Detection and persistence of environmental DNA (eDNA) of a vector mosquito, Culex pipiens pallens

Introduction: Preventing mosquito-borne infectious diseases requires that vector mosquitoes are monitored and controlled. Targeting immature mosquitoes (eggs, larvae, and pupae), which have less mobility than adults, is an effective management approach. However, conducting these surveys is often difficult due to the limitations of morphological classification and survey costs. The application of environmental DNA (eDNA) analysis can solve these issues because it allows easy estimation of species distribution and morphology-independent species identification. Although a few previous studies have reported mosquito eDNA detection, there is a gap in knowledge regarding the dynamics of mosquito eDNA during the developmental stages of immature mosquitoes. Methods: We used Culex pipiens pallens, a vector of West Nile fever, as a model species. First, we developed a species-specific detection assay and confirmed its specificity using in silico and in vitro tests. Next, we conducted laboratory experiments using breeding tanks. Water samples were collected at each developmental stage. In addition, water samples were collected daily until the seventh day after emergence from the pupae. We quantified eDNA using real-time PCR with the developed assay to investigate the dynamics of mosquito eDNA. Results: The specificity of the developed assay was confirmed by in silico and in vitro tests. Mosquito eDNA was detected at all developmental stages and detected up to seven days after emergence of pupae. In particular, high concentrations of eDNA were detected immediately after hatching from eggs and after emergence from pupae. Highly frequent positive eDNA signals were continuously detected between egg hatching and pupa hatching. Conclusions: Mosquito eDNA was detected immediately after the eggs were introduced, and eDNA-positive detections continued until pupae emergence, suggesting that eDNA analysis is useful for monitoring mosquito larvae. The results show that eDNA analysis provides valuable information about the water sources inhabited by immature mosquitoes in mosquito control. In the future, monitoring immature mosquitoes using eDNA analysis will aid in preventing mosquito-borne infectious diseases.

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

Culex 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.

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

CPR63 Promotes Pyrethroid Resistance by Increasing Cuticle Thickness in Culex Pipiens Pallens

The cuticle protein (CP) encoded by CPR63 plays a role in deltamethrin resistance in Culex pipiens pallens. Herein, we investigated the distribution of CPR63 transcripts in this organism, and revealed high expression levels in legs and wings. Furthermore, expression of CPR63 in the legs of deltamethrin-resistant (DR) strains were 1.79-fold higher than in deltamethrin-susceptible (DS) strains. Cuticle analysis of small interfering RNA (siRNA) groups by scanning electron microscopy (SEM) revealed a significantly thinner procuticle of the tarsi in the siCPR63 group than the siNC (negative control (group). Transmission electron microscopy (TEM) revealed that the procuticle, exocuticle and endocuticle thickness of the tarsi were significantly thinner in the siCPR63 group than the siNC group. Our results illuminate the resistance mechanism of CPRs and demonstrate that CPR63 contributes to the resistance phenotype by thickening the cuticle and substantially reducing uptake of insecticides.

Response of Culex pipiens pallens to Visual and Olfactory Stimuli from a Mosquito Trap

ABSTRACT This study examined Culex pipiens pallens responses to different combinations of colors and chemicals employed via a mosquito trap under semifield conditions. Our results indicated that Cx. p. pallens has color and chemical concentration preferences. Culex p. pallens had a 38.0% greater response to white than black color treated traps. Further, Cx. p. pallens showed differences in olfactory attraction depending on the chemical and concentration. Culex p. pallens was 107.6% more attracted to traps employing 500 ppm ammonia than control (i.e., unscented). Similarly, Cx. p. pallens was 117.5%, 128.8%, and 140.3% more attracted to traps employing, respectively, 1,000, 10,000, and 20,000 ppm of ammonia hydrogen carbonate compared to controls. And the response to lactic acid showed that Cx. p. pallens was most attracted to concentrations of 100 and 500 ppm (135.7% and 142.9%, respectively) compared to controls.

Repellency of Veratraldehyde (3,4-Dimethoxy Benzaldehyde) against Mosquito Females and Tick Nymphs

Arthropod-borne infectious diseases cause many deaths and a major economic burden worldwide. Repellents play an important role in protecting people from infectious biting arthropods. The repellency of veratraldehyde, a known food additive, and the WJ-1041 formulation containing 10% veratraldehyde was tested against Aedes albopictus and Culex pipiens pallens females and Haemaphysalis longicornis nymphs using arm-in-cage, indoor or filter paper tests. Veratraldehyde exhibited repellency similar to or lower than that of n,n-diethyl-meta-toluamide (DEET) against A. albopictus, but in H. longicornis, the activity of veratraldehyde was better than that of DEET. The repellency of the 10% veratraldehyde solution was comparable to that of 20% DEET against the two mosquitoes. When comparing repellency between the WJ-1041 formulation (10% veratraldehyde) and 10% DEET against C. pipiens pallens, A. Albopictus and H. longicornis, the two showed similar repellency and complete protection time (CPT) values. However, there was a small difference depending on the tested insects. The absorption of veratraldehyde via skin was minimal, if at all. The pharmacokinetic parameters (Cmax and Tmax) of veratraldehyde in blood samples of rats were not different from those of the control group. Based on these results, veratraldehyde has high potential to be commercialized as a repellent agent against infectious disease-borne pests in the near future.

Physiological characterization of chitin synthase A responsible for the biosynthesis of cuticle chitin in Culex pipiens pallens (Diptera: Culicidae)

Background The pathogens transmitted by mosquitoes to humans and animals cause several emerging and resurgent infectious diseases. Increasing insecticide resistance requires rational action to control the target vector population. Chitin is indispensable for insect growth and development and absent from vertebrates and higher plants. Chitin synthase A (CHSA) is a crucial enzyme in chitin synthesis; therefore, identifying and characterizing how CHSA determines chitin content may contribute to the development of novel vector control strategies. Results The injection of small interfering RNA targeting CHSA (siCHSA) to knockdown CHSA transcripts in larval, pupal and adult stages of Culex pipiens pallens resulted in the appearance of different lethal phenotypes. When larval and pupal stages were injected with siCHSA, CHSA knockdown prevented larval molting, pupation and adult eclosion, and affected the production of chitin and chitin degradation, which resulted in an ecdysis defect phenotype of mosquitoes. When siCHSA was injected into mosquitoes in the adult stage, CHSA knockdown also affected the laminar organization of the mesoderm and the formation of pseudo-orthogonal patterns of the large fibers of the endoderm. Conclusion We provide a systematic and comprehensive description of the effects of CHSA on morphogenesis and metamorphosis. The results show that CHSA not only affects chitin synthesis during molting, but also might be involved in chitin degradation. Our results further show that CHSA is important for the structural integrity of the adult mosquito cuticle. Graphic abstract

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

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.

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

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.