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

Abstract

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.

Keywords: Aedes aegypti; Aedes albopictus; Anopheles gambiae; Culex quinquefasciatus; Larvae; Larvicide; Methionine; RNAi; Saccharomyces cerevisiae; Sex; Yeast.

Fig. 1

Yeast larvicide MtnB.496 induces female-specific mortality. a MtnB transcript is expressed throughout larval development (L1 = first instar, L2 = second instar, L3 = third instar, L4 = fourth instar, Pupae-M = male pupae, Pupae-F = female pupae; error bars denote standard deviations; transcript levels relative to those of L4 larvae are displayed). b Silencing of MtnB following larval consumption of MtnB.496 yeast was confirmed through qRT-PCR (***P < 0.001, Student’s t-test; error bars represent standard deviation). c Oral consumption of MtnB.496 yeast during larval development resulted in significant female larval mortality (***P < 0.001, chi-squared test), but did not impact male survival (P > 0.05, chi-squared test); larval consumption of control interfering RNA strain yeast did not significantly impact survival of female (P > 0.05, chi-squared test) or male (P > 0.05, chi-squared test) larvae. Data, which were compiled from nine replicate container trials per condition (with each container bearing 20 larvae), are represented as mean survival through adulthood, and error bars represent standard errors of the mean

Fig. 2

MtnB.496 yeast facilitates scaled production of fit A. aegypti males. Larval consumption of MtnB.496 yeast does not significantly impact adult male mating capacity (a, P > 0.05, chi-squared test, n = 49), the number of eggs laid by females that mate with these males (b, P > 0.05, Student’s t-test), or the percentage of larvae hatching from these eggs (b, P > 0.05, Student’s t-test). Incorporation of MtnB.496 yeast into the diet used for mosquito mass rearing induced significant female mortality (c, ***P < 0.001 vs. mass-rearing diet, chi-squared test) yet did not significantly impact male survival (c, P > 0.05 vs. mass-rearing diet, chi-squared test; data were compiled from four replicate containers per condition, each bearing 500 larvae) or wing size (d, P > 0.05 vs. mass-rearing diet, t-test; n = 50 wings/treatment), which correlates with fitness. Error bars represent SEM in all panels. NSD: Not significantly different

Fig. 3

MtnB is required for female larval survival in multiple species of mosquitoes. Larval consumption of yeast interfering larvicides corresponding to the A. albopictus (a), A. gambiae (b), and C. quinquefasciatus (c) MtnB genes induce significant female-specific mortality in each species (***P < 0.001, chi-squared test), but have no impact on male survival (a–c, P > 0.05, chi-squared test). Controls in a–c were larvae fed with yeast expressing an interfering RNA with no known target in mosquitoes, which had no significant impact on female or male survival in any of the three mosquitoes species (a–c, P > 0.05, chi-squared test). Data, which were compiled from nine (a and c) or six (b) replicate container trials per treatment (with each container bearing 20 larvae), represent mean survival through adulthood, with error bars corresponding to SEM