MiR-279-3p regulates deltamethrin resistance through CYP325BB1 in Culex pipiens pallens

Background The overuse of insecticides to control insect vectors has promoted extensive insecticide resistance in mosquitoes. In this study, the functions of microRNA (miR)-279-3p and its target CYP325BB1 in the regulation of deltamethrin resistance in Culex pipiens pallens was investigated. Methods Quantitative real-time reverse transcription PCR was used to detect the expression levels of miR-279-3p and CYP325BB1. Then, the dual-luciferase reporter assay system, RNA interference, CDC bottle bioassay and Cell Counting Kit-8 (CCK-8) assay were used to explore the roles of these molecules in deltamethrin resistance both in vivo and in vitro. Results The expression patterns of miR-279-3p and CYP325BB1 were compared between deltamethrin-sensitive (DS-strain) and deltamethrin-resistant (DR-strain) mosquitoes. Luciferase activity was downregulated by miR-279-3p, the effect of which was ablated by a mutation of the putative binding site for CYP325BB1. In DR-strain mosquitoes, the expression of miR-279-3p was increased by microinjection and oral feeding of miR-279-3p agomir (mimic). CYP325BB1 mRNA levels were downregulated, which resulted in a higher mortality of the mosquitoes in miR-279-3p mimic-treated groups. In the DS-strain mosquitoes, microinjection of a miR-279-3p inhibitor decreased miR-279-3p expression, whereas the expression of CYP325BB1 was increased; the mortality of these mosquitoes decreased significantly. In addition, overexpression of pIB/V5-His-CYP325BB1 changed the sensitivity of C6/36 cells to deltamethrin in vitro. Also in DR-strain mosquitoes, downregulation of CYP325BB1 expression by microinjection of si-CYP325BB1 increased mosquito mortality in vivo. Conclusions These findings provide empirical evidence of the involvement of miRNAs in the regulation of insecticide resistance and indicate that miR-279-3p suppresses the expression of CYP325BB1, which in turn decreases deltamethrin resistance, resulting in increased mosquito mortality. Taken together, the results provide important information for use in the development of future mosquito control strategies. Graphical abstract

Key Role of Mitochondrial Mutation Leu107Ser (COX1) in Deltamethrin Resistance in Salmon Lice (Lepeophtheirus Salmonis)

Deltamethrin (DTM) is used to treat Atlantic salmon (Salmon salar) against salmon lice (Lepeophtheirus salmonis) infestations. However, development of DTM resistance has been reported from North Atlantic L. salmonis populations, in which resistance is associated with mitochondrial (mtDNA) mutations. This study investigated the relationship between DTM resistance and mtDNA single nucleotide polymorphisms (SNPs). A total of 188 L. salmonis collected from Scottish aquaculture sites were assessed using DTM bioassays and genotyped at 18 SNP loci. Genotyping further included archived parasites of known DTM susceptibility status. The results identified eleven mtDNA haplotypes, three of which were associated with DTM resistance. Phylogenetic analyses of haplotypes suggested multiple origins of DTM resistance. L. salmonis laboratory strains IoA-00 and IoA-10 showed similarly high levels (~100-fold) of DTM resistance in bioassays. Both strains differed strongly in mtDNA haplotype, but shared the missense mutation Leu107Ser in the mitochondrial gene cytochrome c oxidase subunit 1 (COX1), which was detected in all further DTM resistant L. salmonis isolates assessed. In crossing experiments with a DTM-susceptible strains, maternal inheritance of DTM resistance is apparent with both IoA-10 (this study) and IoA-02 (earlier reports). We conclude that Leu107Ser (COX1) is a main genetic determinant of DTM resistance in L. salmonis.

Insecticides Susceptibility Status of Malaria Vectors in a High Malaria Endemic Tribal District Gadchiroli (Maharashtra) of India

Background and Objective: The current study was undertaken to determine insecticide susceptibility of malaria vectors in various villages of high malaria endemic PHCs of Gadchiroli district of Maharashtra. Methods: Adult malaria vectors were collected from the human dwellings/ cattle sheds of 156 villages of 18 malaria endemic PHCs. Susceptibility tests were carried out for different insecticides against An. culicifacies and An. fluviatilis mosquitoes as per the World Health Organization (WHO) procedure. Cone bioassays were also done to assess the quality and efficacy of indoor residual spray. Results:An. fluviatilis could be collected from 23 villages only and all the populations were fully susceptible to synthetic pyrethroid (deltamethrin) while being tolerant to organophosphorous (malathion). Susceptibility of An. culicifacies from 156 villages indicated that only 3 populations of An. culicifacies were resistant to deltamethrin while 57 populations were fully susceptible and other 96 populations were tolerant to deltamethrin. Resistance was recorded in 25 populations of An. culicifacies against malathion and 30 populations were tolerant to malathion insecticide. Remaining populations of An. fluviatilis and An. culicifacies were highly resistant to organochlorine. Results of cone bioassay revealed the mortality ranged from 32.5-51.1% on cemented and 27.5-43.3% on the mud wall sprayed with lambda cyhalothrin. Conclusion: The current study indicates that resistance has developed to synthetic pyrethroids in the major malaria vector An. culicifacies. Therefore, there is an urgent need for the evaluation of new insecticide molecules for better control of malaria vectors.

Knockdown Resistance (kdr) Mutations I1532T and F1534S Were Identified in Aedes albopictus Field Populations in Zhejiang Province, Central China

Aedes albopictus is the only vector that can transmit the dengue virus in Zhejiang Province, central China, and it can develop insecticide resistance due to long-term exposure to pyrethroids. The presence of knockdown resistance (kdr) mutations is one of the mechanisms responsible for pyrethroid resistance, and has been reported in some Ae. albopictus populations in southern China. However, little is known about the DNA diversity of the voltage-gated sodium channel (VGSC) gene in Ae. albopictus populations in central China. Four Ae. albopictus field populations were collected, in Yiwu (YW), Quzhou (QZ), Wenzhou (WZ), and Jiaxing (JX) from Zhejiang Province, central China. The susceptibility of Ae. albopictus adults to three pyrethroids (beta-cypermethrin, deltamethrin, and permethrin) was tested using the WHO tube assay, and Kdr mutations were identified via PCR and sequencing. The relationship between kdr mutations and pyrethroid phenotypes was also analyzed. Of the four populations, none was sensitive to any pyrethroid tested, and the YW population showed the strongest pyrethroid resistance. Non-synonymous kdr mutations were detected in codons 1532 and 1534, domain III. At codon 1534, one mutant allele, TCC(S), was detected in the four populations with a frequency of 42.08%, while at codon 1532, one mutant allele, ACC(T), was detected in the JX and QZ populations, with frequencies of 4.22 and 3.03%, respectively. The F1534S mutant allele was positively correlated with both beta-cypermethrin and deltamethrin resistance phenotypes (OR > 1, P < 0.05), whereas the I1532T mutant allele was possibly negatively correlated with beta-cypermethrin, deltamethrin, and permethrin resistance phenotypes (OR < 1, P > 0.05). In conclusion, resistance and resistance mutations regarding to three pyrethroids are already present in the Ae. Albopictus populations from Zhejiang, central China, which prompts the need to use non-insecticide-based methods of insect control.

Impact of deltamethrin-resistance in Aedes albopictus on its fitness cost and vector competence

Background Aedes albopictus is one of the most invasive species in the world as well as the important vector for mosquito-borne diseases such as dengue fever, chikungunya fever and zika virus disease. Chemical control of mosquitoes is an effective method to control mosquito-borne diseases, however, the wide and improper application of insecticides for vector control has led to serious resistance problems. At present, there have been many reports on the resistance to pyrethroid insecticides in vector mosquitoes including deltamethrin to Aedes albopictus. However, the fitness cost and vector competence of deltamethrin resistant Aedes albopictus remain unknown. To understand the impact of insecticide resistant mosquito is of great significance for the prevention and control mosquitoes and mosquito-borne diseases. Methodology/Principal findings A laboratory resistant strain (Lab-R) of Aedes albopictus was established by deltamethrin insecticide selecting from the laboratory susceptible strain (Lab-S). The life table between the two strains were comparatively analyzed. The average development time of Lab-R and Lab-S in larvae was 9.7 days and 8.2 days (P < 0.005), and in pupae was 2.0 days and 1.8 days respectively (P > 0.05), indicating that deltamethrin resistance prolongs the larval development time of resistant mosquitoes. The average survival time of resistant adults was significantly shorter than that of susceptible adults, while the body weight of resistant female adults was significantly higher than that of the susceptible females. We also compared the vector competence for dengue virus type-2 (DENV-2) between the two strains via RT-qPCR. Considering the results of infection rate (IR) and virus load, there was no difference between the two strains during the early period of infection (4, 7, 10 day post infection (dpi)). However, in the later period of infection (14 dpi), IR and virus load in heads, salivary glands and ovaries of the resistant mosquitoes were significantly lower than those of the susceptible strain (IR of heads, salivary glands and ovaries: P < 0.005; virus load in heads and salivary glands: P < 0.05; virus load in ovaries: P < 0.001). And then, fourteen days after the DENV-2-infectious blood meal, females of the susceptible and resistant strains were allow to bite 5-day-old suckling mice. Both stains of mosquito can transmit DENV-2 to mice, but the onset of viremia was later in the mice biting by resistant group as well as lower virus copies in serum and brains, suggesting that the horizontal transmission of the resistant strain is lower than the susceptible strain. Meanwhile, we also detected IR of egg pools of the two strains on 14 dpi and found that the resistant strain were less capable of vertical transmission than susceptible mosquitoes. In addition, the average survival time of the resistant females infected with DENV-2 was 16 days, which was the shortest among the four groups of female mosquitoes, suggesting that deltamethrin resistance would shorten the life span of female Aedes albopictus infected with DENV-2. Conclusions/Significance As Aedes albopictus developing high resistance to deltamethrin, the resistance prolonged the growth and development of larvae, shorten the life span of adults, as well as reduced the vector competence of resistant Aedes albopictus for DENV-2. It can be concluded that the resistance to deltamethrin in Aedes albopictus is a double-edged sword, which not only endow the mosquito survive under the pressure of insecticide, but also increase the fitness cost and decrease its vector competence. However, Aedes albopictus resistant to deltamethrin can still complete the external incubation period and transmit dengue virus, which remains a potential vector for dengue virus transmission and becomes a threat to public health. Therefore, we should pay high attention for the problem of insecticide resistance so that to better prevent and control mosquito-borne diseases.

Overabundance of Asaia and Serratia bacteria is associated with deltamethrin insecticide susceptibility in Anopheles coluzzii from Agboville, Côte d’Ivoire

Background Insecticide resistance among mosquito species is now a pervasive phenomenon, which threatens to jeopardise global malaria vector control efforts. Evidence of links between the mosquito microbiota and insecticide resistance is emerging, with significant enrichment of insecticide degrading bacteria and enzymes in resistant populations. Using 16S rRNA amplicon sequencing, we characterised and compared the microbiota of Anopheles (An.) coluzzii in relation to their deltamethrin resistance and exposure profiles. Results Comparisons between 2-3 day old deltamethrin resistant and susceptible mosquitoes, demonstrated significant differences in microbiota diversity (PERMANOVA, pseudo-F = 19.44, p=0.0015). Ochrobactrum, Lysinibacillus and Stenotrophomonas genera, each of which comprised insecticide degrading species, were significantly enriched in resistant mosquitoes. Susceptible mosquitoes had a significant reduction in alpha diversity compared to resistant individuals (Shannon index: H=13.91, q=0.0003, Faith’s phylogenetic diversity: H=6.68, q=0.01), with Asaia and Serratia dominating microbial profiles. There was no significant difference in deltamethrin exposed and unexposed 5-6 day old individuals, suggesting that insecticide exposure had minimal impact on microbial composition. Serratia and Asaia were also dominant in 5-6 day old mosquitoes, regardless of exposure or phenotype, and had reduced microbial diversity compared with 2-3 day old mosquitoes. Conclusions Our findings revealed significant alterations of An. coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for insecticide resistance surveillance. qPCR detection of Serratia and Asaia was consistent with 16S rRNA sequencing, suggesting that population level field screening of the bacterial microbiota may be feasibly integrated into wider resistance monitoring if reliable and reproducible markers associated with phenotype can be identified.

Overabundance of Asaia and Serratia bacteria is associated with deltamethrin insecticide susceptibiltiy in Anopheles coluzzii from Agboville, Cote d'Ivoire

Background: Insecticide resistance among mosquito species is now a pervasive phenomenon, which threatens to jeopardise global malaria vector control efforts. Evidence of links between the mosquito microbiota and insecticide resistance is emerging, with significant enrichment of insecticide degrading bacteria and enzymes in resistant populations. Using 16S rRNA amplicon sequencing, we characterised and compared the microbiota of Anopheles (An.) coluzzii in relation to their deltamethrin resistance and exposure profiles. Results: Comparisons between 2-3 day old deltamethrin resistant and susceptible mosquitoes, demonstrated significant differences in microbiota diversity (PERMANOVA, pseudo-F = 19.44, p=0.0015). Ochrobactrum, Lysinibacillus and Stenotrophomonas genera, each of which comprised insecticide degrading species, were significantly enriched in resistant mosquitoes. Susceptible mosquitoes had a significant reduction in alpha diversity compared to resistant individuals (Shannon index: H=13.91, q=0.0003, Faiths phylogenetic diversity: H=6.68, q=0.01), with Asaia and Serratia dominating microbial profiles. There was no significant difference in deltamethrin exposed and unexposed 5-6 day old individuals, suggesting that insecticide exposure had minimal impact on microbial composition. Serratia and Asaia were also dominant in 5-6 day old mosquitoes, regardless of exposure or phenotype, and had reduced microbial diversity compared with 2-3 day old mosquitoes. Conclusions: Our findings revealed significant alterations of An. coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for insecticide resistance surveillance. qPCR detection of Serratia and Asaia was consistent with 16S rRNA sequencing, suggesting that population level field screening of the bacterial microbiota may be feasibly integrated into wider resistance monitoring if reliable and reproducible markers associated with phenotype can be identified.