A conserved female-specific larval requirement for MtnB function facilitates sex separation in multiple species of disease vector mosquitoes

Background: Clusters of sex-specific loci are predicted to shape the boundaries of the M/m sex-determination locus of the dengue vector mosquito Aedes aegypti, but the identities of these genes are not known. Identification and characterization of these loci could promote a better understanding of mosquito sex chromosome evolution and lead to the elucidation of new strategies for male mosquito sex separation, a requirement for several emerging mosquito population control strategies that are dependent on the mass rearing and release of male mosquitoes. This investigation revealed that the methylthioribulose-1-phosphate dehydratase (MtnB) gene, which resides adjacent to the M/m locus and encodes an evolutionarily conserved component of the methionine salvage pathway, is required for survival of female larvae. Results: Larval consumption of Saccharomyces cerevisiae (yeast) strains engineered to express interfering RNA corresponding to MtnB resulted in target gene silencing and significant female death, yet had no impact on A. aegypti male survival or fitness. Integration of the yeast larvicides into mass culturing protocols permitted scaled production of fit adult male mosquitoes. Moreover, silencing MtnB orthologs in Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus revealed a conserved female-specific larval requirement for MtnB among different species of mosquitoes. Conclusions: The results of this investigation, which may have important implications for the study of mosquito sex chromosome evolution, indicate that silencing MtnB can facilitate sex separation in multiple species of disease vector insects

A functional requirement for sex-determination M/m locus region lncRNA genes in Aedes aegypti female larvae

Although many putative long non-coding RNA (lncRNA) genes have been identified in insect genomes, few of these genes have been functionally validated. A screen for female-specific larvicides that facilitate Aedes aegypti male sex separation uncovered multiple interfering RNAs with target sites in lncRNA genes located in the M/m locus region, including loci within or tightly linked to the sex determination locus. Larval consumption of a Saccharomyces cerevisiae (yeast) strain engineered to express interfering RNA corresponding to lncRNA transcripts resulted in significant female death, yet had no impact on male survival or fitness. Incorporation of the yeast larvicides into mass culturing protocols facilitated scaled production and separation of fit adult males, indicating that yeast larvicides could benefit mosquito population control strategies that rely on mass releases of male mosquitoes. These studies functionally verified a female-specific developmental requirement for M/m locus region lncRNA genes, suggesting that sexually antagonistic lncRNA genes found within this highly repetitive pericentromeric DNA sequence may be contributing to the evolution of A. aegypti sex chromosomes

Oral RNAi for Gene Silencing in Mosquitoes: From the Bench to the Field

RNA interference (RNAi) has played a key role in the field of insect functional genomics, a discipline that has enhanced the study of developmental, evolutionary, physiological, and molecular biological phenomena in a wide variety of insects, including disease vector mosquitoes. Here we introduce a recently optimized RNAi procedure in which adult mosquitoes are fed with a colored sugar bait containing small interfering RNA (siRNA). This procedure effectively and economically leads to gene silencing, is technically straightforward, and has been successfully used to characterize a number of genes in adult mosquitoes. We also discuss how, in addition to laboratory applications, this oral RNAi procedure might one day be used in the field for controlling insect pests

A Conserved Female-Specific Requirement for the <i>GGT</i> Gene in Mosquito Larvae Facilitates RNAi-Mediated Sex Separation in Multiple Species of Disease Vector Mosquitoes

Although several emerging mosquito control technologies are dependent on mass releases of adult males, methods of sex-sorting that can be implemented globally have not yet been established. RNAi screens led to the discovery of siRNA, which targets gamma-glutamyl transpeptidase (GGT), a gene which is well conserved in multiple species of mosquitoes and located at the sex-determining M locus region in Aedes aegypti. Silencing the A. aegypti, Aedes albopictus, Anopheles gambiae, Culex pipiens, and Culex quinquefasciatus GGT genes resulted in female larval death, with no significant impact on male survival. Generation of yeast strains that permitted affordable expression and oral delivery of shRNA corresponding to mosquito GGT genes facilitated larval target gene silencing and generated significantly increased 5 males:1 female adult ratios in each species. Yeast targeting a conserved sequence in Culex GGT genes was incorporated into a larval mass-rearing diet, permitting the generation of fit adult male C. pipiens and C. quinquefasciatus, two species for which labor-intensive manual sex separation had previously been utilized. The results of this study indicate that female-specific yeast-based RNAi larvicides may facilitate global implementation of population-based control strategies that require releases of sterile or genetically modified adult males, and that yeast RNAi strategies can be utilized in various species of mosquitoes that have progressed to different stages of sex chromosome evolution

Characterization of a yeast interfering RNA larvicide with a target site conserved in the synaptotagmin gene of multiple disease vector mosquitoes.

New mosquito control strategies are vitally needed to address established and emerging arthropod-borne infectious diseases. Here we describe the characterization of a yeast interfering RNA larvicide that was developed through the genetic engineering of Saccharomyces cerevisiae (baker's yeast) to express a short hairpin RNA targeting the Aedes aegypti synaptotagmin (Aae syt) gene. The larvicide effectively silences the Aae syt gene, causes defects at the larval neural synapse, and induces high rates of A. aegypti larval mortality in laboratory, simulated-field, and semi-field trials. Conservation of the interfering RNA target site in multiple mosquito species, but not in humans or other non-target species, suggested that it may function as a broad-range mosquito larvicide. In support of this, consumption of the yeast interfering RNA larvicide was also found to induce high rates of larval mortality in Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus mosquito larvae. The results of these studies suggest that this biorational yeast interfering RNA larvicide may represent a new intervention that can be used to combat multiple mosquito vectors of human diseases

Correction: Mysore et al. A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting <i>Rbfox1</i> Represses <i>Notch</i> Signaling and Kills Both Larvae and Adult Mosquitoes. <i>Pathogens</i> 2021, <i>10</i>, 1251

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A Yeast RNA-Interference Pesticide Targeting the Irx Gene Functions as a Broad-Based Mosquito Larvicide and Adulticide

Concerns for widespread insecticide resistance and the unintended impacts of insecticides on nontarget organisms have generated a pressing need for mosquito control innovations. A yeast RNAi-based insecticide that targets a conserved site in mosquito Irx family genes, but which has not yet been identified in the genomes of nontarget organisms, was developed and characterized. Saccharomyces cerevisiae constructed to express short hairpin RNA (shRNA) matching the target site induced significant Aedes aegypti larval death in both lab trials and outdoor semi-field evaluations. The yeast also induced high levels of mortality in adult females, which readily consumed yeast incorporated into an attractive targeted sugar bait (ATSB) during simulated field trials. A conserved requirement for Irx function as a regulator of proneural gene expression was observed in the mosquito brain, suggesting a possible mode of action. The larvicidal and adulticidal properties of the yeast were also verified in Aedes albopictus, Anopheles gambiae, and Culexquinquefasciatus mosquitoes, but the yeast larvicide was not toxic to other nontarget arthropods. These results indicate that further development and evaluation of this technology as an ecofriendly control intervention is warranted, and that ATSBs, an emerging mosquito control paradigm, could potentially be enriched through the use of yeast-based RNAi technology