Strong resistance to β-cyfluthrin in a strain of the beetle Alphitobius diaperinus: a de novo transcriptome analysis

Abstract

The lesser mealworm, Alphitobius diaperinus, is an invasive tenebrionid beetle and a vector of pathogens. Due to the emergence of insecticide resistance and consequent outbreaks that generate significant phytosanitary and energy costs for poultry farmers, it has become a major insect pest worldwide. To better understand the molecular mechanisms behind this resistance, we studied a strain of A. diaperinus from a poultry house in Brittany that was found to be highly resistant to the β-cyfluthrin. The strain survived β-cyfluthrin exposures corresponding to more than 100 times the recommended dose. We used a comparative de novo RNA-Seq approach to explore genes expression in resistant versus sensitive strains. Our de novo transcriptomic analyses showed that responses to β-cyfluthrin likely involved a whole set of resistance mechanisms. Genes related to detoxification, metabolic resistance, cuticular hydrocarbon biosynthesis and proteolysis were found to be constitutively overexpressed in the resistant compared to the sensitive strain. Follow-up enzymatic assays confirmed that the resistant strain exhibited high basal activities for detoxification enzymes such as cytochrome P450 monooxygenase and glutathione-S-transferase. The in-depth analysis of differentially expressed genes suggests the involvement of complex regulation of signaling pathways. Detailed knowledge of these resistance mechanisms is essential for the establishment of effective pest control.

Keywords: Alphitobius diaperinus; P450; de novo transcriptome; insecticide resistance; xenobiotic metabolism.

Fig. 1

Susceptibility of SENS and RES strains to β‐cyfluthrin. SENS and RES insects were exposed to 4RD β‐cyfluthrin (SENS‐4RD, RES‐4RD). Exposure to 100RD β‐cyfluthrin was only applied to resistant beetles (RES‐100RD). At each sampling time (from 1 to 9 days), beetles were scored as alive and active (“Active”) or alive but knocked down (“KD”) and the total number of living insects, called “Alive,” included the total of “Active” and “KD.” Each dot represents a mean of three replicates with 95% CI.

Fig. 2

Venn diagram representing the common and specific DEGs according to the different comparisons. (A) Number of detected DEGs between the SENS strain in response to the 4RD β‐cyfluthrin (SENS‐4RD/SENS‐0RD) and the RES strain in response to the 4RD (RES‐4RD/RES‐0RD) and 100RD (RES‐100RD/RES‐0RD) β‐cyfluthrin. (B) Number of detected DEGs between the untreated RES strain and the untreated SENS strain (RES‐0RD/SENS‐0RD) and the 4RD treated RES strain compared to the 4RD treated SENS strain (RES‐4RD/SENS‐4RD).

Fig. 3

Effects of β‐cyfluthrin treatments on detoxification enzyme activities. (A) Glutathione S‐transferase (GST) expressed in units corresponding to the production of μmol of conjugated CDNB per min per mg of protein, (B) Cytochrome P450 (Cyp) expressed in units corresponding to the production of μmol of P450 equivalent (oxidized TMBZ) per min per mg of protein, and (C) esterase (Est) activity expressed in units corresponding to the production of μmol of beta‐naphthol per min per mg of protein in the sensitive (SENS, blue) and the resistant (RES, red) strains nonexposed or exposed to 4 time or 100 time the recommended dose (RD). Boxplot represents the lower quartile (Q1), the median, and the upper quartile (Q3). The different letters indicate significant differences resulting from Holm‐adjusted pairwise comparisons at P‐value < 0.05.

Fig. 4

Differential expression of DEGs involved in detoxification, proteolysis and related to fatty acids. Each dot in each volcano plot represents the values of expression of one contig. Y‐axis shows values of (−Log₁₀(FDR)) and X‐axis shows Log₂FC according to RES‐0RD/SENS‐0RD (red), RES‐4RD/SENS‐4RD (yellow) and SENS‐4RD/SENS‐0RD (blue) comparisons. The colored dots represent DEGs annotated as involved in detoxification (A), proteolysis (B), and related to fatty acids (C). Gray dots correspond to all other contigs according to their fold‐change and FDR in the three comparisons. The dashed line shows the FDR cutoff with points above the line having an FDR < 0.05. Vertical dashed lines represent the threshold level for 3 fold changes. Heat map (D) of contigs differentially expressed in RES‐0RD/SENS‐0RD (1), RES‐4RD/SENS‐4RD (2), and SENS‐4RD/SENS‐0RD (3) comparisons are represented in map as Log₂FC. Red, blue, and black colors indicate upregulated or downregulated, or unchanged expression, respectively. The colored sidebar legend corresponding to the description of the contigs is displayed at the bottom. CPR, NADPH‐cytochrome P450 reductase; Cyp, Cytochrome P450; Cyt, Cytochrome b5; UGT, UDP‐glycosyltransferase; GST, Glutathione S‐transferase; COX, Cytochrome c oxidase; GGCT, Gamma‐glutamylcyclotransferase.