Unveiling the Role of Two Rhodopsin-like GPCR Genes in Insecticide-Resistant House Flies, Musca domestica

Abstract

Insecticide resistance in insects, driven by the overexpression of P450 enzymes, presents a significant challenge due to the enhanced metabolic detoxification of insecticides. Although the transcriptional regulation of P450 genes is not yet fully understood, G-protein-coupled receptor (GPCR) genes have emerged as key regulators in this process. This study is the first to associate GPCR genes with insecticide resistance in Musca domestica. We identified two key rhodopsin-like GPCR genes, ALHF_02706.g1581 and ALHF_04422.g2918, which were significantly overexpressed in the resistant ALHF strain compared to sensitive strains. Notably, both ALHF_02706.g1581 and ALHF_04422.g2918 were mapped to autosome 2, where critical but unidentified regulatory factors controlling resistance and P450 gene regulation are located. This supports our hypothesis that GPCRs function as trans-regulatory factors for P450-mediated resistance. Functional analysis using transgenic Drosophila demonstrated that overexpression of these rhodopsin-like GPCR genes increased permethrin resistance by approximately two-fold. Specifically, ALHF_02706.g1581 overexpression significantly upregulated the Drosophila resistance-related P450 genes CYP12D1, CYP6A2, and CYP6A8, while ALHF_04422.g2918 increased CYP6G1 and CYP6A2 expression, thereby enhancing insecticide detoxification in rhodopsin-like GPCR transgenic Drosophila lines. These findings suggest that these rhodopsin-like GPCR genes on autosome 2 may act as trans-regulatory factors for P450-mediated resistance, underscoring their critical role in insecticide detoxification and resistance development in M. domestica.

Keywords: G-protein coupled receptors (GPCRs); cytochrome P450s; gene mapping; house flies; insecticide resistance.

Figure 1

The expression of GPCR genes in house flies, Musca domestica. The expression levels of GPCR genes ALHF_02706.g1581 and ALHF_04422.g2918 were assessed in the susceptible house fly strains aabys and CS, as well as in a resistant strain. mRNA levels of both GPCR genes were quantitatively measured using qRT-PCR. Results are presented as mean ± S.E. (n ≥ 3). Statistical significance in gene expression among samples, indicated by different letters (a, b, or c), was determined using one-way ANOVA with a p-value ≤ 0.05.

Figure 2

Allele-specific RT-PCR for autosomal mapping of GPCR gene ALHF_02706.g1581 in M. domestica. The full length of the GPCR gene ALHF_02706.g1581 is 1116 bp in both the ALHF and aabys strains. (A), PCR fragments were generated using an allele-specific primer set designed according to the ALHF sequence of ALHF_02706.g1581. (B). The absence of a PCR product in a house fly line indicates that the gene is located on the corresponding autosome from aabys (e.g., the absence of a band in the A1345 line indicates that the gene is located on autosome 2).

Figure 3

Effects of overexpressed GPCR genes on permethrin resistance and P450 gene expression in Drosophila melanogaster. (A) RT-PCR amplification of the GPCR genes ALHF_02706.g1581 and ALHF_04422.g2918 in transgenic Drosophila melanogaster lines. “−” indicates the non-transgenic control D. melanogaster line, while “+” represents the transgenic D. melanogaster lines containing house fly GPCR genes. The 1 kb DNA Plus ladder (Biolabs) was used as the molecular size reference, with numbers indicating DNA band sizes in bp. (B) Toxicity of permethrin to non-transgenic (control) and transgenic Drosophila melanogaster lines. Resistance ratios were calculated as LD50 of D. melanogaster lines/LD50 of the control line. Statistical significance in gene expression among samples, indicated by different letters (a, b, or c), was determined using one-way ANOVA with a p-value ≤ 0.05. No significant differences in expression levels were observed between groups labeled with the same letter (p < 0.05).

Figure 4

Relative expression levels of four Drosophila resistance-related P450 genes in control and GPCR transgenic lines. The expression patterns of four P450 genes (CYP6A2, CYP6A8, CYP12D1, CYP6G1) were analyzed in control and transgenic Drosophila lines using qRT-PCR. (A) Expressing of the GPCR genes ALHF_02706.g1581 and (B) Expressing of the GPCR ALHF_04422.g2918. The relative expression levels in transgenic flies are shown relative to their expression in the control line. Results are presented as mean ± S.E. (n ≥ 3). Statistical significance in gene expression among samples, indicated by different letters (a or b), was determined using one-way ANOVA with p ≤ 0.05.