RNAi-Mediated Knockdown of Acidic Ribosomal Stalk Protein P1 Arrests Egg Development in Adult Female Yellow Fever Mosquitoes, Aedes aegypti.

After taking a blood meal, the fat body of the adult female yellow fever mosquito, Aedes aegypti, switches from a previtellogenic state of arrest to an active state of synthesizing large quantities of yolk protein precursors (YPPs) that are crucial for egg development. The synthesis of YPPs is regulated at both the transcriptional and translational levels. Previously, we identified the cytoplasmic protein general control nonderepressible 1 (GCN1) as a part of the translational regulatory pathway for YPP synthesis. In the current study, we used the C-terminal end of GCN1 to screen for protein-protein interactions and identified 60S acidic ribosomal protein P1 (P1). An expression analysis and RNAi-mediated knockdown of P1 was performed to further investigate the role of P1 in mosquito reproduction. We showed that in unfed (absence of a blood meal) adult A. aegypti mosquitoes, P1 was expressed ubiquitously in the mosquito organs and tissues tested. We also showed that the RNAi-mediated knockdown of P1 in unfed adult female mosquitoes resulted in a strong, transient knockdown with observable phenotypic changes in ovary length and egg deposition. Our results suggest that 60S acidic ribosomal protein P1 is necessary for mosquito reproduction and is a promising target for mosquito population control.

Transcriptomics analysis of ethanol treatment of male Aedes aegypti reveals a small set of putative radioprotective genes.

Introduction: Sterile Insect Technique (SIT) is based on releasing sterilized male insects into wild insect populations to compete for mating with wild females. Wild females mated with sterile males will produce inviable eggs, leading to a decline in population of that insect species. Sterilization with ionizing radiation (x-rays) is a commonly used mechanism for sterilization of males. Since irradiation can cause damage to both, somatic and germ cells, and can severely reduce the competitiveness of sterilized males relative to wild males, means to minimize the detrimental effects of radiation are required to produce sterile, competitive males for release. In an earlier study, we identified ethanol as a functional radioprotector in mosquitoes. Methods: Here, we used Illumina RNA-seq to profile changes in gene expression of male Aedes aegypti mosquitoes fed on 5% ethanol for 48 hours prior to receiving a sterilizing x-ray dose, compared to males fed on water prior to sterilization. Results: RNA-seq revealed a robust activation of DNA repair genes in both ethanol-fed and water-fed males after irradiation, but surprisingly few differences in gene expression between ethanol-fed and water-fed males regardless of radiation treatment. Discussion: While differences in gene expression due to ethanol exposure were minimal, we identified a small group of genes that may prime ethanol-fed mosquitoes for improved survivability in response to sterilizing radiation.

Repellent efficacy of 20 essential oils on Aedes aegypti mosquitoes and Ixodes scapularis ticks in contact-repellency assays.

Cases of mosquito- and tick-borne diseases are rising worldwide. Repellent products can protect individual users from being infected by such diseases. In a previous study, we identified five essential oils that display long-distance mosquito repellency using a Y-tube olfactometer assay. In the current study, the contact repellent efficacy of 20 active ingredients from the Environmental Protection Agency's (EPA) Minimum Risk Pesticides list were tested using Aedes aegypti and Ixodes scapularis. We utilized an arm-in-cage assay to measure complete protection time from mosquito bites for these active ingredients. To measure tick repellency, we used an EPA-recommended procedure to measure the complete protection time from tick crossings. We found that of the 20 ingredients tested, 10% v/v lotion emulsions with clove oil or cinnamon oil provided the longest protection from both mosquito bites and tick crossings. We conclude that in a 10% v/v emulsion, specific active ingredients from the EPA Minimum Risk Pesticides list can provide complete protection from mosquito bites and tick crossings for longer than one hour.

Knockdown of the Sodium/Potassium ATPase Subunit Beta 2 Reduces Egg Production in the Dengue Vector, Aedes aegypti.

The Na+/K+ ATPase (NKA) is present in the cellular membrane of most eukaryotic cells. It utilizes energy released by ATP hydrolysis to pump sodium ions out of the cell and potassium ions into the cell, which establishes and controls ion gradients. Functional NKA pumps consist of three subunits, alpha, beta, and FXYD. The alpha subunit serves as the catalytic subunit while the beta and FXYD subunits regulate the proper folding and localization, and ion affinity of the alpha subunit, respectively. Here we demonstrate that knockdown of NKA beta subunit 2 mRNA (nkaß2) reduces fecundity in female Ae. aegypti. We determined the expression pattern of nkaß2 in several adult mosquito organs using qRT-PCR. We performed RNAi-mediated knockdown of nkaß2 and assayed for lethality, and effects on female fecundity. Tissue expression levels of nkaß2 mRNA were highest in the ovaries with the fat body, midgut and thorax having similar expression levels, while Malpighian tubules had significantly lower expression. Survival curves recorded post dsRNA injection showed a non-significant decrease in survival of nkaß2 dsRNA-injected mosquitoes compared to GFP dsRNA-injected mosquitoes. We observed a significant reduction in the number of eggs laid by nkaß2 dsRNA-injected mosquitoes compared to control mosquitoes. These results, coupled with the tissue expression profile of nkaß2, indicate that this subunit plays a role in normal female Ae. aegypti fecundity. Additional research needs to be conducted to determine the exact role played by NKAß2 in mosquito post-blood meal nutrient sensing, transport, yolk precursor protein (YPP) synthesis and yolk deposition.

General control nonderepressible 1 interacts with cationic amino acid transporter 1 and affects Aedes aegypti fecundity.

BACKGROUND: The amino acid transporter protein cationic amino acid transporter 1 (CAT1) is part of the nutrient sensor in the fat body of mosquitoes. A member of the SLC7 family of cationic amino acid transporters, it is paramount for the detection of elevated amino acid levels in the mosquito hemolymph after a blood meal and the subsequent changes in gene expression in the fat body. METHODS: We performed a re-annotation of Aedes aegypti cationic amino acid transporters (CATs) and selected the C-terminal tail of CAT1 to perform a yeast two-hybrid screen to identify putative interactors of this protein. One interesting interacting protein we identified was general control nonderepressible 1 (GCN1). We determined the expression pattern of GCN1 in several adult organs and structures using qRT-PCR and western blots. Finally, we knocked down GCN1 using double-stranded RNA and identified changes in downstream signaling intermediates and the effects of knockdown on vitellogenesis and fecundity. RESULTS: In a screen for Ae. aegypti CAT1-interacting proteins we identified GCN1 as a putative interactor. GCN1 is highly expressed in the ovaries and fat body of the mosquito. We provide evidence that eukaryotic translation initiation factor 2 subunit alpha (eIF2α) phosphorylation changed during vitellogenesis and that RNA interference knockdown of GCN1 in whole mosquitoes reduced egg clutch sizes of treated mosquitoes relative to controls. CONCLUSIONS: Aedes aegypti CAT1 and GCN1 are likely interacting partners and GCN1 is likely necessary for proper egg development. Our data suggest that GCN1 is part of a nutrient sensor mechanism in various mosquito tissues involved in vitellogenesis.

Exploratory phosphoproteomics profiling of Aedes aegypti Malpighian tubules during blood meal processing reveals dramatic transition in function.

Malpighian tubules, the renal organs of mosquitoes, facilitate the rapid dehydration of blood meals through aquaporin-mediated osmosis. We performed phosphoproteomics analysis of three Malpighian tubule protein-libraries (1000 tubules/sample) from unfed female mosquitoes as well as one and 24 hours after a blood meal. We identified 4663 putative phosphorylation sites in 1955 different proteins. Our exploratory dataset reveals blood meal-induced changes in phosphorylation patterns in many subunits of V-ATPase, proteins of the target of rapamycin signaling pathway, vesicle-mediated protein transport proteins, proteins involved in monocarboxylate transport, and aquaporins. Our phosphoproteomics data suggest the involvement of a variety of new pathways including nutrient-signaling, membrane protein shuttling, and paracellular water flow in the regulation of urine excretion. Our results support a model in which aquaporin channels translocate from intracellular vesicles to the cell membrane of stellate cells and the brush border membrane of principal cells upon blood feeding.

Olfaction-Related Gene Expression in the Antennae of Female Mosquitoes From Common Aedes aegypti Laboratory Strains.

Adult female mosquitoes rely on olfactory cues like carbon dioxide and other small molecules to find vertebrate hosts to acquire blood. The molecular physiology of the mosquito olfactory system is critical for their host preferences. Many laboratory strains of the yellow fever mosquito Aedes aegypti have been established since the late 19th century. These strains have been used for most molecular studies in this species. Some earlier comparative studies have identified significant physiological differences between different laboratory strains. In this study, we used a Y-tube olfactometer to determine the attraction of females of seven different strains of Ae. aegypti to a human host: UGAL, Rockefeller, Liverpool, Costa Rica, Puerto Rico, and two odorant receptor co-receptor (Orco) mutants Orco2 and Orco16. We performed RNA-seq using antennae of Rockefeller, Liverpool, Costa Rica, and Puerto Rico females. Our results showed that female Aedes aegypti from the Puerto Rico strain had significantly reduced attraction rates toward human hosts compared to all other strains. RNA-seq analyses of the antenna transcriptomes of Rockefeller, Liverpool, Costa Rica, and Puerto Rico strains revealed distinct differences in gene expression between the four strains, but conservation in gene expression patterns of known human-sensing genes. However, we identified several olfaction-related genes that significantly vary between strains, including receptors with significantly different expression in mosquitoes from the Puerto Rico strain and the other strains.

A novel Tick Carousel Assay for testing efficacy of repellents on Amblyomma americanum L.

Ticks are important vectors of human and veterinary diseases. A primary way ticks gain access to human hosts is by engaging to clothing. Repellents or acaricides sprayed onto fabric are used to deter ticks' access to human hosts. However, there are a limited amount of standardized laboratory assays that can determine the potency and efficacy of repellents. We present a novel fabric-engagement assay referred to as the 'Tick Carousel Assay'. This assay utilizes fabric brushing past ticks located on an artificial grass patch and measures tick engagements to fabric over time. After screening a variety of tick species, we used the lone star tick (Amblyomma americanum) to test the efficacy of four commonly used active ingredients in repellents: DEET, Picaridin, IR3535, and Oil of Lemon Eucalyptus. Repellency was tested immediately, after three hours, and six hours post application to fabric. Our data show that each repellent we tested significantly reduced the number of tick engagements to fabric for at least 6 hours. We did not find significant differences in repellent efficacy between the four active ingredients tested directly and three hours after application. After six hours, Oil of Lemon Eucalyptus repelled ticks more than the other active ingredients. We show that our Tick Carousel Assay provides an affordable, repeatable, and standardized way to compare and test repellent efficacy on treated fabrics. Our results confirm that commonly used repellents applied to fabric are an effective way to reduce tick engagement.

Fat and Happy: Profiling Mosquito Fat Body Lipid Storage and Composition Post-blood Meal.

The fat body is considered the insect analog of vertebrate liver and fat tissue. In mosquitoes, a blood meal triggers a series of processes in the fat body that culminate in vitellogenesis, the process of yolk formation. Lipids are stored in the fat body in specialized organelles called lipid droplets that change in size depending on the nutritional and metabolic status of the insect. We surveyed lipid droplets in female Aedes aegypti fat body during a reproductive cycle using confocal microscopy and analyzed the dynamic changes in the fat body lipidome during this process using LC/MS. We found that lipid droplets underwent dynamic changes in volume after the mosquito took a blood meal. The lipid composition found in the fat body is quite complex with 117 distinct lipids that fall into 19 classes and sublcasses. Our results demonstrate that the lipid composition of the fat body is complex as most lipid classes underwent significant changes over the course of the vitellogenic cycle. This study lays the foundation for identifying unknown biochemical pathways active in the mosquito fat body, that are high-value targets for the development of novel mosquito control strategies.

Long-Term Mosquito culture with SkitoSnack, an artificial blood meal replacement.

The reliance on blood is a limiting factor for mass rearing of mosquitoes for Sterile-Insect-Technique (SIT) and other mosquito-based control strategies. To solve this problem, we have developed SkitoSnack, a formulated diet for Aedes aegypti (L) mosquitoes, as an alternative for vertebrate blood. Here we addressed the question if long-term yellow fever mosquito culture with SkitoSnack resulted in changed life history traits and fitness of the offspring compared to blood-raised mosquitoes. We also explored if SkitoSnack is suitable to raise Asian tiger mosquitos, Aedes albopictus (L.), and the human bed bug, Cimex lectularius (L). We measured life history traits for 30th generation SkitoSnack-raised Ae. aegypti and 11th generation SkitoSnack-raised Ae. albopictus, and compared them with control mosquitoes raised on blood only. We compared meal preference, flight performance, and reproductive fitness in Ae. aegypti raised on SkitoSnack or blood. We also offered SkitoSnack to bed bug nymphs. We found that long-term culture with SkitoSnack resulted in mosquitoes with similar life history traits compared to bovine blood-raised mosquitoes in both species we studied. Also, Ae. aegypti mosquitoes raised on SkitoSnack had similar flight performance compared to blood raised mosquitoes, were still strongly attracted by human smell and had equal mating success. Minimal feeding occurred in bed bugs. Our results suggest that long-term culture with the blood-meal replacement SkitoSnack results in healthy, fit mosquitoes. Therefore, artificial diets like SkitoSnack can be considered as a viable alternative for vertebrate blood in laboratory mosquito culture as well as for mosquito mass production for Sterile-Insect-Technique mosquito control interventions. SkitoSnack was not suitable to induce engorgement of bed bugs.

Low Levels of Pyrethroid Resistance in Hybrid Offspring of a Highly Resistant and a More Susceptible Mosquito Strain.

The use of insecticides has been a central approach to control disease-transmitting mosquitoes for the last century. The high prevalence of pyrethroid use as public health insecticides has resulted in the evolution of pyrethroid resistance in many populations of Aedes aegypti (Linnaeus) (Diptera: Culicidae), throughout its global distribution range. Insecticide resistance is often correlated with an associated fitness cost. In this project, we studied the phenotypes of hybrid mosquitoes derived from crossing a pyrethroid-resistant strain of Ae. aegypti (Puerto Rico [PR]) with a more susceptible one (Rockefeller [ROCK]). We first sequenced and compared the para gene of both original strains. We then crossed males from one strain with females of the other, creating two hybrids (Puertofeller, Rockorico). We used a Y-tube choice assay to measure the attraction of these strains towards a human host. We then compared the levels of pyrethroid resistance in the different strains. We found three known resistance mutations in the para gene sequence of the PR strain. In our attraction assays, PR females showed lower attraction to humans, than the ROCK females. Both hybrid strains showed strong attraction to a human host. In the insecticide resistance bottle assays, both hybrid strains showed marginal increases in resistance to permethrin compared to the more susceptible ROCK strain. These results suggest that hybrids of sensitive and permethrin-resistant mosquitoes have an incremental advantage compared to more susceptible mosquitoes when challenged with permethrin. This explains the rapid spread of permethrin resistance that was observed many times in the field.

Efficacy of Active Ingredients From the EPA 25(B) List in Reducing Attraction of Aedes aegypti (Diptera: Culicidae) to Humans.

Mosquitoes of the Aedes genus are vectors for dengue, chikungunya, Zika, and yellow fever viruses. Mosquito repellents are an effective way to prevent mosquito bites and reduce the spread of mosquito-borne diseases. In the early 90s, the U.S. Environmental Protection Agency (EPA) published a list of active ingredients that pose minimum risk to human health that can be used as pesticides or repellents without passing the EPA registration process. The present study examined the efficacy of 21 of the active ingredients listed by the EPA 25 (B) exempt list and five commercially available sprays that only contained active ingredients from the EPA 25(B) list in repelling female Aedes aegypti (L.) females. We performed choice bioassays in a controlled laboratory environment, using a Y-tube olfactometer to determine attraction rates of humans to female Ae. aegypti in the presence of one of the 21 active ingredients and five commercially available repellent sprays. We found that cinnamon oil, peppermint oil, spearmint oil, lemongrass oil, and garlic oil reduced mosquito attraction to human odor. Of the five commercial repellent sprays, only one reduced mosquito attraction for up to 30 min in our assay. The EPA 25 (B) list contains active ingredients that under the conditions of our assay repel Ae. aegypti.

Wolbachia pipientis occurs in Aedes aegypti populations in New Mexico and Florida, USA.

The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever, and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but rarely detected in Ae. aegypti. In this study, we used a two-step PCR assay to detect Wolbachia in wild-collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A loop-mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild Ae. aegypti specimens from various cities in New Mexico, and in 2/46 (4.3%) from St. Augustine, Florida. Wolbachia was not detected in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia-positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico, in 2018. The infected females of this strain transmitted Wolbachia to their progeny when crossed with males of Rockefeller strain of Ae. aegypti, which does not carry Wolbachia. In contrast, none of the progeny of Las Cruces males mated to Rockefeller females were infected with Wolbachia.

Widespread insecticide resistance in Aedes aegypti L. from New Mexico, U.S.A.

BACKGROUND: Aedes aegypti mosquitoes are vectors of a variety of emerging viral pathogens, including yellow fever, dengue, chikungunya, and Zika virus. This species has established endemic populations in all cities across southern New Mexico sampled to date. Presently, control of Aedes-borne viruses relies on deployment of insecticides to suppress mosquito populations, but the evolution of insecticide resistance threatens the success of vector control programs. While insecticide resistance is quite common in Ae. aegypti field populations across much of the U.S., the resistance status of this species in populations from New Mexico has not previously been assessed. RESULTS: First, we collected information on pesticide use in cities in southern New Mexico and found that the most commonly used active ingredients were pyrethroids. The use of insecticides with the same mode-of-action over multiple years is likely to promote the evolution of resistance. To determine if there was evidence of resistance in some cities in southern New Mexico, we collected Ae. aegypti from the same cities and established laboratory strains to assess resistance to pyrethroid insecticides and, for a subset of populations, to organophosphate insecticides. F2 or F4 generation mosquitoes were assessed for insecticide resistance using bottle test bioassays. The majority of the populations from New Mexico that we analyzed were resistant to the pyrethroids permethrin and deltamethrin. A notable exception to this trend were mosquitoes from Alamogordo, a city that did not report using pyrethroid insecticides for vector control. We screened individuals from each population for known knock down resistance (kdr) mutations via PCR and found a strong association between the presences of the F1534C kdr mutation in the para gene of Ae. aegypti (homologue to F1534C in Musca domestica L.) and pyrethroid resistance. CONCLUSION: High-level pyrethroid resistance is common in Ae. aegypti from New Mexico and geographic variation in such resistance is likely associated with variation in usage of pyrethroids for vector control. Resistance monitoring and management is recommended in light of the potential for arbovirus outbreaks in this state. Also, alternative approaches to mosquito control that do not involve insecticides should be explored.

Toward Implementation of Mosquito Sterile Insect Technique: The Effect of Storage Conditions on Survival of Male Aedes aegypti Mosquitoes (Diptera: Culicidae) During Transport.

Sterile insect technique (SIT) is a promising, environmentally friendly alternative to the use of pesticides for insect pest control. However, implementing SIT with Aedes aegypti (Linnaeus) mosquitoes presents unique challenges. For example, during transport from the rearing facility to the release site and during the actual release in the field, damage to male mosquitoes should be minimized to preserve their reproductive competitiveness. The short flight range of male Ae. aegypti requires elaborate release strategies such as release via Unmanned Aircraft Systems, more commonly referred to as drones. Two key parameters during transport and release are storage temperature and compaction rate. We performed a set of laboratory experiments to identify the optimal temperatures and compaction rates for storage and transport of male Ae. aegypti. We then conducted shipping experiments to test our laboratory-derived results in a 'real-life' setting. The laboratory results indicate that male Ae. aegypti can survive at a broad range of storage temperatures ranging from 7 to 28°C, but storage time should not exceed 24 h. Male survival was high at all compaction rates we tested with a low at 40 males/cm3. Interestingly, results from our 'real-life' shipping experiment showed that high compaction rates were beneficial to survival. This study advances key understudied aspects of the practicalities of moving lab-reared insects into the field and lies the foundation for further studies on the effect of transport conditions on male reproductive fitness.

Colonized Sabethes cyaneus, a Sylvatic New World Mosquito Species, Shows a Low Vector Competence for Zika Virus Relative to Aedes aegypti.

The introduction of Zika virus (ZIKV) to the Americas raised concern that the virus would spill back from human transmission, perpetuated by Aedes aegypti, into a sylvatic cycle maintained in wildlife and forest-living mosquitoes. In the Americas, Sabethes species are vectors of sylvatic yellow fever virus (YFV) and are therefore candidate vectors of a sylvatic ZIKV cycle. To test the potential of Sabethes cyaneus to transmit ZIKV, Sa. cyaneus and Ae. aegypti were fed on A129 mice one or two days post-infection (dpi) with a ZIKV isolate from Mexico. Sa. cyaneus were sampled at 3, 4, 5, 7, 14, and 21 days post-feeding (dpf) and Ae. aegypti were sampled at 14 and 21 dpf. ZIKV was quantified in mosquito bodies, legs, and saliva to measure infection, dissemination, and potential transmission, respectively. Of 69 Sa. cyaneus that fed, ZIKV was detected in only one, in all body compartments, at 21 dpf. In contrast, at 14 dpf 100% of 20 Ae. aegypti that fed on mice at 2 dpi were infected and 70% had virus in saliva. These data demonstrate that Sa. cyaneus is a competent vector for ZIKV, albeit much less competent than Ae. aegypti.

The Effect of SkitoSnack, an Artificial Blood Meal Replacement, on Aedes aegypti Life History Traits and Gut Microbiota.

Public health research and vector control frequently require the rearing of large numbers of vector mosquitoes. All target vector mosquito species are anautogenous, meaning that females require vertebrate blood for egg production. Vertebrate blood, however, is costly, with a short shelf life. To overcome these constraints, we have developed SkitoSnack, an artificial blood meal replacement for the mosquito Aedes aegypti, the vector of dengue, Zika and chikungunya virus. SkitoSnack contains bovine serum albumin and hemoglobin as protein source as well as egg yolk and a bicarbonate buffer. SkitoSnack-raised females had comparable life history traits as blood-raised females. Mosquitoes reared from SkitoSnack-fed females had similar levels of infection and dissemination when orally challenged with dengue virus type 2 (DENV-2) and significantly lower infection with DENV-4. When SkitoSnack was used as a vehicle for DENV-2 delivery, blood-raised and SkitoSnack-raised females were equally susceptible. The midgut microbiota differed significantly between mosquitoes fed on SkitoSnack and mosquitoes fed on blood. By rearing 20 generations of Aedes exclusively on SkitoSnack, we have proven that this artificial diet can replace blood in mosquito mass rearing.

Correction: Dengue virus serotype 2 infection alters midgut and carcass gene expression in the Asian tiger mosquito, Aedes albopictus.

[This corrects the article DOI: 10.1371/journal.pone.0171345.].

Corrigendum: RNA-Seq Comparison of Larval and Adult Malpighian Tubules of the Yellow Fever Mosquito Aedes aegypti Reveals Life Stage-Specific Changes in Renal Function.

[This corrects the article on p. 283 in vol. 8, PMID: 28536536.].

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus.

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall. The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics. In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.