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.

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.

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.

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. Graphical Abstract

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 30 th generation SkitoSnack-raised Ae. aegypti and 11 th 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.