A new allele conferring resistance to Lysinibacillus sphaericus is detected in low frequency in Culex quinquefasciatus field populations

Abstract

Background: The Cqm1 α-glucosidase of Culex quinquefasciatus larvae acts as the midgut receptor for the binary toxin of the biolarvicide Lysinibacillus sphaericus. Mutations within the cqm1 gene can code for aberrant polypeptides that can no longer be properly expressed or bind to the toxin, leading to insect resistance. The cqm1 REC and cqm1 REC-2 alleles were identified in a laboratory selected colony and both displayed mutations that lead to equivalent phenotypes of refractoriness to L. sphaericus. cqm1 REC was first identified as the major resistance allele in this colony but it was subsequently replaced by cqm1 REC-2 , suggesting the better adaptive features of the second allele. The major aim of this study was to evaluate the occurrence of cqm1 REC-2 and track its origin in field populations where cqm1 REC was previously identified.

Methods: The screening of the cqm1 REC-2 allele was based on more than 2000 C. quinquefasciatus larvae from five localities in the city of Recife, Brazil and used a multiplex PCR assay that is also able to identify cqm1 REC . Full-length sequencing of the cqm1 REC-2 and selected cqm1 samples was performed to identify further polymorphisms between these alleles.

Results: The cqm1 REC-2 allele was found in field samples, specifically in two heterozygous individuals from a single locality with an overall frequency and distribution much lower than that observed for cqm1 REC . The full-length sequences from these two cqm1 REC-2 copies were almost identical to the cqm1 REC-2 derived from the resistant colony but displayed more than 30 SNPs when compared with cqm1 and cqm1 REC . The cqm1 REC and cqm1 REC-2 resistant alleles were found to be associated with two distinct sets of wild-type cqm1 variants found in field populations.

Conclusions: The cqm1 REC-2 allele occurs in populations in Recife and was probably already present in the samples used to establish the laboratory resistant colony. The data generated indicates that cqm1 REC-2 can be selected in field populations, although its low frequency and distribution in Recife suggest that cqm1 REC-2 presents a lower risk of selection compared to cqm1 REC .

Fig. 1

Fragments of Culex quinquefasciatus cqm1 alleles amplified from larvae of susceptible (S) and Lysinibacillus sphaericus-resistant colonies (REC, REC-2). Profile of fragments in base pairs (bp): S (ab, 376-257 bp), REC (a’b’, 357-238 bp) and REC-2 homozygous (ac, 376-172 bp); S/REC (aa’bb’) and S/REC-2 (abc) heterozygous; sample without DNA (C-). Molecular markers (M) in base pairs

Fig. 2

Fragments of Culex quinquefasciatus cqm1 alleles amplified from Jaboatão field samples. cqm1 homozygotes (S), cqm1/cqm1 _REC-2 heterozygote (S/REC-2), cqm1 _REC (C_REC) and cqm1 _REC-2 (C_REC-2) internal positive controls for the respective alleles, samples without DNA (C-). Molecular markers (M) in base pairs

Fig. 3

Number of single nucleotide polymorphisms (SNPs) between Jaboatão cqm1 _REC-2 sequence (cqm1 _REC-2 J1) versus other cqm1 sequences. cqm1 reference (Ref) (GeneBank DQ333335), cqm1 from Jaboatão (J), cqm1 _REC and cqm1 _REC-2, from the respective REC and REC-2 laboratory resistant colonies