Small RNA regulatory pathways (SRRPs) control key aspects of development and anti-viral defense in metazoans. Members of the Argonaute family of catalytic enzymes degrade target RNAs in each of these pathways. SRRPs include the microRNA, small interfering RNA (siRNA) and PIWI-type gene silencing pathways. Mosquitoes generate viral siRNAs when infected with RNA arboviruses. However, in some mosquitoes, arboviruses survive antiviral RNA interference (RNAi) and are transmitted via mosquito bite to a subsequent host. Increased knowledge of these pathways and functional components should increase understanding of the limitations of anti-viral defense in vector mosquitoes. To do this, we compared the genomic structure of SRRP components across three mosquito species and three major small RNA pathways.

Aedes aegyptiis the primary urban mosquito vector of viruses causing dengue, Zika and chikungunya fevers –for which vaccines and effective pharmaceuticals are still lacking. Current strategies to suppress arbovirus outbreaks include removal of larval-breeding sites and insecticide treatment of larval and adult populations. Insecticidal control ofAe. aegyptiis challenging, due to a recent rapid global increase in knockdown-resistance (kdr) to pyrethroid insecticides. Widespread, heavy use of pyrethroid space-sprays has created an immense selection pressure for kdr, which is primarily under the control of the voltage-gated sodium channel gene (vgsc). To date, eleven replacements invgschave been discovered, published and shown to be associated with pyrethroid resistance to varying degrees. In Mexico, F1,534C and V1,016I have co-evolved in the last 16 years acrossAe. aegyptipopulations. Recently, a novel replacement V410L was identified in Brazil and its effect onvgscwas confirmed by electrophysiology. Herein, we screened V410L in 25Ae. aegyptihistorical collections from Mexico, the first heterozygote appeared in 2002 and frequencies have increased in the last 16 years alongside V1,016I and F1,534C. Knowledge of the specificvgscreplacements and their interaction to confer resistance is essential to predict and to develop strategies for resistance management.

Arthropod-borne viruses (arboviruses) infect mosquito salivary glands and then escape to saliva prior to virus transmission. Arbovirus transmission from mosquitoes can be modulated by salivary gland infection barriers (SGIBs) and salivary gland escape barriers (SGEBs). We determined the influence of SGIBs and SGEBs by estimating the quantitative genetic contributions of Aedes aegypti half-sib families (Mapastepec, Mexico) infected with three dengue 2 (DENV2), two chikungunya (CHIKV), and two Zika (ZIKV) genotypes. We determined virus titer per salivary gland and saliva at seven days post-infection and virus prevalence in the half-sib population. CHIKV or ZIKV genotypes did not present SGIB, whereas DENV2 genotypes showed low rates of SGIB. However, virus titer and prevalence due to additive genetic factors in the half-sib family displayed a significant narrow-sense heritability (h²) for SGIB in two of the three DENV2 genotypes and one CHIKV and one ZIKV genotype. SGEBs were detected in all seven virus strains: 60–88% of DENV2 and 48–62% of CHIKV or ZIKV genotype infections. SGEB h² was significant for all CHIKV or ZIKV genotypes but not for any of the DENV2 genotypes. SGIBs and SGEBs exhibited classical gene-by-gene interaction dynamics and are influenced by genetic factors in the mosquito and the virus.

In urban settings, chikungunya, Zika, and dengue viruses are transmitted by Aedes aegypti mosquitoes. Since these viruses co-circulate in several regions, coinfection in humans and vectors may occur, and human coinfections have been frequently reported. Yet, little is known about the molecular aspects of virus interactions within hosts and how they contribute to arbovirus transmission dynamics. We have previously shown that Aedes aegypti exposed to chikungunya and Zika viruses in the same blood meal can become coinfected and transmit both viruses simultaneously. However, mosquitoes may also become coinfected by multiple, sequential feeds on single infected hosts. Therefore, we tested whether sequential infection with chikungunya and Zika viruses impacts mosquito vector competence. We exposed Ae. aegypti mosquitoes first to one virus and 7 days later to the other virus and compared infection, dissemination, and transmission rates between sequentially and single infected groups. We found that coinfection rates were high after sequential exposure and that mosquitoes were able to co-transmit both viruses. Surprisingly, chikungunya virus coinfection enhanced Zika virus transmission 7 days after the second blood meal. Our data demonstrate heterologous arbovirus synergism within mosquitoes, by unknown mechanisms, leading to enhancement of transmission under certain conditions.

Background: Aedes aegyptiis arguably the most studied of all mosquito species in the laboratory and is the primary vector of both Dengue and Yellow Fever flaviviruses in the field. A large number of transcriptional studies have been made in the species and these usually report transcript quantities observed at a certain age or stage of development. However, circadian oscillation is an important characteristic of gene expression in many animals and plants, modulating both their physiology and behavior. Circadian gene expression in mosquito species has been previously reported but for only a few genes directly involved in the function of the molecular clock.Results: Herein we analyze the transcription profiles of 21,494 messenger RNAs using anAe. aegyptiAgilent^®microarray. Transcripts were quantified in adult female heads at 24 hours and then again at 72 hours and eight subsequent time points spaced four hours apart. We document circadian rhythms in multiple molecular pathways essential for growth, development, immune response, detoxification/pesticide resistance. Circadian rhythms were also noted in ribosomal protein genes used for normalization in reverse transcribed PCR (RT-PCR) to determine transcript abundance. We report pervasive oscillations and intricate synchronization patterns relevant to all known biological pathways.Conclusion: These results argue strongly that transcriptional analyses either need to be made over time periods rather than confining analyses to a single time point or development stage or exceptional care needs to be made to synchronize all mosquitoes to be analyzed and compared among treatment groups.

ZIKV is a new addition to the arboviruses circulating in the New World, with more than 1 million cases since its introduction in 2015. A growing number of studies have reported vector competence (VC) of Aedes mosquitoes from several areas of the world for ZIKV transmission. Some studies have used New World mosquitoes from disparate regions and concluded that these have a variable but relatively low competence for the Asian lineage of ZIKV. Ten Aedes aegypti (L) and three Ae. albopictus (Skuse) collections made in 2016 from throughout Mexico were analyzed for ZIKV (PRVABC59—Asian lineage) VC. Mexican Ae. aegypti had high rates of midgut infection (MIR), dissemination (DIR) and salivary gland infection (SGIR) but low to moderate transmission rates (TR). It is unclear whether this low TR was due to heritable salivary gland escape barriers or to underestimating the amount of virus in saliva due to the loss of virus during filtering and random losses on surfaces when working with small volumes. VC varied among collections, geographic regions and whether the collection was made north or south of the Neovolcanic axis (NVA). The four rates were consistently lower in northeastern Mexico, highest in collections along the Pacific coast and intermediate in the Yucatan. All rates were lowest north of the NVA. It was difficult to assess VC in Ae. albopictus because rates varied depending upon the number of generations in the laboratory. Mexican Ae. aegypti and Ae. albopictus are competent vectors of ZIKV. There is however large variance in vector competence among geographic sites and regions. At 14 days post infection, TR varied from 8–51% in Ae. aegypti and from 2–26% in Ae. albopictus. Aedes aegypti is an efficient vector for arboviruses which is partially determined by its vector competence (VC) status, a highly variable trait. Based on previous reports, the VC of New World mosquitoes is limited for ZIKV. However, a VC assessment from additional geographical sources was lacking. Therefore, we evaluated the VC for ZIKV using recently colonized mosquitoes from Mexico. Aedes aegypti and Ae. albopictus were highly susceptible to ZIKV infection but varied greatly in their transmission rates. As with previous studies of other flaviviruses; VC for ZIKV was highly variable in both Ae. aegypti and Ae. albopictus but was greatest in Ae. aegypti, supporting its role as the main ZIKV vector.