), have provided insights into proteins and cellular processes involved with LGTV infection. Methods: RNA interference (RNAi)-induced knockdown of transcripts was used to investigate the role of ten tick proteins in the LGTV infection cycle in ISE6 cells. LGTV-infected cells were separately transfected with dsRNA corresponding to each gene of interest and the effect on LGTV genome replication and release of infectious virus was assessed by RT-qPCR and plaque assays, respectively. Results: RNAi-induced knockdown of transcripts for two enzymes that likely function in amino acid, carbohydrate, lipid, terpenoid/polykeytide and vitamin metabolism, and a transcript for one protein of unknown function were associated with decreased replication of the LGTV genome and release of infectious virus from cells. The knockdown of transcripts for five enzymes predicted to function in metabolism, a protein likely associated with folding, sorting and degradation, and a protein of unknown function was associated with a decrease only in the amount of infectious LGTV released from cells. Conclusions: These data suggest tick proteins potentially associated with metabolism and protein processing may be involved in LGTV infection of ISE6 cells. Our study provides information to begin to elucidate the function of these proteins and identify targets for the development of new interventions aimed at controlling the transmission of TBFs. [ABSTRACT FROM AUTHOR]

Aedes aegypti is an anautogenous mosquito that must blood feed on a vertebrate host to produce and lay a clutch of eggs. The rockpool mosquito, Georgecraigius atropalpus , is related to A. aegypti but is a facultatively autogenous species that produces its first clutch of eggs shortly after emerging without blood feeding. Consumption of a blood meal by A. aegypti triggers the release of ovary ecdysteroidogenic hormone (OEH) and insulin-like peptide 3 (ILP3) from the brain, which stimulate egg formation . OEH and ILP3 also stimulate egg formation in G. atropalpus but are released at eclosion independently of blood feeding. These results collectively suggest that blood meal dependent release of OEH and ILP3 is one factor that prevents A. aegypti from reproducing autogenously. Here, we examined two other factors that potentially inhibit autogeny in A. aegypti : teneral nutrient reserves and the ability of OEH and ILP3 to stimulate egg formation in the absence of blood feeding. Measures of nutrient reserves showed that newly emerged A. aegypti females had similar wet weights but significantly lower protein and glycogen reserves than G. atropalpus females when larvae were reared under identical conditions. OEH stimulated non-blood fed A. aegypti females to produce ecdysteroid hormone and package yolk into oocytes more strongly than ILP3. OEH also reduced host seeking and blood feeding behavior, yet females produced few mature eggs. Overall, our results indicate that multiple factors prevent A. aegypti from reproducing autogenously. [ABSTRACT FROM AUTHOR]

The rockpool mosquito, Georgecraigius atropalpus, is a facultatively autogenous species that produces its first egg clutch without a blood meal shortly after emergence. Several days after depositing this clutch, females must take a blood meal to produce a second egg clutch. Decapitation of females shortly after emergence or blood ingestion prevents egg maturation. Here, we report that a single injected dose of the neuropeptide ovary ecdysteroidogenic hormone (OEH) fully restored egg maturation in decapitated females in both circumstances. This neuropeptide and two insulin-like peptides (ILPs) are potent gonadotropins in the related yellow fever mosquito, Aedes aegypti. ILP3 was marginally restorative in decapitated G. atropalpus, and ILP4 had no effect. Egg maturation in non- and blood-fed G. atropalpus was dependent on the enzymatic mobilization of amino acids from stored protein or the blood meal for yolk protein (vitellogenin, VG) synthesis and uptake by oocytes. We further show that OEH stimulates serine protease activity in the fat body of newly eclosed females or in the midgut of blood-fed ones, and ecdysteroid hormone production by the ovaries of both females. In contrast, only 20-hydroxyecdysone stimulated VG synthesis in the fat body of non- and blood-fed females. Using RNA interference to knock down expression of the insulin receptor, we found that OEH still fully restored autogenous egg maturation. In summary, our results identify OEH as a primary regulator of egg maturation in both autogenous and blood-fed G. atropalpus females and suggest the shift from blood meal-dependent to blood meal-independent release of OEH is a key factor in the evolution of autogeny in this species. [ABSTRACT FROM AUTHOR]

Background: Mosquitoes are insects that vector many serious pathogens to humans and other vertebrates. Most mosquitoes must feed on the blood of a vertebrate host to produce eggs. In turn, multiple cycles of blood feeding promote frequent contacts with hosts and make mosquitoes ideal disease vectors. Both hormonal and nutritional factors are involved in regulating egg development in the mosquito, Aedes aegypti. However, the processes that regulate digestion of the blood meal remain unclear. Methodology/Principal Findings: Here we report that insulin peptide 3 (ILP3) directly stimulated late phase trypsin-like gene expression in blood fed females. In vivo knockdown of the mosquito insulin receptor (MIR) by RNA interference (RNAi) delayed but did not fully inhibit trypsin-like gene expression in the midgut, ecdysteroid (ECD) production by ovaries, and vitellogenin (Vg) expression by the fat body. In contrast, in vivo treatment with double-stranded MIR RNA and rapamycin completely blocked egg production. In vitro experiments showed that amino acids did not simulate late phase trypsin-like gene expression in the midgut or ECD production by the ovaries. However, amino acids did enhance ILP3-mediated stimulation of trypsin-like gene expression and ECD production. Conclusions/Significance: Overall, our results indicate that ILPs from the brain synchronize blood meal digestion and amino acid availability with ovarian ECD production to maximize Vg expression by the fat body. The activation of digestion by ILPs may also underlie the growth promoting effects of insulin and TOR signaling in other species. [ABSTRACT FROM AUTHOR]

Adult male and female mosquitoes consume sugar as floral and extrafloral nectar. Earlier work demonstrated that mosquito populations and their vector potential are dependent upon the availability of sugar sources. Thus, a novel method of vector control may involve targeting sugar-feeding mosquitoes. Multiple human-safe sugar substitutes are already approved by the U.S. Food and Drug Administration and are readily available. However, plant-based sugar substitutes such as stevia (erythritol) have been shown to affect lifespan in other flies. Therefore, the current study was carried out to test the potential of commercially available sugar substitutes to adversely affect the survival, fecundity, and metabolism of adult Aedes aegypti mosquitoes. Of the four sugar substitutes tested, erythritol (Stevia), sucralose (Splenda), aspartame (Equal), and saccharin (Sweet'N Low), only erythritol negatively affected mosquito longevity and fecundity. The effect on fecundity was probably due in part to a corresponding decrease in glycogen and lipid levels over time in mosquitoes fed on erythritol. Comparative mosquito head transcriptomes indicated upregulation of a gene in the mannose biosynthesis pathway in females fed on erythritol, suggesting that N-linked glycosylation might be responsible for the negative impact of erythritol feeding in mosquitoes. Mosquitoes preferred sucrose when a choice was given but were not averse to erythritol. Our results suggest the possibility of using erythritol alone or in combination with sucrose as a component of attractive toxic sugar baits for a human-safe approach for mosquito control. [ABSTRACT FROM AUTHOR]

Lymphatic filariasis, commonly known as elephantiasis, is a painful and profoundly disfiguring disease. Wuchreria bancrofti (Wb) is responsible for >90% of infections and the remainder are caused by Brugia spp. Mosquitoes of the genera Culex (in urban and semi-urban areas), Anopheles (in rural areas of Africa and elsewhere), and Aedes (in Pacific islands) are the major vectors of W. bancrofti. A preventive chemotherapy called mass drug administration (MDA), including albendazole with ivermectin or diethylcarbamazine citrate (DEC) is used in endemic areas. Vector control strategies such as residual insecticide spraying and long-lasting insecticidal nets are supplemental to the core strategy of MDA to enhance elimination efforts. However, increasing insecticide resistance in mosquitoes and drug resistance in parasite limit the effectiveness of existing interventions, and new measures are needed for mosquito population control and disruption of mosquito-parasite interactions to reduce transmission. Mosquito insulin signaling regulates nutrient metabolism and has been implicated in reduced prevalence and intensity of malaria parasite, Plasmodium falciparum, infection in mosquitoes. Currently no data are available to assess how insulin signaling in mosquitoes affects the development of multi-cellular parasites, such as filarial nematodes. Here, we show that insulin receptor knockdown in blood fed C. quinquefasciatus, the major vector of Wb in India, completely blocks the development of filarial nematode parasite to the infective L3 stage, and results in decreased ecdysteroid production and trypsin activity leading to fewer mosquito eggs. These data indicate that a functional mosquito insulin receptor (IR) is necessary for filarial parasite development and mosquito reproduction. Therefore, insulin signaling may represent a new target for the development of vector control or parasite blocking strategies. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Abstract: Most mosquito species must feed on the blood of a vertebrate host to produce eggs. In the yellow fever mosquito, Aedes aegypti, blood feeding triggers medial neurosecretory cells in the brain to release insulin-like peptides (ILPs) and ovary ecdysteroidogenic hormone (OEH). Theses hormones thereafter directly induce the ovaries to produce ecdysteroid hormone (ECD), which activates the synthesis of yolk proteins in the fat body for uptake by oocytes. ILP3 stimulates ECD production by binding to the mosquito insulin receptor (MIR). In contrast, little is known about the mode of action of OEH, which is a member of a neuropeptide family called neuroparsin. Here we report that OEH is the only neuroparsin family member present in the Ae. aegypti genome and that other mosquitoes also encode only one neuroparsin gene. Immunoblotting experiments suggested that the full-length form of the peptide, which we call long OEH (lOEH), is processed into short OEH (sOEH). The importance of processing, however, remained unclear because a recombinant form of lOEH (rlOEH) and synthetic sOEH exhibited very similar biological activity. A series of experiments indicated that neither rlOEH nor sOEH bound to ILP3 or the MIR. Signaling studies further showed that ILP3 activated the MIR but rlOEH did not, yet both neuropeptides activated Akt, which is a marker for insulin pathway signaling. Our results also indicated that activation of TOR signaling in the ovaries required co-stimulation by amino acids and either ILP3 or rlOEH. Overall, we conclude that OEH activates the insulin signaling pathway independently of the MIR, and that insulin and TOR signaling in the ovaries is coupled. [Copyright &y& Elsevier]

Simple Summary: Even after decades of control interventions, mosquito-borne diseases still pose a huge threat to humans. Understanding gene functions is important for discovering new targets for mosquito and mosquito-borne disease control. One of the well-established and widely used methods for understanding gene function is RNA-interference (RNAi). The use of RNAi is, however, restricted mostly to adult mosquitoes. A few studies have shown its applicability in mosquito larvae, and just one in pupae. The current double-stranded RNA (dsRNA) delivery methods for RNAi are labor-intensive and require microinjections into mosquitoes (adults, larvae, or pupae). In this study, we present a simple, fast, and less labor-intensive technique for RNAi in the pupal stage by soaking pupae in water containing dsRNA. This method will be useful in studying genes expressed in immature life stages of the mosquitoes and will hopefully open new avenues for identifying mosquito control targets in early life stages. RNA-interference (RNAi) is a standard technique for functional genomics in adult mosquitoes. However, RNAi in immature, aquatic mosquito stages has been challenging. Several studies have shown successful larval RNAi, usually in combination with a carrier molecule. Except for one study in malaria mosquito, Anopheles gambiae, none of the previous studies has explored RNAi in mosquito pupae. Even in the study that used RNAi in pupae, double stranded RNA (dsRNA) was introduced by microinjection. Here, we describe a successful method by soaking pupae in water containing dsRNA without any carrier or osmotic challenge. The knockdown persisted into adulthood. We expect that this simple procedure will be useful in the functional analysis of genes that highly express in pupae or newly emerged adults. [ABSTRACT FROM AUTHOR]

Simple Summary: Human malaria is transmitted by mosquitoes in the genus Anopheles—particularly species that prefer to feed on humans. The mosquito's sense of smell drives this preference; however, most studies have focused on species native to Sub-Saharan Africa. Malaria vectors in other parts of the world may use different odorants to choose hosts. We, therefore, focused on Anopheles stephensi, the south Asian malaria mosquito, in this study. Mosquitoes have different organs specialized for odor perception, such as the antennae; however, we focused on the mouthparts (primarily the maxillary palp and labella) in this study. We used the RNAseq technique to determine which odor receptors are present in the mouthparts and then focused on one of these receptors: Or8. Using a technique known as the Drosophila empty neuron system, we tested this receptor's ability to detect different odorants, particularly chemicals emitted by humans. This receptor in An. stephensi detected similar odors to a homologous receptor in an African species, Anopheles gambiae, with the exception of the chemical sulcatone. This chemical is an important attractant in other disease-transmitting mosquitoes and suggests that different mosquito species may be using odors differently to find hosts. Several mosquito species within the genus Anopheles are vectors for human malaria, and the spread of this disease is driven by the propensity of certain species to feed preferentially on humans. The study of olfaction in mosquitoes is important to understand dynamics of host-seeking and host-selection; however, the majority of these studies focus on Anopheles gambiae or An. coluzzii, both vectors of malaria in Sub-Saharan Africa. Other malaria vectors may recognize different chemical cues from potential hosts; therefore, in this study, we investigated An. stephensi, the south Asian malaria mosquito. We specifically focused on the mouthparts (primarily the maxillary palp and labella) that have been much less investigated compared to the antennae but are also important for host-seeking. To provide a broad view of chemoreceptor expression, RNAseq was used to examine the transcriptomes from the mouthparts of host-seeking females, blood-fed females, and males. Notably, AsOr8 had a high transcript abundance in all transcriptomes and was, therefore, cloned and expressed in the Drosophila empty neuron system. This permitted characterization with a panel of odorants that were selected, in part, for their presence in the human odor profile. The responsiveness of AsOr8 to odorants was highly similar to An. gambiae Or8 (AgOr8), except for sulcatone, which was detected by AsOr8 but not AgOr8. Subtle differences in the receptor sensitivity to specific odorants may provide clues to species- or strain-specific approaches to host-seeking and host selection. Further exploration of the profile of An. stephensi chemosensory proteins may yield a better understanding of how different malaria vectors navigate host-finding and host-choice. [ABSTRACT FROM AUTHOR]

Ixodes scapularis is the major vector of Lyme disease in the Eastern United States. Each active life stage (larva, nymph, and adult) takes a blood meal either for developing and molting to the next stage (larvae and nymphs) or for oviposition (adult females). This protein-rich blood meal is the only food taken by Ixodes ticks and therefore efficient blood digestion is critical for survival. Studies in partially engorged ticks have shown that the initial stages of digestion are carried out by cathepsin proteases within acidic digestive cells. In this study, we investigated the potential role of serine proteases in blood digestion in replete ticks. RNA interference was used for functional analysis and a trypsin-benzoyl-D, L-arginine 4-nitoanilide assay was used to measure active trypsin levels. Hemoglobinolytic activity was determined in vitro, with or without a serine protease inhibitor. Our data suggest that trypsin levels increase significantly after repletion. Knockdown of serine proteases negatively impacted blood feeding, survival, fecundity, levels of active trypsin in the midgut, and resulted in lower hemoglobin degradation. Incubation of midgut extract with a trypsin inhibitor resulted in 65% lower hemoglobin degradation. We provide evidence of the serine proteases as digestive enzymes in fully engorged, replete females. Understanding the digestive profile of trypsin during blood meal digestion in I. scapularis improves our understanding of the basic biology of ticks and may lead to new methods for tick control. [ABSTRACT FROM AUTHOR]

Mosquitoes have distinct developmental and adult life history, and the vectorial capacity of females has been shown to be affected by the larval nutritional environment. However, little is known about the effect of developmental nutrition on insulin-signaling and nutrient storage. In this study, we used Aedes aegypti, the yellow fever mosquito, to determine whether larval nutrition affects insulin gene expression. We also determined the traits regulated by insulin signaling, such as stored-nutrient levels and fecundity. We raised mosquito larvae on two different diets, containing either high protein or high carbohydrates. Development on a high-carbohydrate diet resulted in several life-history phenotypes indicative of suboptimal conditions, including increased developmental time and decreased fecundity. Additionally, our data showed that insulin transcript levels are affected by a high-carbohydrate diet during development. Females, not males, reared on high-carbohydrate diets had much higher transcript levels of insulin-like peptide 3 (ILP3), a mosquito equivalent of human insulin, and these females more readily converted sugar meals into lipids. We also found that AaILP4, not AaILP3, transcript levels were much higher in the males after a sugar meal, suggesting sex-specific differences in the insulin-signaling pathway. Our findings suggest a conserved mechanism of carbohydrate-mediated hyperinsulinemia in animals. [ABSTRACT FROM AUTHOR]