Transgenic Mosquitoes Research Articles

Mimicking superinfection exclusion disrupts alphavirus infection and transmission in the yellow fever mosquito Aedes aegypti

Multiple viruses, including pathogenic viruses, bacteriophages, and even plant viruses, cause a phenomenon termed superinfection exclusion whereby a currently infected cell is resistant to secondary infection by the same or a closely related virus. In alphaviruses, this process is thought to be mediated, at least in part, by the viral protease (nsP2) which is responsible for processing the nonstructural polyproteins (P123 and P1234) into individual proteins (nsP1-nsP4), forming the viral replication complex. Taking a synthetic biology approach, we mimicked this naturally occurring phenomenon by generating a superinfection exclusion-like state in Aedes aegypti mosquitoes, rendering them refractory to alphavirus infection. By artificially expressing Sindbis virus (SINV) and chikungunya virus (CHIKV) nsP2 in mosquito cells and transgenic mosquitoes, we demonstrated a reduction in both SINV and CHIKV viral replication rates in cells following viral infection as well as reduced infection prevalence, viral titers, and transmission potential in mosquitoes.

Evaluation of Aedes aegypti Feeding Status.

Female mosquitoes need vertebrate blood for egg development. Evaluating mosquito behavior is essential for determining the ability of a mosquito to blood feed. Blood feeding experiments are often performed using artificial membrane feeders; however, such experiments do not represent realistic scenarios in which a mosquito injects saliva into the host to prevent host hemostatic responses. Vertebrate animal models are therefore more representative of a natural blood feeding event. Here, we describe a methodology to evaluate mosquito blood feeding success that can be used to compare blood feeding between mosquito groups-for instance, wild-type versus transgenic mosquitoes lacking salivary proteins or field-collected versus laboratory-reared mosquitoes. We also include a simple procedure to measure blood meal size, allowing for a more quantitative assessment of feeding status. The volume of ingested blood directly affects mosquito fecundity and fertility, important markers of fitness. The methods described herein can be used to evaluate transmission-blocking vaccines, insecticides, or fitness costs associated with transgenic mosquitoes.

Mathematical modeling of the performance of wild and transgenic mosquitoes in malaria transmission.

A mathematical model that simulates malaria transmission under the influence of transgenic mosquitoes refractory to malaria is presented in this paper. The zygosity of transgenic mosquitoes is taken into account and, consequently, the total population of mosquitoes is comprised of wild type and heterozygous and homozygous transgenic mosquitoes. These three mosquito varieties interact by mating and competition, and the genetic characteristics of their resulting offspring are in accordance with Mendelian genetics or the mutagenic chain reaction (MCR) technique. Although the incorporation of transgenic mosquitoes into the ecosystem reduces the incidence of malaria, the model also takes into account the importance of completing treatment in individuals with confirmed infection and the imminent risk of increased environmental temperature.

CRISPR-Cas9 Methods and Key Considerations in the Production of Aedes aegypti Mutant Strains.

Since the characterization of the CRISPR-Cas9 system in prokaryotes, it has become the prime choice in gene editing because of its exceptional flexibility, ease of use, high efficiency, and superior specificity. As a result, CRISPR-Cas9-mediated gene-editing technologies have enabled researchers not only to engineer transgenic animal strains with site-directed insertions more efficiently but also to generate desired mutants for previously intractable species. One such species is the invasive yellow fever mosquito, Aedes aegypti, which is notorious for its ability to transmit many blood-borne human pathogens. Methods for developing new transgenic strains of the yellow fever mosquito may aid in the effort to control its populations and provide significant benefits for the public. Here, we provide an overview of injection and noninjection methods for generating transgenic mosquitoes and also highlight important experimental design features.

Fine-scale spatial distribution of deltamethrin resistance and population structure of Anopheles funestus and Anopheles arabiensis populations in Southern Mozambique

BackgroundInsecticide resistance in malaria vectors can be spatially highly heterogeneous, yet population structure analyses frequently find relatively high levels of gene flow among mosquito populations. Few studies have contemporaneously assessed phenotypic, genotypic and population structure analysis on mosquito populations and none at fine geographical scales. In this study, genetic diversity, population structure, and insecticide resistance profiles of Anopheles funestus and Anopheles arabiensis were examined across mosquito populations from and within neighbouring villages.MethodsMosquitoes were collected from 11 towns in southern Mozambique, as well as from different neighbourhoods within the town of Palmeira, during the peak malaria transmission season in 2016. CDC bottle bioassay and PCR assays were performed with Anopheles mosquitoes at each site to determine phenotypic and molecular insecticide resistance profiles, respectively. Microsatellite analysis was conducted on a subsample of mosquitoes to estimate genetic diversity and population structure.ResultsPhenotypic insecticide resistance to deltamethrin was observed in An. funestus sensu stricto (s.s.) throughout the area, though a high level of mortality variation was seen. However, 98% of An. funestus s.s. were CYP6P9a homozygous resistant. An. arabiensis was phenotypically susceptible to deltamethrin and 99% were kdr homozygous susceptible. Both Anopheles species exhibited high allelic richness and heterozygosity. Significant deviations from Hardy–Weinberg equilibrium were observed, and high linkage disequilibrium was seen for An. funestus s.s., supporting population subdivision. However, the FST values were low for both anophelines (− 0.00457 to 0.04213), Nm values were high (9.4–71.8 migrants per generation), AMOVA results showed almost 100% genetic variation among and within individuals, and Structure analysis showed no clustering of An. funestus s.s. and An. arabiensis populations. These results suggest high gene flow among mosquito populations.ConclusionDespite a relatively high level of phenotypic variation in the An. funestus population, molecular analysis shows the population is admixed. These data indicate that CYP6P9a resistance markers do not capture all phenotypic variation in the area, but also that resistance genes of high impact are likely to easily spread in the area. Conversely, other strategies, such as transgenic mosquito release programmes will likely not face challenges in this locality.

Genetically modified mosquitoes for a better world

Discoveries of the scientific world have made our life better. Commencing genetically modified mosquitoes will help us avoid mosquito-borne diseases such as malaria. Mosquitoes cause more human suffering than any other organisms as they carry dreadful diseases like malaria, West Nile virus, yellow fever, dengue and over a million people die worldwide from mosquito-borne diseases every year. They are debilitating and deadly in tropics and subtropical areas. The malarial parasite in particular has started to develop resistance to currently available drugs. Even insecticide spraying is now a challenge in many mosquito species like Aedes egypti which is a vector for viruses such as chikungunya, dengue, zika. These transgenic mosquitoes will be released to interact with wild-type populations and through interspecific mating, the mutated gene will be transferred which will impede the plasmodium evolution with its species.

Evolutionary outlook on the impending endgame of malaria

Malaria is a highly debilitating vector-borne parasitic infection which has been known to be mortal and morbid to human beings for centuries now. As of today, the success rate of effective control strategies is less; however there have been several riveting developments. These include: 1. A global incidence of decrease in malaria cases; 2. The development of several non-malaria transmissible transgenic mosquitoes and 3. The launch of the vaccine has been a game changer as it is now being given to children with the success rate being high. In this review, we discuss if the current control strategies in place and the launch of the vaccine and its speculated success in children has become/been a game changer. Our take on the coevolution of parasites and vector can be the answer to the questions above. We highlight that parasites and vectors have evolved together for millions of years now, and to break this liaison will not be as easy.

Two-Photon Calcium Imaging in the Brain of Aedes aegypti Mosquitoes.

Understanding the neural basis of mosquito behavior is critical for designing effective vector control strategies and can potentially shed new light on basic nervous system function. Because mosquitoes are a non-model species, however, functional studies of mosquito nervous systems have long been restricted to electrophysiological recording from peripheral sensory organs such as the antenna. This is now changing with the advent of CRISPR-Cas9 gene editing and the development of other powerful new genetic tools. Transgenic mosquitoes that carry genetically encoded calcium sensors, for example, open the door to optical recording of neural activity with two-photon calcium imaging. Compared with electrophysiology, calcium imaging permits continuous monitoring of neural activity from large populations of neurons, even deep in the brain. When combined with selective neural drivers, it also allows targeted recording from specific neuronal types. Here, we describe a calcium imaging protocol we use in our laboratory to study neural activity in the brain of Aedes aegypti mosquitoes.

The impact of transgenesis on mosquito fitness: A review.

Transgenic mosquitoes developed by genetic manipulation, offer a promising strategy for the sustainable and effective control of mosquito-borne diseases. This strategy relies on the mass release of transgenic mosquitoes into the wild, where their transgene is expected to persist in the natural environment, either permanently or transiently, within the mosquito population. In such circumstances, the fitness of transgenic mosquitoes is an important factor in determining their survival in the wild. The impact of transgene expression, insertional mutagenesis, inbreeding depression related to laboratory adaptation, and the hitchhiking effect involved in developing homozygous mosquito lines can all have an effect on the fitness of transgenic mosquitoes. Therefore, real-time estimation of transgene-associated fitness cost is imperative for modeling and planning transgenic mosquito release programs. This can be achieved by directly comparing fitness parameters in individuals homozygous or hemizygous for the transgene and their wild-type counterparts, or by cage invasion experiments to monitor the frequency of the transgenic allele over multiple generations. Recent advancements such as site-specific integration systems and gene drives, provide platforms to address fitness issues in transgenic mosquitoes. More research on the fitness of transgenic individuals is required to develop transgenic mosquitoes with a low fitness cost.

Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium sporogonic development.

Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two exogenous antimicrobial peptides, magainin 2 and melittin. This small genetic modification, capable of efficient nonautonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium berghei. It delays the release of infectious sporozoites, while it simultaneously reduces the life span of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of transmission intensities.

Manipulation of pantothenate kinase in Anopheles stephensi suppresses pantothenate levels with minimal impacts on mosquito fitness

Pantothenate (Pan) is an essential nutrient required by both the mosquito vector and malaria parasite. We previously demonstrated that increasing pantothenate kinase (PanK) activity and co-enzyme A (CoA) biosynthesis led to significantly decreased parasite infection prevalence and intensity in the malaria mosquito Anopheles stephensi. In this study, we demonstrate that Pan stores in A. stephensi are a limited resource and that manipulation of PanK levels or activity, via small molecule modulators of PanK or transgenic mosquitoes, leads to the conversion of Pan to CoA and an overall reduction in Pan levels with minimal to no effects on mosquito fitness. Transgenic A. stephensi lines with repressed insulin signaling due to PTEN overexpression or repressed c-Jun N-terminal kinase (JNK) signaling due to MAPK phosphatase 4 (MKP4) overexpression exhibited enhanced PanK levels and significant reductions in Pan relative to non-transgenic controls, with the PTEN line also exhibiting significantly increased CoA levels. Provisioning of the PTEN line with the small molecule PanK modulator PZ-2891 increased CoA levels while provisioning Compound 7 decreased CoA levels, affirming chemical manipulation of mosquito PanK. We assessed effects of these small molecules on A. stephensi lifespan, reproduction and metabolism under optimized laboratory conditions. PZ-2891 and Compound 7 had no impact on A. stephensi survival when delivered via bloodmeal throughout mosquito lifespan. Further, PZ-2891 provisioning had no impact on egg production over the first two reproductive cycles. Finally, PanK manipulation with small molecules was associated with minimal impacts on nutritional stores in A. stephensi mosquitoes under optimized rearing conditions. Together with our previous data demonstrating that PanK activation was associated with significantly increased A. stephensi resistance to Plasmodium falciparum infection, the studies herein demonstrate a lack of fitness costs of mosquito Pan depletion as a basis for a feasible, novel strategy to control parasite infection of anopheline mosquitoes.

Intron-derived small RNAs for silencing viral RNAs in mosquito cells.

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster pre-miRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3’ UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes.

Transgenic Anopheles mosquitoes expressing human PAI-1 impair malaria transmission

In mammals, the serine protease plasmin degrades extracellular proteins during blood clot removal, tissue remodeling, and cell migration. The zymogen plasminogen is activated into plasmin by two serine proteases: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), a process regulated by plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor that specifically inhibits tPA and uPA. Plasmodium gametes and sporozoites use tPA and uPA to activate plasminogen and parasite-bound plasmin degrades extracellular matrices, facilitating parasite motility in the mosquito and the mammalian host. Furthermore, inhibition of plasminogen activation by PAI-1 strongly blocks infection in both hosts. To block parasite utilization of plasmin, we engineered Anopheles stephensi transgenic mosquitoes constitutively secreting human PAI-1 (huPAI-1) in the midgut lumen, in the saliva, or both. Mosquitoes expressing huPAI-1 strongly reduced rodent and human Plasmodium parasite transmission to mosquitoes, showing that co-opting plasmin for mosquito infection is a conserved mechanism among Plasmodium species. huPAI-1 expression in saliva induced salivary gland deformation which affects sporozoite invasion and P. berghei transmission to mice, resulting in significant levels of protection from malaria. Targeting the interaction of malaria parasites with the fibrinolytic system using genetically engineered mosquitoes could be developed as an intervention to control malaria transmission.

Generation of Transgenic Mosquitoes Harboring a Replication-Restricted Virus

Live microbe vaccines are designed to elicit strong cellular and antibody responses without developing the symptoms of the disease, and these are effective in preventing infectious diseases. A flying vaccinator (also known as a flying syringe) is a conceptual, genetically engineered hematophagous insect that is used to deliver vaccines such as an antigen from a parasite produced in mosquito saliva; bites from such insects may elicit antibody production by immunizing the host with an antigen through blood-feeding. In addition to a simple vaccine antigen, a flying vaccinator may potentially load a live attenuated microbe with an appropriate mechanism for sustaining its constitutive proliferation in the insect. In this study, a recombinant vesicular stomatitis virus (VSV) lacking the glycoprotein gene (VSV-G) was used to produce replication-restricted VSV (rrVSV) containing GFP. Transgenic Anopheles stephensi mosquitoes, in which the salivary glands expressed a VSV-G gene driven by an aapp salivary gland-specific promoter, were generated and injected intraperitoneally with rrVSV. The injected rrVSV entered the cells of the salivary gland and stimulated endogenous production of progeny rrVSV particles, as seen in rrVSV-infected Drosophila melanogaster expressing VSV-G. These data suggested the possibility of developing a valuable tool for delivering genetically attenuated virus vaccines via mosquito saliva, although efficient replication-restricted virus production is required.

Engineered anti-viral shRNAs are more effective than lhRNAs in transgenic Aedes aegypti

The Aedes aegypti mosquito is a major vector of chikungunya virus (CHIKV), which has no licensed vaccine. Engineered mosquitoes expressing long RNA hairpins (lhRNA) or small RNAs against selected arboviruses have been developed to limit virus replication, but their silencing efficiency has not been compared. We developed lhRNA and short hairpin RNA (shRNA) arrays against CHIKV non-structural protein nsP2. We used a Tet-response element (TRE) and a tTA transactivator to control expression of lhRNAs (TRE-lhRNA) and shRNAs (TRE-shRNA). Constitutive expression in Aag2 cells was assessed with a PUb-tTA driverand midgut specific expression in transgenic mosquitoes was assessed using Carboxypeptidase A (AeCPA)-tTA as driver. In vitrointerference ability was determined with a CHIKV split replication system, and a synthetic luciferase reporter with mRNA containing the targeted CHIKV sequence (CHIK-FF). In vivo interference was tested by inserting the TRE-lhRNA and TRE-shRNA constructs into a AeCPA-tTA line so both constructs expressed from the same locus, and a TRE reporter line that expressed an AmCyan reporter with an N terminal CHIKV fusion (CHIK-AmC). In Aag2 cells, shRNAs were more effective than lhRNA in silencing both the CHIKV split replication system (99.7%, S. D. ± 0.47 and 72.8%, S. D. ± 9.50, respectively, P<0.001) and CHIK-FF (98.8%, S. D. ± 0.71 and 50.9%, S. D. ± 7.92, respectively, P<0.05). Similar results were observed in transgenic mosquitoes when comparing AmC expression in the midgut. This study demonstrates that, in mosquitoes, effectively chosen shRNAs can induce greater interference of the desired viral target than the corresponding lhRNA.

Preparing an Insectary in Burkina Faso to Support Research in Genetic Technologies for Malaria Control.

The Institut de Recherche en Sciences de la Santé (IRSS) of Burkina Faso, West Africa, was the first African institution to import transgenic mosquitoes for research purposes. A shift from the culture of mosquito research to regulated biotechnology research and considerable management capacity is needed to set up and run the first insectary for transgenic insects in a country that applied and adapted the existing biosafety framework, first developed for genetically modified (GM) crops, to this new area of research. The additional demands arise from the separate regulatory framework for biotechnology, referencing the Cartagena Protocol on Biosafety, and the novelty of the research strain, making public understanding and acceptance early in the research pathway important. The IRSS team carried out extensive preparations following recommendations for containment of GM arthropods and invested efforts in local community engagement and training with scientific colleagues throughout the region. Record keeping beyond routine practice was established to maintain evidence related to regulatory requirements and risk assumptions. The National Biosafety Agency of Burkina Faso, Agence Nationale de Biosécurité (ANB), granted the permits for import of the self-limiting transgenic mosquito strain, which took place in November 2016, and for conducting studies in the IRSS facility in Bobo-Dioulasso. Compliance with permit terms and conditions of the permits and study protocols continued until the conclusion of studies, when the transgenic colonies were terminated. All this required close coordination between management and the insectary teams, as well as others. This article outlines the experiences of the IRSS to support others undertaking such studies. The IRSS is contributing to the ongoing development of genetic technologies for malaria control, as a partner of Target Malaria. The ultimate objective of the innovation is to reduce malaria transmission by using GM mosquitoes of the same species released to reduce the disease-vectoring native populations of Anopheles gambiae s.l.

From the Lab to the Last Mile: Deploying Transgenic Approaches Against Mosquitoes

Ingenious exploitation of transgenic approaches to produce malaria resistant or sterile mosquitoes, or hypervirulent mosquito pathogens, has produced many potential solutions to vector borne diseases. However, in spite of technological feasibility, it has not been determined how well these new methods will work, and how they should be tested and regulated. Some self-limiting transgenic fungal pathogens and mosquitoes are almost field ready, and may be easier to regulate than self-sustaining strategies. However, they require repeat sales and so must show business viability; low-cost mass production is just one of a number of technical constraints that are sometimes treated as an afterthought in technology deployment. No transgenic self-sustaining approach to anopheline control has ever been deployed because of unresolved ethical, social and regulatory issues. These overlapping issues include: 1) the transparency challenge, which requires public discourse, particularly in Africa where releases are proposed, to determine what society is willing to risk given the potential benefits; 2) the transboundary challenge, self-sustaining mosquitoes or pathogens are potentially capable of crossing national boundaries and irreversibly altering ecosystems, and 3) the risk assessment challenge. The polarized debate as to whether these technologies will ever be used to save lives is ongoing; they will founder without a political answer as to how do we interpret the precautionary principle, as exemplified in the Cartagena protocol, in the global context of technological changes.

Transgenic refractory Aedes aegypti lines are resistant to multiple serotypes of dengue virus

The areas where dengue virus (DENV) is endemic have expanded rapidly, driven in part by the global spread of Aedes species, which act as disease vectors. DENV replicates in the mosquito midgut and is disseminated to the mosquito’s salivary glands for amplification. Thus, blocking virus infection or replication in the tissues of the mosquito may be a viable strategy for reducing the incidence of DENV transmission to humans. Here we used the mariner Mos1 transposase to create an Aedes aegypti line that expresses virus-specific miRNA hairpins capable of blocking DENV replication. These microRNA are driven by the blood-meal-inducible carboxypeptidase A promoter or by the polyubiquitin promoter. The transgenic mosquitoes exhibited significantly lower infection rates and viral titers for most DENV serotypes 7 days after receiving an infectious blood meal. The treatment was also effective at day 14 post infection after a second blood meal had been administered. In viral transmission assay, we found there was significantly reduced transmission in these lines. These transgenic mosquitoes were effective in silencing most of the DENV genome; such an approach may be employed to control a dengue fever epidemic.

Becoming Without

AbstractThe Aedes aegypti mosquito, known as the vector for Zika, dengue, chikungunya, and yellow fever viruses, has historically been targeted by public health campaigns as an enemy to be eliminated. However, new strategies, such as the transgenic approach, biologically modify the A. aegypti so that they can be deployed to control their own population—here, mosquito breeding and mating is operationalized as an insecticide. In this case, the insect must be simultaneously a friend and an enemy, cared for and killed, and it must establish encounters and nonencounters. Drawing on ethnographic fieldwork at a “biofactory” in the northeast of Brazil dedicated to mass-producing these transgenic mosquitoes, this article investigates the new forms of labor and value produced through these contrasting human-mosquito relations. The author also examines how the project is implemented within broader geopolitics of experimentation and more-than-human gendered conceptions. Analyzing the multispecies relationships engendered under the premise that it is possible to produce nonencounters, she identifies the historical conditions and promissory claims of transforming the A. aegypti ’s reproductive capacity into labor for killing. Such recasting yields what the author calls the “nonencounter value” within the scientific remaking of mosquitoes, their becoming and being.

Increased insulin signaling in the Anopheles stephensi fat body regulates metabolism and enhances the host response to both bacterial challenge and Plasmodium falciparum infection

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.