Established populations of Aedes aegypti, a mosquito vector of multiple major arthropod-borne viruses, were first found in three California (CA) cities in 2013. From 2013 to April 2021, Ae. aegypti thwarted almost all control efforts to stop its spread and expanded its range to 308 cities, including Exeter, in 22 counties in CA. Population genomic analyses have suggested that multiple genetically distinct Ae. aegypti populations were introduced into CA. However Ae. aegypti collected for the first time in 2014 in Exeter, appeared to be different from three major genetic clusters found elsewhere in CA. Due to intense control efforts by the Delta Vector Control District (DVCD), Ae. aegypti was thought to have been eliminated from Exeter in 2015. Unfortunately, it was recollected in 2018. It was not clear if the reemergence of Ae. aegypti in Exeter was derived from the bottlenecked remnants of the original 2014 Exeter population or from an independent invasion from a different population derived from surrounding areas. The goal of this work was to determine which of these scenarios occurred (recovery after bottleneck or reintroduction after elimination) and if elimination and reintroduction occurred to identify the origin of the invading population using a population genomic approach. Our results support the reintroduction after elimination hypothesis. The source of reintroduction, however, was unexpectedly from the southern CA cluster rather than from other two geographically closer central CA genetic clusters. We also conducted a knockdown resistance mutation profile, which showed Exeter 2014 had the lowest level of resistant alleles compared to the other populations, could have contributed towards DVCD’s ability to locally eliminate Ae. aegypti in 2014.

Background Since their detection in 2013, Aedes aegypti has become a widespread urban pest in California. The availability of cryptic larval breeding sites in residential areas and resistance to insecticides pose significant challenges to control efforts. Resistance to pyrethroids is largely attributed to mutations in the voltage gated sodium channels (VGSC), the pyrethroid site of action. However, past studies have indicated that VGSC mutations may not be entirely predictive of the observed resistance phenotype. Methods To investigate the frequencies of VGSC mutations and the relationship with pyrethroid insecticide resistance in California, we sampled Ae. aegypti from four locations in the Central Valley, and the Greater Los Angeles area. Mosquitoes from each location were subjected to an individual pyrethrum bottle bioassay to determine knockdown times. A subset of assayed mosquitoes from each location was then analyzed to determine the composition of 5 single nucleotide polymorphism (SNP) loci within the VGSC gene. Results The distribution of knockdown times for each of the five Californian populations sampled was non-parametric with potentially bimodal distributions. One group succumbs to insecticidal effects around 35–45 min and the second group lasts up to and beyond the termination of the assay (120+ min). We detected 5 polymorphic VGSC SNPs within the sampled California populations. One is potentially new and alternatively spliced (I915K), and four are documented and associated with resistance: F1534C, V1016I, V410L and S723T. The Central Valley populations (Clovis, Dinuba, Sanger and Kingsburg) are fairly homogenous with only 5% of the mosquitoes showing heterozygosity at any given position. In the Greater LA mosquitoes, 55% had at least one susceptible allele at any of the five SNP loci. The known resistance allele F1534C was detected in almost all sampled mosquitoes (99.4%). We also observe significant heterogeneity in the knockdown phenotypes of individuals with the identical VGSC haplotypes suggesting the presence of additional undefined resistance mechanisms. Conclusions Resistance associated VGSC SNPs are prevalent, particularly in the Central Valley. Interestingly, among mosquitoes carrying all 4 resistance associated SNPs, we observe significant heterogeneity in bottle bioassay profiles suggesting that other mechanisms are important to the individual resistance of Ae. aegypti in California. Keywords: Aedes aegypti, Resistance, Pyrethroid, IPLEX genotyping, Voltage gated sodium channel, California.

Tsetse flies (genus Glossina), the sole vectors of African trypanosomiasis, are distinct from most other insects, due to dramatic morphological and physiological adaptations required to support their unique biology. These adaptations are driven by demands associated with obligate hematophagy and viviparous reproduction. Obligate viviparity entails intrauterine larval development and the provision of maternal nutrients for the developing larvae. The reduced reproductive capacity/rate associated with this biology results in increased inter- and intra-sexual competition. Here, we use phase contrast microcomputed tomography (pcMicroCT) to analyze morphological adaptations associated with viviparous biology. These include (1) modifications facilitating abdominal distention required during blood feeding and pregnancy, (2) abdominal and uterine musculature adaptations for gestation and parturition of developed larvae, (3) reduced ovarian structure and capacity, (4) structural features of the male-derived spermatophore optimizing semen/sperm delivery and inhibition of insemination by competing males and (5) structural features of the milk gland facilitating nutrient incorporation and transfer into the uterus. Three-dimensional analysis of these features provides unprecedented opportunities for examination and discovery of internal morphological features not possible with traditional microscopy techniques and provides new opportunities for comparative morphological analyses over time and between species.

The worldwide expansion of mosquito-borne pathogens necessitates improved control measures, including approaches to restrict infection and transmission by mosquito vectors. Reducing transmission is challenging because determinants of vector competence for viruses like Zika (ZIKV) are poorly understood. Our previous work established thatAedes (Ae.) aegyptilarvae reared in microbe-rich environmental water are less susceptible to ZIKV as adults compared to cohorts reared in microbe-deficient laboratory tap water. Here, we explain the association by identifying a mechanism by which environment-derived microbes reduce susceptibility ofAe. aegyptifor ZIKV. Provided that the midgut represents the first barrier to mosquito infection, we hypothesized that microbial exposure modulates midgut infection by ZIKV. Since mosquitoes live in water as larvae and pupae and then transition to air as adults, we also define the stage in the life of a mosquito when microbial exposure reduces ZIKV susceptibility.Ae. aegyptilarvae were reared in microbe-rich water and then treated with antibiotics during the pupal and adult stages, adult stage only, or provided no antibiotics at any stage. Vector competence was next evaluated in mosquitoes that ingested ZIKV-spiked bloodmeals. Antibiotic treatment enhanced ZIKV infection and dissemination rates, especially inAe. aegyptitreated as both pupae and adults. Antibiotic treated adult mosquitoes also had increased midgut epithelium permeability, higher numbers of ZIKV-infected midgut cells, and impaired bloodmeal digestion. Consistent with these changes,Ae. aegyptitreated with antibiotics as pupae and adults that ingested ZIKV in bloodmeals showed reduced expression of genes associated with bloodmeal digestion and metabolism relative to mosquitoes that were not antibiotic treated. Together, these data show that exposure to microbes throughout the life ofAe. aegyptirestricts ZIKV dissemination by facilitating blood digestion and limiting midgut cell infection. Understanding the roles mosquito microbiota play in determining midgut physiology and arbovirus susceptibility can lead to novel approaches to decrease mosquito transmission and will improve understanding of vector competence in microbe-rich environmental habitats.Author SummaryMosquito-transmitted viruses like Zika continue to threaten human health. Absent vaccines or treatments, controlling mosquitoes or limiting their ability to transmit viruses represents a primary way to prevent mosquito-borne viral diseases. The role mosquito microbiota play in shaping transmission of Zika virus has been limited to association-based studies. Our prior work showed thatAedes aegyptimosquito larvae that develop in bacteria-rich water are less susceptible to Zika virus compared to larvae reared in microbe-poor laboratory tap water. Here we identify a mechanism that explains this association. Since mosquitoes are aquatic as larvae and pupae and terrestrial as adults, we also define the life stage when microbes need be present to reduce Zika virus susceptibility. We used antibiotics to reduce environmental water-derived microbes at pupal and adult or only adult stages and observed that, compared to mosquitoes with microbes, antibiotic treatment increases Zika virus dissemination, increases permeability and infection of the midgut, and impairs bloodmeal digestion. These data show that microbial exposure throughout the life of a mosquito restricts Zika virus dissemination by facilitating blood digestion and limiting midgut cell infection. These findings advance understanding of microbiota-mosquito-virus interactions by defining how microbes reduce susceptibility ofAedes aegyptito Zika virus.

Tsetse flies (genus Glossina), the sole vectors of African trypanosomiasis, are distinct from other disease vectors, and most other insects, due to dramatic evolutionary adaptations required to support their unique life history. These morphological and physiological adaptations are driven by demands associated with their strict dietary and reproductive requirements. Tsetse reproduce by obligate viviparity which entails obligate intrauterine larval development and provisioning of nutrients for the developing larvae. Viviparous reproduction reduces reproductive capacity/rate which also drives increased inter-and intra-sexual competition. This work describes three-dimensional (3D) analysis of viviparity associated morphological adaptations of tsetse female reproductive tract as well as that of male seminal secretions by phase contrast microcomputed tomography (pcMicroCT). Structural features of note include abdominal modifications facilitating the extreme abdominal distention required during blood feeding and pregnancy; abdominal and uterine musculature required for parturition of developed larvae; reduction of ovarian structure and capacity; structural features of the male seminal spermatophore that enhance sperm delivery and inhibition of insemination by competing males; uterine morphological features facilitating expansion and contraction before, during and after pregnancy; analysis of structural optimizations of the milk gland facilitating nutrient incorporation and transfer into the uterus. The use of pcMicroCT provides unprecedented opportunities for examination and discovery of internal morphological features not possible with traditional microscopy techniques and new opportunities for comparative morphological analyses over time and between species.3