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

Error in Figure [...].

A Conserved Female-Specific Requirement for the GGT 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.

Sugar-Baited Delivery of Small Interfering RNA for Gene Silencing in Adult Mosquitoes.

RNA interference (RNAi), an innate regulatory mechanism that is conserved across many eukaryotic species, has been harnessed for experimental gene silencing in many organisms, including mosquitoes. This protocol describes an optimized method for inducing RNAi in adult Aedes aegypti and Anopheles gambiae mosquitoes that involves feeding them a red-colored sugar bait containing small interfering RNA (siRNA). This oral delivery method is less physically disruptive than delivery by subcutaneous injection, and the use of siRNAs (in contrast to long dsRNAs) for RNAi enables the design of molecules that target conserved sites so that gene function can be studied in multiple species. After feeding, the behavioral and morbidity phenotypes that result from the suppression of target gene expression can then be analyzed.

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 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.

A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting Rbfox1 Represses Notch Signaling and Kills Both Larvae and Adult Mosquitoes.

Prevention of mosquito-borne infectious diseases will require new classes of environmentally safe insecticides and novel mosquito control technologies. Saccharomyces cerevisiae was engineered to express short hairpin RNA (shRNA) corresponding to mosquito Rbfox1 genes. The yeast induced target gene silencing, resulting in larval death that was observed in both laboratory and outdoor semi-field trials conducted on Aedes aegypti. High levels of mortality were also observed during simulated field trials in which adult females consumed yeast delivered through a sugar bait. Mortality correlated with defects in the mosquito brain, in which a role for Rbfox1 as a positive regulator of Notch signaling was identified. The larvicidal and adulticidal activities of the yeast were subsequently confirmed in trials conducted on Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus, yet the yeast had no impact on survival of select non-target arthropods. These studies indicate that yeast RNAi pesticides targeting Rbfox1 could be further developed as broad-based mosquito larvicides and adulticides for deployment in integrated biorational mosquito control programs. These findings also suggest that the species-specificity of attractive targeted sugar baits, a new paradigm for vector control, could potentially be enhanced through RNAi technology, and specifically through the use of yeast-based interfering RNA pesticides.

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.

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.

Allopatric genetic origins for sympatric host-plant shifts and race formation in Rhagoletis.

Tephritid fruit flies belonging to the Rhagoletis pomonella sibling species complex are controversial because they have been proposed to diverge in sympatry (in the absence of geographic isolation) by shifting and adapting to new host plants. Here, we report evidence suggesting a surprising source of genetic variation contributing to sympatric host shifts for these flies. From DNA sequence data for three nuclear loci and mtDNA, we infer that an ancestral, hawthorn-infesting R. pomonella population became geographically subdivided into Mexican and North American isolates approximately 1.57 million years ago. Episodes of gene flow from Mexico subsequently infused the North American population with inversion polymorphism affecting key diapause traits, forming adaptive clines. Sometime later (perhaps +/-1 million years), diapause variation in the latitudinal clines appears to have aided North American flies in adapting to a variety of plants with differing fruiting times, helping to spawn several new taxa. Thus, important raw genetic material facilitating the adaptive radiation of R. pomonella originated in a different time and place than the proximate ecological host shifts triggering sympatric divergence.

Evidence for inversion polymorphism related to sympatric host race formation in the apple maggot fly, Rhagoletis pomonella.

Evidence suggests that the apple maggot, Rhagoletis pomonella (Diptera: Tephritidae) is undergoing sympatric speciation (i.e., divergence without geographic isolation) in the process of shifting and adapting to a new host plant. Prior to the introduction of cultivated apples (Malus pumila) in North America, R. pomonella infested the fruit of native hawthorns (Crataegus spp.). However, sometime in the mid-1800s the fly formed a sympatric race on apple. The recently derived apple-infesting race shows consistent allele frequency differences from the hawthorn host race for six allozyme loci mapping to three different chromosomes. Alleles at all six of these allozymes correlate with the timing of adult eclosion, an event dependent on the duration of the overwintering pupal diapause. This timing difference differentially adapts the univoltine fly races to an approximately 3- to 4-week difference in the peak fruiting times of apple and hawthorn trees, partially reproductively isolating the host races. Here, we report finding substantial gametic disequilibrium among allozyme and complementary DNA (cDNA) markers encompassing the three chromosomal regions differentiating apple and hawthorn flies. The regions of disequilibrium extend well beyond the previously characterized six allozyme loci, covering substantial portions of chromosomes 1, 2, and 3 (haploid n = 6 in R. pomonella). Moreover, significant recombination heterogeneity and variation in gene order were observed among single-pair crosses for each of the three genomic regions, implying the existence of inversion polymorphism. We therefore have evidence that genes affecting diapause traits involved in host race formation reside within large complexes of rearranged genes. We explore whether these genomic regions (inversions) constitute coadapted gene complexes and discuss the implications of our findings for sympatric speciation in Rhagoletis.

A FIELD TEST FOR HOST-PLANT DEPENDENT SELECTION ON LARVAE OF THE APPLE MAGGOT FLY, RHAGOLETIS POMONELLA.

Host-plant dependent fitness trade-offs refer to traits that enhance the performance of an insect on one plant species to its detriment on others. Such trade-offs are central to models of sympatric speciation via host shifts, but have proven difficult to empirically demonstrate. Here, we test for host-plant dependent selection on larvae of apple (Malus pumila L.)- and hawthorn (Crataegus mollis L. spp.)-infesting races of Rhagoletis pomonella (Walsh). Samples of larvae were reared in the field and under protective conditions in a garage. Our rationale was that the garage should slow rates of fruit rot relative to the field, relaxing selection pressures associated with declining fruit quality. Four findings emerged from the study. (1) Larvae suffered higher mortality in fruits in the field than the garage. (2) The increase in mortality was greater for larvae in haws. (3) Larvae possessing the alleles Me 100, Acon-2 95, and Mpi 37, three allozymes displaying host-related differentiation in R. pomonella that map to linkage group II in the fly, left fruits earlier than other genotypes. (4) Allele frequencies for Me 100, Acon-2 95, and Mpi 37 were significantly higher in both apple and haw larvae surviving the field versus the garage treatment. Our results suggested that field conditions favored larvae that rapidly developed and left rotting fruits. Since these individuals tended to possess the alleles Me 100, Acon-2 95, and Mpi 37, frequencies of these allozymes were higher in the field. Selection on larvae was directional for Me 100, Acon-2 95, and Mpi 37 (or linked genes) in both host races. We previously showed that these same alleles can be disfavored in the pupal stage, especially in the apple race, where they correlate with premature diapause termination. Fitness trade-offs in Rhagoletis may therefore be due as much to differences in the relative strengths of directional selection pressures acting on different life stages as to disruptive selection affecting any one particular stage. The necessity to consider details of the entire life-cycle highlights one of the many challenges posed to documenting fitness trade-offs for phytophagous insects.

THE EFFECTS OF WINTER LENGTH ON THE GENETICS OF APPLE AND HAWTHORN RACES OF RHAGOLETIS POMONELLA (DIPTERA: TEPHRITIDAE).

Host plant-associated fitness trade-offs are central to models of sympatric speciation proposed for certain phytophagous insects. But empirical evidence for such trade-offs is scant, which has called into question the likelihood of nonallopatric speciation. Here, we report on the second in a series of studies testing for host-related selection on pupal life-history characteristics of apple- (Malus pumila L.) and hawthorn- (Crataegus mollis L. spp.) infesting races of the Tephritid fruit fly, Rhagoletis pomonella (Walsh). In particular, we examine the effects of winter length on the genetics of these flies. We have previously found that the earlier fruiting phenology of apple trees exposes apple-fly pupae to longer periods of warm weather preceding winter than hawthorn-fly pupae. Because R. pomonella has a facultative diapause, we hypothesized that this selects for pupae with more recalcitrant pupal diapauses (or slower metabolic/development rates) in the apple-fly race. A study in which we experimentally manipulated the length of the prewintering period for hawthorn-origin pupae supported this prediction. If the period preceding winter is important for apple- and hawthorn-fly pupae, then so too should be the length (duration) of winter; the rationale for this prediction is that "fast developing" pupae that break diapause too early will deplete their energy reserves and disproportionately die during long winters. To test this possibility, we chilled apple- and hawthorn-origin pupae collected from a field site near Grant, Michigan, in a refrigerator at 4°C for time periods ranging from one week to two years. Our a priori expectation was that longer periods of cold storage would select against allozyme markers that were associated with faster rates of development in our earlier study. Since these electromorphs are typically found at higher frequencies in hawthorn flies, extending the overwintering period should favor "apple-fly alleles" in both races. The results from this "overwinter" experiment supported the diapause hypothesis. The anticipated genetic response was observed in both apple and hawthorn races, as allele frequencies became significantly more "apple-fly-like" in eclosing adults surviving longer chilling periods. This indicates that it is the combination of environmental conditions before and during winter that selects on the host races. Many tests for trade-offs fail to adequately consider the interplay between insect development, host plant phenology, and local climatic conditions. Our findings suggest that such oversight may help to explain the paucity of reported fitness trade-offs.