In Vitro and In Vivo Validation of CYP6A14 and CYP6N6 Participation in Deltamethrin Metabolic Resistance in Aedes albopictus.

The extensive use of chemical insecticides for public health and agricultural purposes has increased the occurrence and development of insecticide resistance. This study used transcriptome sequencing to screen 10 upregulated metabolic detoxification enzyme genes from Aedes albopictus resistant strains. Of these, CYP6A14 and CYP6N6 were found to be substantially overexpressed in the deltamethrin-induced expression test, indicating their role in deltamethrin resistance in Ae. albopictus. Furthermore, the corresponding 60-kDa recombinant proteins, CYP6A14 and CYP6N6, were successfully expressed using the Escherichia coli expression system. Enzyme activity studies revealed that CYP6A14 (5.84 U/L) and CYP6N6 (6.3 U/L) have cytochrome P450 (CYP450) enzyme activity. In vitro, the metabolic analysis revealed that the recombinant proteins degraded deltamethrin into 1-oleoyl-sn-glycero-3-phosphoethanolamine and 2',2'-dibromo-2'-deoxyguanosine. Subsequently, the CYP450 genes in larvae of Ae. albopictus were silenced by RNA interference technology to study deltamethrin resistance in vivo. The silencing of CYP6A14 and CYP6N6 increased the mortality rate of mosquitoes without affecting their survival time, spawning quantity, hatching rate, and other normal life activities. Altogether, CYP6A14 and CYP6N6 belong to the CYP6 family and mutually increase deltamethrin resistance in Ae. albopictus.

Interactions between the gut micro-community and transcriptome of Culex pipiens pallens under low-temperature stress.

BACKGROUND: Culex pipiens pallens (Diptera: Culicidae) can survive at low temperature for long periods. Understanding the effects of low-temperature stress on the gut microflora and gene expression levels in Cx. pipiens pallens, as well as their correlation, will contribute to the study of the overwintering mechanism of Cx. pipiens pallens. METHODS: The gut bacteria were removed by antibiotic treatment, and the survival of Cx. pipiens pallens under low-temperature stress was observed and compared with the control group. Then, full-length 16S rRNA sequencing and the Illumina HiSeq X Ten sequencing platform were used to evaluate the gut microflora and gene expression levels in Cx. pipiens pallens under low-temperature stress. RESULTS: Under the low-temperature stress of 7 °C, the median survival time of Cx. pipiens pallens in the antibiotic treatment group was significantly shortened by approximately 70% compared to that in the control group. The species diversity index (Shannon, Simpson, Ace, Chao1) of Cx. pipiens pallens decreased under low-temperature stress (7 °C). Non-metric multidimensional scaling (NMDS) analysis divided all the gut samples into two groups: control group and treatment group. Pseudomonas was the dominant taxon identified in the control group, followed by Elizabethkingia and Dyadobacter; in the treatment group, Pseudomonas was the dominant taxon, followed by Aeromonas and Comamonas. Of the 2417 differentially expressed genes (DEGs), 1316 were upregulated, and 1101 were downregulated. Functional GO terms were enriched in 23 biological processes, 20 cellular components and 21 molecular functions. KEGG annotation results showed that most of these genes were related to energy metabolism-related pathways. The results of Pearson's correlation analysis showed a significant correlation between the gut microcommunity at the genus level and several DEGs. CONCLUSIONS: These results suggest that the mechanism of adaptation of Cx. pipiens pallens to low-temperature stress may be the result of interactions between the gut bacterial community and transcriptome.

A symbiotic gut bacterium enhances Aedes albopictus resistance to insecticide.

BACKGROUND: The increasing insecticide resistance of Aedes albopictus puts many countries in Asia and Africa, including China, at great risk of a mosquito-borne virus epidemic. To date, a growing number of researches have focused on the relationship between intestinal symbiotic bacteria and their hosts' resistance to insecticides. This provides a novel aspect to the study of resistant mechanisms. METHODS/FINDINGS: This study reveals significant composition and dynamic changes in the intestinal symbiotic bacteria of Ae. albopictus between the resistant and susceptible strains based on full-length sequencing technology. The relative abundance of Serratia oryzae was significantly higher in the resistance strain than in the susceptible strains; also, the relative abundance of S. oryzae was significantly higher in deltamethrin-induced Ae. albopictus than in their counterpart. These suggested that S. oryzae may be involved in the development of insecticide resistance in Ae. albopictus. To explore the insecticide resistance mechanism, adult mosquitoes were fed with GFP-tagged S. oryzae, which resulted in stable bacterial enrichment in the mosquito gut without affecting the normal physiology, longevity, oviposition, and hatching rates of the host. The resistance measurements were made based on bioassays as per the WHO guidelines. The results showed that the survival rate of S. oryzae-enriched Ae. albopictus was significantly higher than the untreated mosquitoes, indicating the enhanced resistance of S. oryzae-enriched Ae. albopictus. Also, the activities of three metabolic detoxification enzymes in S. oryzae-enriched mosquitoes were increased to varying degrees. Meanwhile, the activity of extracellular enzymes released by S. oryzae was measured, but only carboxylesterase activity was detected. HPLC and UHPLC were respectively used to measure deltamethrin residue concentration and metabolite qualitative analysis, showing that the deltamethrin degradation efficiency of S. oryzae was positively correlated with time and bacterial amount. Deltamethrin was broken down into 1-Oleoyl-2-hydroxy-sn-glycero-3-PE and 2',2'-Dibromo-2'-deoxyguanosine. Transcriptome analysis revealed that 9 cytochrome P450s, 8 GSTs and 7 CarEs genes were significantly upregulated. CONCLUSIONS: S. oryzae can be accumulated into adult Ae. albopictus by artificial feeding, which enhances deltamethrin resistance by inducing the metabolic detoxification genes and autocrine metabolic enzymes. S. oryzae is vertically transmitted in Ae. albopictus population. Importantly, S. oryzae can degrade deltamethrin in vitro, and use deltamethrin as the sole carbon source for their growths. Therefore, in the future, S. oryzae may also be commercially used to break down the residual insecticides in the farmland and lakes to protect the environment.