High Antennal Expression of CYP6K1 and CYP4V2 Participate in the Recognition of Alarm Pheromones by Solenopsis invicta Buren

Abstract

Insects have highly developed olfactory systems in which cytochrome P450s (CYPs) were involved as odor-degrading enzymes throughout the olfactory recognition of odor compounds by insects to avoid continuous stimulation of signaling molecules and thus damage to the olfactory nervous. To understand whether the highly expressed CYPs in the antennae play an olfactory function in Solenopsis invicta worker, in this study, we find six highly expressed antennal CYPs from the transcriptome of S. invicta. Multiple sequence alignment and phylogenetic analysis divided them into two families: the CYP3 family (SinvCYP6K1, SinvCYP6K1-1) and the CYP4 family (SinvCYP4C1, SinvCYP4C1-1, SinvCYP4C1-2, SinvCYP4V2). The expression patterns of these six CYPs were analyzed by RT-qPCR, which revealed that SinvCYP6K1 and SinvCYP4V2 were only highly expressed in the antennae of adult workers. The expression of SinvCYP6K1 and SinvCYP4V2 in workers was markedly diminished after feeding with dsRNA. The electroantennography (EAG) assay demonstrated that the silencing of either SinvCYP6K1 or SinvCYP4V2 resulted in a notable reduction in the EAG response of workers to 2-ethyl-3,6(5)-dimethylpyrazine (EDMP). Furthermore, the trajectory behavior assay showed that the worker's range and speed of movement in response to EDMP significant decreased after the silencing of SinvCYP6K1 and SinvCYP4V2. The findings indicated that both SinvCYP6K1 and SinvCYP4V2 were implicated in the recognition of EDMP by S. invicta.

Keywords: RNA interference; alarm pheromone; cytochrome P450s; electroantennography; red imported fire ant.

Figure 1

Amino acid sequence analysis of CYPs from SinvCYP4C1, SinvCYP4C1-1, SinvCYP4C1-2, SinvCYP4V2, SinvCYP6K1, and SinvCYP6K1-1, and their comparison with CYPs from S. litura, A. mellifera, and D. ponderosae. Black-labeled regions represent cytochrome P450 conserved regions, which include the following patterns: Helix C: WXXXR, Helix I: GXE/DTT/S, Helix: EXXR, Meander: PXXFXPEX/DF, and heme-binding domain: PFXXGXRXCXG/A. Gene bank accession numbers of other insect CYPs utilized for amino acid sequence analysis are presented in Table S5.

Figure 2

Phylogenetic analysis of SinvCYP4C1, SinvCYP4C1-1, SinvCYP4C1-2, SinvCYP4V2, SinvCYP6K1, and SinvCYP6K1-1 in conjunction with CYPs from other insects (blue indicates CYP3 family, green indicates CYP4 family, red indicates mitochondrial CYP family, and yellow indicates CYP2 family). The resulting developmental trees were constructed using the maximum likelihood method, with values indicating percentages based on 1000 replicates. Gene bank accession numbers of other insect CYPs utilized for phylogenetic analysis are presented in Table S5.

Figure 3

Expression pattern of SinvCYPs in antenna, head, thorax, and abdomen of S. invicta. (A) Expression of SinvCYP6K1. (B) Expression of SinvCYP6K1-1. (C) Expression of SinvCYP4C1. (D) Expression of SinvCYP4C1-1. (E) Expression of SinvCYP4C1-2. (F) Expression of SinvCYP4V2. Columns and error bars represent mean ± SD (n = 4). Different letters above bars indicate significant differences between different tissues according to Tukey’s HSD test (one-way ANOVA, p < 0.05).

Figure 4

Expression patterns of SinvCYP6K1 and SinvCYP4V2 at different developmental stages. (A) Expression of SinvCYP6K1. (B) Expression of SinvCYP4V2. Mean ± SD (n = 4). Different letters above bars indicate significant differences between different developmental stage according to Tukey’s HSD test (one-way ANOVA, p < 0.05).

Figure 5

Interference efficiency of SinvCYP6K1 and SinvCYP4V2 following 12, 24, and 36 h of feeding specific dsRNAs. (A) Expression of SinvCYP6K1 following feeding of dsCYP6K1. (B) Expression of SinvCYP4V2 following feeding of dsCYP4V2. (C) Expression of SinvCYP6K1 after mixed feeding of dsCYP6K1 and dsCYP4V2. (D) Expression of SinvCYP4V2 after mixed feeding of dsCYP6K1 and dsCYP4V2. Data are means ± SD of 3 biological repeats. Asterisks (*) on bars indicate significant differences (ns, p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test).

Figure 6

Response of workers to varying concentrations of EDMP following 24 h of dsRNA feeding. Concentrations tested were 0.1, 1.0, and 10.0 μg/μL. Columns and error bars represent mean ± SD (n = 3). Different letters above bars indicate significant differences between different tissues according to Tukey’s HSD test (one-way ANOVA, p < 0.05).

Figure 7

Behavioral response of workers to exposure to EDMP was assessed following a 24 h feeding period with double-stranded RNA (dsRNA). (A) Trajectory thermograms of S. invicta fed dsGFP, dsCYP6K1, dsCYP4V2, and dsCYP6K1 + 4V2 for 24 h in response to 0.1, 1.0, and 10.0 μg/μL EDMP. (B) Movement velocities of worker ants in response to 0.1, 1.0, and 10.0 μg/μL EDMP after 24 h of feeding with dsGFP, dsCYP6k1, dsCYP4V2, and dsCYP6K1 + 4V2. Columns and error bars represent mean ± SD (n = 3). Different letters above bars indicate significant differences between different tissues according to Tukey’s HSD test (one-way ANOVA, p < 0.05).