Diverse sensitivities of TRPA1 from different mosquito species to thermal and chemical stimuli

AbstractTemperature and odors profoundly affect the behavior of animals. Transient receptor potential channel, subfamily A, member 1 (TRPA1) functions as a polymodal nociceptor for sensing both vital environmental cues in insects. Mosquitoes are recognized as disease vectors, and many efforts have been devoted to investigations of their host-seeking behaviors and repellents. However, the physiological characteristics of mosquito TRPA1 have not been systematically studied. We identified multiple alternative splice variants of the TrpA1 gene from Anopheles gambiae, Anopheles stephensi, Aedes aegypti and Culex pipiens pallens mosquitoes. And we performed comparative analyses of the responses of mosquito TRPA1s to heat or chemical stimuli with calcium-imaging and whole-cell patch-clamp methods. Comparison of TRPA1 among four mosquito species from different thermal niches revealed that TRPA1 of Culex pipiens pallens inhabiting the temperate zone had a lower temperature threshold for heat-evoked activation, which was supported by the in vivo heat-avoidance test. Notably, the chemosensitivity of mosquito TRPA1 channels revealed differences not only between variants but also among species. Moreover, we discovered three novel mosquito TRPA1 agonists. Thermal niches selection and evolutionary trajectories significantly affect the functional properties of mosquito TRPA1, which represents a hallmark of the behaviors that may permit the design of improved mosquito control methods.

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MiR-4448 is involved in deltamethrin resistance by targeting CYP4H31 in Culex pipiens pallens

Parasites & Vectors ◽

10.1186/s13071-021-04665-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Xixi Li ◽

Shengli Hu ◽

Haitao Yin ◽

Hongbo Zhang ◽

Dan Zhou ◽

...

Keyword(s):

Culex Pipiens ◽

Oral Feeding ◽

Cell Counting ◽

Luciferase Reporter ◽

Culex Pipiens Pallens ◽

Deltamethrin Resistance ◽

Regulatory Functions ◽

Pipiens Pallens

Abstract Background Culex pipiens (Cx. pipiens) complex, which acts as a vector of viruses and is widespread and abundant worldwide, including West Nile virus, Japanese encephalitis virus, and Sindbis virus, can cause serious vector-borne diseases affecting human health. Unfortunately, mosquitoes have developed deltamethrin resistance because of its long-term overuse, representing a major challenge to mosquito control. Understanding the molecular regulatory mechanisms of resistance is vital to control mosquitoes. MicroRNAs (miRNAs) are short non-coding RNAs that have been demonstrated to be important regulators of gene expression across a wide variety of organisms, which might function in mosquito deltamethrin resistance. In the present study, we aimed to investigate the regulatory functions of miR-4448 and CYP4H31 in the formation of insecticidal resistance in mosquito Culex pipiens pallens. Methods We used quantitative real-time reverse transcription PCR to measure miR-4448 and CYP4H31 (encoding a cytochrome P450) expression levels. The regulatory functions of miR-4448 and CYP4H31 were assessed using dual-luciferase reporter assays. Then, oral feeding, RNA interference, and the American Centers for Disease Control and Prevention bottle bioassay were used to determine miR-4448’s association with deltamethrin resistance by targeting CYP4H31in vivo. Cell Counting Kit-8 (CCK-8) was also used to detect the viability of pIB/V5-His-CYP4H31-transfected C6/36 cells after deltamethrin treatment in vitro. Results MiR-4448 was downregulated in the deltamethrin-resistant strain (DR strain), whereas CYP4H31 was downregulated in deltamethrin-susceptible strain. CYP4H31 expression was downregulated by miR-4448 recognizing and binding to its 3′ untranslated region. Functional verification experiments showed that miR-4448 overexpression resulted in lower expression of CYP4H31. The mortality of miR-4448 mimic-injected DR strain mosquitoes was higher than that of the controls. CCK-8 assays showed that CYP4H31 decreased cellular resistance to deltamethrin in vitro and the mortality of the DR strain increased when CYP4H31 was knocked down in vivo. Conclusions In mosquitoes, miR-4448 participates in deltamethrin resistance by targeting CYP4H31. The results of the present study increase our understanding of deltamethrin resistance mechanisms.

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Microbiota and transcriptome changes of Culex pipiens pallens larvae exposed to Bacillus thuringiensis israelensis

Scientific Reports ◽

10.1038/s41598-021-99733-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ruiling Zhang ◽

Wenjuan Liu ◽

Qian Zhang ◽

Xinyu Zhang ◽

Zhong Zhang

Keyword(s):

Bacillus Thuringiensis ◽

Culex Pipiens ◽

Mosquito Control ◽

Sphingolipid Metabolism ◽

Glutathione Metabolism ◽

Control Group ◽

Culex Pipiens Pallens ◽

Bacillus Thuringiensis Israelensis ◽

Epidemic Encephalitis ◽

Pipiens Pallens

AbstractCulex pipiens pallens is an important vector of lymphatic filariasis and epidemic encephalitis. Mosquito control is the main strategy used for the prevention of mosquito-borne diseases. Bacillus thuringiensis israelensis (Bti) is an entomopathogenic bacterium widely used in mosquito control. In this study, we profiled the microbiota and transcriptional response of the larvae of Cx. pipiens pallens exposed to different concentrations of Bti. The results demonstrated that Bti induced a significant effect on both the microbiota and gene expression of Cx. pipiens pallens. Compared to the control group, the predominant bacteria changed from Actinobacteria to Firmicutes, and with increase in the concentration of Bti, the abundance of Actinobacteria was gradually reduced. Similar changes were also detected at the genus level, where Bacillus replaced Microbacterium, becoming the predominant genus in Bti-exposed groups. Furthermore, alpha diversity analysis indicated that Bti exposure changed the diversity of the microbota, possibly because the dysbiosis caused by the Bti infection inhibits some bacteria and provides opportunities to other opportunistic taxa. Pathway analysis revealed significant enhancement for processes associated with sphingolipid metabolism, glutathione metabolism and glycerophospholipid metabolism between all Bti-exposed groups and control group. Additionally, genes associated with the Toll and Imd signaling pathway were found to be notably upregulated. Bti infection significantly changed the bacterial community of larvae of Cx. pipiens pallens.

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Transcription factor FTZ-F1 regulates mosquito cuticular protein CPLCG5 conferring resistance to pyrethroids in Culex pipiens pallens

Parasites & Vectors ◽

10.1186/s13071-020-04383-w ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Yang Xu ◽

Xiaoshan Yang ◽

Xiaohong Sun ◽

Xixi Li ◽

Zhihan Liu ◽

...

Keyword(s):

Culex Pipiens ◽

Pyrethroid Resistance ◽

Luciferase Reporter ◽

Cuticular Protein ◽

Culex Pipiens Pallens ◽

Pyrethroid Insecticides ◽

Cuticle Thickness ◽

Pipiens Pallens ◽

Cuticular Resistance

Abstract Background Culex pipiens pallens poses a serious threat to human health because of its widespread distribution, high carrier capacity for several arboviruses, frequent human-biting, and growth in urban environments. Pyrethroid insecticides have been mainly used to control adult Cx. pipiens pallens during outbreaks of mosquito-borne diseases. Unfortunately, mosquitoes have developed resistance, rendering the insecticides ineffective. Cuticular resistance is the primary mechanism of pyrethroid resistance. Previously, we revealed that cuticular protein of low complexity CPLCG5 is a major cuticular protein associated with deltamethrin resistance in Cx. pipiens pallens, which is enriched in the cuticle of mosquitoes’ legs and participates in pyrethroid resistance by forming a rigid matrix. However, the regulatory mechanisms of its transcription remain unknown. Results First, qRT-PCR analysis revealed that the expression of FTZ-F1 (encoding Fushi tarazu-Factor 1) was ~ 1.8-fold higher in the deltamethrin-resistant (DR) than deltamethrin-susceptible (DS) strains at 24 h post-eclosion (PE) and ~ 2.2-fold higher in the DR strain than in the DS strain at 48 h PE. CPLCG5 and FTZ-F1 were co-expressed in the legs, indicating that they might play an essential role in the legs. Dual luciferase reporter assays and EMSA (electrophoretic mobility shift experiments) revealed that FTZ-F1 regulates the transcription of CPLCG5 by binding to the FTZ-F1 response element (− 870/− 864). Lastly, knockdown of FTZ-F1 not only affected CPLCG5 expression but also altered the cuticle thickness and structure of the legs, increasing the susceptibility of the mosquitoes to deltamethrin in vivo. Conclusions The results revealed that FTZ-F1 regulates the expression of CPLCG5 by binding to the CPLCG5 promoter region, altering cuticle thickness and structure, and increasing the susceptibility of mosquitoes to deltamethrin in vivo. This study revealed part of the mechanism of cuticular resistance, providing a deeper understanding of insecticide resistance.

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

Parasites & Vectors ◽

10.1186/s13071-021-05033-5 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Xixi Li ◽

Shengli Hu ◽

Hongbo Zhang ◽

Haitao Yin ◽

Huan Wang ◽

...

Keyword(s):

Insecticide Resistance ◽

Culex Pipiens ◽

Oral Feeding ◽

Luciferase Reporter ◽

Culex Pipiens Pallens ◽

Mosquito Mortality ◽

Deltamethrin Resistance ◽

Pipiens Pallens

Abstract 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

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Comparative proteomics reveals mechanisms that underlie insecticide resistance in Culex pipiens pallens Coquillett

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009237 ◽

2021 ◽

Vol 15 (3) ◽

pp. e0009237

Author(s):

Chongxing Zhang ◽

Qiqi Shi ◽

Tao Li ◽

Peng Cheng ◽

Xiuxia Guo ◽

...

Keyword(s):

Cytochrome P450 ◽

Insecticide Resistance ◽

Culex Pipiens ◽

Mosquito Control ◽

Expression Profiles ◽

Susceptible Strain ◽

Culex Pipiens Pallens ◽

Metabolic Resistance ◽

Tandem Mass Spectrometric ◽

Pipiens Pallens

Mosquito control based on chemical insecticides is considered as an important element of the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. In contrast to target site resistance, other mechanisms are far from being fully understood. Global protein profiles among cypermethrin-resistant, propoxur-resistant, dimethyl-dichloro-vinyl-phosphate-resistant and susceptible strain of Culex pipiens pallens were obtained and proteomic differences were evaluated by using isobaric tags for relative and absolute quantification labeling coupled with liquid chromatography/tandem mass spectrometric analysis. A susceptible strain of Culex pipiens pallens showed elevated resistance levels after 25 generations of insecticide selection, through iTRAQ data analysis detected 2,502 proteins, of which 1,513 were differentially expressed in insecticide-selected strains compared to the susceptible strain. Finally, midgut differential protein expression profiles were analyzed, and 62 proteins were selected for verification of differential expression using iTRAQ and parallel reaction monitoring strategy, respectively. iTRAQ profiles of adaptation selection to three insecticide strains combined with midgut profiles revealed that multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Culex pipiens pallens. Significant molecular resources were developed for Culex pipiens pallens, potential candidates were involved in metabolic resistance and reducing penetration or sequestering insecticide. Future research that is targeted towards RNA interference of the identified metabolic targets, such as cuticular proteins, cytochrome P450s, glutathione S-transferases and ribosomal proteins proteins and biological pathways (drug metabolism—cytochrome P450, metabolism of xenobiotics by cytochrome P450, oxidative phosphorylation, ribosome) could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Culex pipiens pallens.

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Naturally Occurring Incompatibilities between Different Culex pipiens pallens Populations as the Basis of Potential Mosquito Control Measures

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0002030 ◽

2013 ◽

Vol 7 (1) ◽

pp. e2030 ◽

Cited By ~ 16

Author(s):

Lin Chen ◽

Changliang Zhu ◽

Donghui Zhang

Keyword(s):

Culex Pipiens ◽

Mosquito Control ◽

Control Measures ◽

Culex Pipiens Pallens ◽

Naturally Occurring ◽

Pipiens Pallens

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Coagulation-Flocculation: A potential Application for Mosquito Larval Source Management (LSM) with No Insecticide Resistance

10.21203/rs.3.rs-36948/v1 ◽

2020 ◽

Author(s):

XinRan Li ◽

Minsheng Huang ◽

PeiEn Leng ◽

XinYu Lu ◽

Bing Xiao

Keyword(s):

Insecticide Resistance ◽

Culex Pipiens ◽

Control Method ◽

Mosquito Control ◽

Tap Water ◽

Oviposition Preference ◽

Mosquito Larvae ◽

Culex Pipiens Pallens ◽

Lethal Time ◽

Pipiens Pallens

Abstract Background: Mosquito control is an essential step to eliminate mosquito-borne diseases. Larval mosquitoes have a more limited home range and lower resistance to adverse environment than adults, thus can be ideal targets for vector control in some cases. Coagulation-flocculation technology, which could be used for water treatment in breeding sites of several vector mosquito species, can significantly change both the distribution of organic particles and surface sediment characteristics in water environment. The aim of this study was to explore the effect, principle and possibility of using coagulation-flocculation technology in immature mosquitoes killing.Method: In laboratory, chlorine-free tap water was treated with Poly Aluminum Chloride (PACl, sewage treatment using). The oviposition preference of gravid Culex pipiens pallens, the hatching of mosquito eggs and the survival amount of mosquito larvae were observed, and the pupa amounts were recorded each day.Results: Coagulation-flocculation treatment could improve the oviposition preference of Culex pipiens pallens to some extent (compared with ordinary chlorine-free tap water), but not significantly (p=0.345). After treatment, mosquitoes laid eggs in chlorine-free tap water were 31.88% more than those laid in untreated water. Coagulation-flocculation affected the larvae’s survival by physical means: Ⅰ) alum floc layer increases the difficulty of larvae foraging, leads larvae starving to death; (Ⅱ) the little floc particles adhere to the surface of larvae, which stops larvae from floating upward to breathe. As a result, the alum floc layer had a good killing effect on the mosquito larvae, presented the half lethal time (LT50) of 2d, the 90% lethal time (LT90) of 8.7±7.3 ~ 14±4.5 d, and the pupation rate of 0 ~ (6.5±0.5)%, respectively.Conclusions: PACl coagulation-flocculation produced lots of alum flocs, which may attract more gravid mosquitoes for laying eggs, and was shown to be highly active against 1st~2nd instar larvae. The principle of this technology illustrates this method won’t develop insecticide resistance. In this study, coagulation-flocculation technology is considered to be a new potential approach to a sustainable, low-impact and low-cost mosquito control method.

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