Synergistic effect of Lysinibacillus sphaericus and glyphosate on temephos-resistant larvae of Aedes aegypti.

BACKGROUND: Glyphosate-based herbicides are one of the most commonly used compounds to control perennial weeds around the world. This compound is very persistent in the environment and tends to filter into aquatic ecosystems, affecting non-target species such as mosquito larvae. Aedes aegypti mosquitoes are vectors of multiple arboviruses such as dengue and Zika. Glyphosate can be degraded into non-harmful environmental compounds by Lysinibacillus sphaericus, a spore forming bacterium which can also kill Ae. aegypti larvae. In this study, we assessed the effect of glyphosate concentrations, typically used in Colombia, on the entomopathogenic activity of L. sphaericus against Ae. aegypti larvae. METHODS: Bioassays and toxicity curves were performed to compare the larval mortality between different treatments with and without bacteria and glyphosate (Roundup 747®). Larvae were exposed to both bacteria and glyphosate by adding the compound on chloride-free water. Comparisons were made using both probit regression and ANOVA analysis. RESULTS: ANOVA showed a significant difference in larval mortality when adding glyphosate and L. sphaericus at the same time. Thus, a positive synergic effect on larval mortality was found when L. sphaericus and glyphosate were mixed. According to probit analysis, median lethal dose (LD50) for bacterial mixture was of 106.23 UFC/ml and for glyphosate was 2.34 g/l. CONCLUSIONS: A positive synergic effect on the mortality of larval Ae. aegypti when exposed to L. sphaericus mixture and glyphosate was found. Molecular studies focusing on the toxin production of L. sphaericus are required to understand more about this synergistic effect.

Co-selection and replacement of resistance alleles to Lysinibacillus sphaericus in a Culex quinquefasciatus colony.

The Cqm1 α-glucosidase, expressed within the midgut of Culex quinquefasciatus mosquito larvae, is the receptor for the Binary toxin (Bin) from the entomopathogen Lysinibacillus sphaericus. Mutations of the Cqm1 α-glucosidase gene cause high resistance levels to this bacterium in both field and laboratory populations, and a previously described allele, cqm1REC, was found to be associated with a laboratory-resistant colony (R2362). This study described the identification of a novel resistance allele, cqm1REC-2, that was co-selected with cqm1REC within the R2362 colony. The two alleles display distinct mutations but both generate premature stop codons that prevent the expression of midgut-bound Cqm1 proteins. Using a PCR-based assay to monitor the frequency of each allele during long-term maintenance of the resistant colony, cqm1REC was found to predominate early on but later was replaced by cqm1REC-2 as the most abundant resistance allele. Homozygous larvae for each allele were then generated that displayed similar high-resistance phenotypes with equivalent low levels of transcript and lack of protein expression for both cqm1REC and cqm1REC-2. In progeny from a cross of homozygous individuals for each allele at a 1 : 1 ratio, analyzed for ten subsequent generations, cqm1REC showed a higher frequency than cqm1REC-2. The replacement of cqm1REC by cqm1REC -2 observed in the R2362 colony, kept for 210 generations, indicates changes in fitness related to traits that are unknown but linked to these two alleles, and constitutes a unique example of evolution of resistance within a controlled laboratory environment.