Mutations and intron polymorphisms in voltage-gated sodium channel genes of different geographic populations of Culex pipiens pallens/Culex pipiens quinquefasciatus in China.

BACKGROUND: Culex pipiens pallens and Culex pipiens quinquefasciatus are the dominant species of Culex mosquitoes in China and important disease vectors. Long-term use of insecticides can cause mutations in the voltage-gated sodium channel (vgsc) gene of mosquitoes, but little is known about the current status and evolutionary origins of vgsc gene in different geographic populations. Therefore, this study aimed to determine the current status of vgsc genes in Cx. p. pallens and Cx. p. quinquefasciatus in China and to investigate the evolutionary inheritance of neighboring downstream introns of the vgsc gene to determine the impact of insecticides on long-term evolution. METHODS: Sampling was conducted from July to September 2021 in representative habitats of 22 provincial-level administrative divisions in China. Genomic DNA was extracted from 1308 mosquitoes, the IIS6 fragment of the vgsc gene on the nerve cell membrane was amplified using polymerase chain reaction, and the sequence was used to evaluate allele frequency and knockdown resistance (kdr) frequency. MEGA 11 was used to construct neighbor-joining (NJ) tree. PopART was used to build a TCS network. RESULTS: There were 6 alleles and 6 genotypes at the L1014 locus, which included the wild-type alleles TTA/L and CTA/L and the mutant alleles TTT/F, TTC/F, TCT/S and TCA/S. The geographic populations with a kdr frequency less than 20.00% were mainly concentrated in the regions north of 38° N, and the geographic populations with a kdr frequency greater than 80.00% were concentrated in the regions south of 30° N. kdr frequency increased with decreasing latitude. And within the same latitude, the frequency of kdr in large cities is relatively high. Mutations were correlated with the number of introns. The mutant allele TCA/S has only one intron, the mutant allele TTT/F has three introns, and the wild-type allele TTA/L has 17 introns. CONCLUSIONS: Cx. p. pallens and Cx. p. quinquefasciatus have developed resistance to insecticides in most regions of China. The neighboring downstream introns of the vgsc gene gradually decreased to one intron with the mutation of the vgsc gene. Mutations may originate from multiple mutation events rather than from a single origin, and populations lacking mutations may be genetically isolated.

Monitoring density of mosquitoes and resistance of major vector mosquitoes to insecticides in Zigong, Sichuan / 中国热带医学

@#Abstract: Objective 　To investigate the population densities composition and seasonal change of mosquitoes, and resistance of Culex pipiens quinquefasciatus and Aedes albopictus several commonly used insecticides in Zigong, so as to provide a theoretical basis for guiding the scientific rational selection and use of chemical insecticides, as well as the prevention and control of mosquito-borne diseases. Methods　The surveillance data on mosquitoes by traps were collected from 2018 to 2020 in Zigong. The wild Culex pipiens quinquefasciatus and Aedes albopictus were collected throughout the city and reared to F1 generation in lab. The resistance of adult Culex pipiens quinquefasciatus and Aedes albopictus to these commonly insecticides was detected by contact tube method recommended. The statistical analysis on the monitoring results was used by Excel 2010. Results　The annual average mosquito densities of monitoring sites were 1.11, 10.71, and 4.81 mosquitoes/(lamp·h), respectively from 2018 to 2020. The seasonal density of mosquitoes showed a single peak curve distribution throughout the year from 2018 to 2020 in Zigong, with the highest in July. There were obvious differences in mosquito density between different habitats, with the highest density of mosquitoes in barn. The 24 h mortality of Aedes albopictus and Culex pipiens quinquefasciatus to beta-cypermethrin, lambda-cyhalothrin, deltamethrin, permethrin, malathion, fenitrothion, chlorpyrifos, propoxur, and bendiocarb were 70.71% and 3.85%, 26.53% and 1.68%, 19.79% and 1.94%, 40.80% and 0.81%, 97.98% and 3.85%, 100.00% and 7.52%, 100.00% and 66.34%, 96.15% and 33.60%, 100.00% and 71.43%, respectively. The adult Aedes albopictus larvae in Zigong was sensitive to fenitrothion, chlorpyrifos, bendiocarb, showed resistance to beta-cypermethrin, lambda-cyhalothrin, deltamethrin, permethrin, and showed resistance dubiously to malathion and propoxur. The adult Culex pipiens quinquefasciatus larvae in Zigong have developed certain resistance to these commonly insecticides. Conclusion　Both Culex pipiens quinquefasciatus and Aedes albopictus had high resistance to pyrethroid insecticides, which suggested that we should strengthen the patriotic hygiene campaigns, reduce pyrethroid insecticides, use alternately different types of insecticides for delay the development of resistance and better mosquito prevention and control.

Gene regulation of the avian malaria parasite Plasmodium relictum, during the different stages within the mosquito vector.

The malaria parasite Plasmodium relictum is one of the most widespread species of avian malaria. As in the case of its human counterparts, bird Plasmodium undergoes a complex life cycle infecting two hosts: the arthropod vector and the vertebrate host. In this study, we examined transcriptomes of P. relictum (SGS1) during crucial timepoints within its vector, Culex pipiens quinquefasciatus. Differential gene-expression analyses identified genes linked to the parasites life-stages at: i) a few minutes after the blood meal is ingested, ii) during peak oocyst production phase, iii) during peak sporozoite phase and iv) during the late-stages of the infection. A large amount of genes coding for functions linked to host-immune invasion and multifunctional genes was active throughout the infection cycle. One gene associated with a conserved Plasmodium membrane protein with unknown function was upregulated throughout the parasite development in the vector, suggesting an important role in the successful completion of the sporogonic cycle. Gene expression analysis further identified genes, with unknown functions to be significantly differentially expressed during the infection in the vector as well as upregulation of reticulocyte-binding proteins, which raises the possibility of the multifunctionality of these RBPs. We establish the existence of highly stage-specific pathways being overexpressed during the infection. This first study of gene-expression of a non-human Plasmodium species in its vector provides a comprehensive insight into the molecular mechanisms of the common avian malaria parasite P. relictum and provides essential information on the evolutionary diversity in gene regulation of the Plasmodium's vector stages.

Metagenome Sequencing Reveals the Midgut Microbiota Makeup of Culex pipiens quinquefasciatus and Its Possible Relationship With Insecticide Resistance.

Midgut microbiota can participate in the detoxification and metabolism processes in insects, but there are few reports on the relationship between midgut microbiota and insecticide resistance in mosquitoes. In this study, we performed metagenomic sequencing on a susceptible strain (SS), a field-collected Hainan strain (HN), and a deltamethrin-resistant strain (RR) of Culex pipiens quinquefasciatus to understand the diversity and functions of their midgut microbiota. The results revealed differences in midgut microbiota among the three strains of Cx. pipiens quinquefasciatus. At the phylum level, Proteobacteria was the most prominent, accounting for nearly 70% of their midgut microbes. At the genus level, Aeromonas made up the highest proportion. In addition, Aeromonas, Morganella, Elizabethkingia, Enterobacter, Cedecea, and Thorsellia showed significant differences between strains. At the species level, Bacillus cereus, Enterobacter cloacae complex sp. 4DZ3-17B2, Streptomyces sp. CNQ329, and some species of Pseudomonas and Wolbachia were more abundant in the two resistant strains. Principal component analysis (PCA) showed that the SS strain had significantly different metagenomic functions than the two deltamethrin-resistant strains (HN and RR strain). The HN and RR strains differed from the SS strain in more than 10 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The analysis of species abundance and functional diversity can provide directions for future studies.

The potential distribution and dynamics of important vectors Culex pipiens pallens and Culex pipiens quinquefasciatus in China under climate change scenarios: an ecological niche modelling approach.

BACKGROUND: Intense studies have been carried out on the effects of climate change on vector-borne diseases and vectors. Culex pipiens pallens and Culex pipiens quinquefasciatus are two medically concerned mosquito species in temperate and tropical areas, which serve as important disease-transmitting pests of a variety of diseases. The ongoing geographical expansion of these mosquitoes has brought an increasing threat to public health. RESULTS: Based on mosquito occurrence records and high-resolution environmental layers, an ecological niche model was established to model their current and future potential distribution in China. Our model showed that the current suitable area for Cx. p. pallens is distributed in the central, eastern and northern parts of China, while Cx. p. quinquefasciatus is distributed in vast areas in southern China. Under future climate change scenarios, both species are predicted to expand their range to varying degrees and RCP 8.5 provides the largest expansion. Northward core shifts will occur in ranges of both species. Environmental variables which have significant impact on the distribution of mosquitoes were also revealed by our model. CONCLUSION: Severe habitat expansion of vectors is likely to occur in the future 21st century. Our models mapped the high-risk areas and risk factors which needs to be paid attention. The results of our study can be referenced in further ecological surveys and will guide the development of strategies for the prevention and control of vector-borne diseases. © 2020 Society of Chemical Industry.

Effects of Propoxur Exposure on Insecticidal Susceptibility and Developmental Traits in Culex pipiens quinquefasciatus.

Propoxur-sel strains of Culex pipiens quinquefasciatus were derived from a lab-bred strain following 16 generations of propoxur exposure under sublethal concentrations of LC25 (lethal concentration of 25%) and LC50 (lethal concentration of 50%), respectively. This resulted in resistance development in F16 with ratios of 8.8× and 6.3×, respectively, compared with F0. The fecundity, longevity, sex ratio (F/M), and hatchability of the propoxur-exposed Cx. quinquefasciatus adult survivors and their offspring were decreased, with no effect on the emergence ratio and pupa survival rate. In addition, the intrinsic rates of increase (r), the net reproduction (R0), and the finite rate of increase (λ) of the Cx. quinquefasciatus offspring generations were also decreased significantly compared to F0. Correspondingly, the mean generation time (T) and the population double time (DT) in propoxur-sels were increased. Enhanced activities of cytochrome P450 monooxygenase and esterase were also observed in propoxur-sels, indicating that a detoxification mechanism might be responsible for resistance development in Cx. quinquefasciatus. Except for the three genes cyp4d42v1, cyp4c52v1, and cyp6aa9 which displayed a coincidence in some degree in different treatments, induction by different doses of propoxur and constitutive expression in different generations of propoxur-sel strains resulted in an inconsistent identification of the P450 genes probably related with resistance.

[Effect of several human metabolic compounds for trapping Culex pipiens quinquefasciatus].

OBJECTIVE: To evaluate the indoor effect of six human metabolic compounds for trapping adult Culex pipiens quinquefasciatus. METHODS: The effects of six human metabolic compounds alone (acetic acid, propionic acid, octanoic acid, lactic acid, 1-octene-3-alcohol and urea alone), liquid lactic acid, acetic acid, propionic acid, octanoic acid, lactic acid or 1-octene-3-alcohol in combination with urea at an equal mass ratio, and lactic acid-urea combinations at various mass ratios, for trapping Cx. p. quinquefasciatus were examined using the trapping method, while the dechlorinated water served as a control. RESULTS: The indoor mosquito-trapping efficacy of the six human metabolic compounds was all superior to the dechlorinated water. Acetic acid, propionic acid, octanoic acid, or 1-octene-3-alcohol combined with urea at a mass ratio of 1∶1 had a comparable mosquito-trapping efficacy with acetic acid, propionic acid, octanoic acid, or 1-octene-3-alcohol alone (all P values > 0.05). The lactic acidurea combination at a mass ratio of 1∶1 had a significantly higher mean cumulative trapping capacity [(35.60 ± 8.11) mosquitoes] than lactic acid [(20.80 ± 8.53) mosquitoes], urea [(17.00 ± 7.18) mosquitoes] or dechlorinated water alone (7.20 ± 2.68) (all P values < 0.05). In addition, the lactic acid-urea combinations at mass ratios of 1∶1, 1∶2, 1∶3, 1∶4 or 1∶5 all had significantly greater mosquito-trapping efficacies than lactic acid, urea or dechlorinated water alone (all P values < 0.05), and the optimal combination (lactic acid-urea at a 1∶4 mass ratio) had a mean cumulative trapping capacity of (56.20 ± 9.88) mosquitoes, which was significantly superior to lactic acid [(17.00 ± 3.94) mosquitoes], urea [(16.40 ± 3.78) mosquitoes] or dechlorinated water alone [(7.40 ± 3.44) mosquitoes] (all P values < 0.05). CONCLUSIONS: The lactic acid-urea combination remarkably increases the indoor trapping capability of Cx. p. quinquefasciatus, and this combination has a weak smell, which is suitable to be used at home and office environments.

Effect of several human metabolic compounds for trapping Culex pipiens quinquefasciatus / 中国血吸虫病防治杂志

Objective To evaluate the indoor effect of six human metabolic compounds for trapping adult Culex pipiens quinquefasciatus. Methods The effects of six human metabolic compounds alone (acetic acid, propionic acid, octanoic acid, lactic acid, 1-octene-3-alcohol and urea alone), liquid lactic acid, acetic acid, propionic acid, octanoic acid, lactic acid or 1-octene-3-alcohol in combination with urea at an equal mass ratio, and lactic acid-urea combinations at various mass ratios, for trapping Cx. p. quinquefasciatus were examined using the trapping method, while the dechlorinated water served as a control. Results The indoor mosquito-trapping efficacy of the six human metabolic compounds was all superior to the dechlorinated water. Acetic acid, propionic acid, octanoic acid, or 1-octene-3-alcohol combined with urea at a mass ratio of 1∶1 had a comparable mosquito-trapping efficacy with acetic acid, propionic acid, octanoic acid, or 1-octene-3-alcohol alone (all P values > 0.05). The lactic acidurea combination at a mass ratio of 1∶1 had a significantly higher mean cumulative trapping capacity [(35.60 ± 8.11) mosquitoes] than lactic acid [(20.80 ± 8.53) mosquitoes], urea [(17.00 ± 7.18) mosquitoes] or dechlorinated water alone (7.20 ± 2.68) (all P values < 0.05). In addition, the lactic acid-urea combinations at mass ratios of 1∶1, 1∶2, 1∶3, 1∶4 or 1∶5 all had significantly greater mosquito-trapping efficacies than lactic acid, urea or dechlorinated water alone (all P values < 0.05), and the optimal combination (lactic acid-urea at a 1∶4 mass ratio) had a mean cumulative trapping capacity of (56.20 ± 9.88) mosquitoes, which was significantly superior to lactic acid [(17.00 ± 3.94) mosquitoes], urea [(16.40 ± 3.78) mosquitoes] or dechlorinated water alone [(7.40 ± 3.44) mosquitoes] (all P values < 0.05). Conclusions The lactic acid-urea combination remarkably increases the indoor trapping capability of Cx. p. quinquefasciatus, and this combination has a weak smell, which is suitable to be used at home and office environments.

Phylogenetic analysis of the ATP-binding cassette transporter family in three mosquito species.

The ATP-binding cassette (ABC) transporter family functions in the ATP-dependent transportation of various substrates across biological membranes. ABC proteins participate in various biological processes and insecticide resistance in insects, and are divided into eight subfamilies (A-H). Mosquitoes are important vectors of human diseases, but the mechanism by which the ABC transporter family evolves in mosquitoes is unknown. In this study, we classified and compared the ABC transporter families of three mosquitoes, namely, Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus. The three mosquitoes have 55, 69, and 70 ABC genes, respectively. The C. p. quinquefasciatus had approximately 40% and 65% expansion in the ABCG subfamily, mainly in ABCG1/G4, compared with the two other mosquito species. The ABCB, ABCD, ABCE, and ABCF subfamilies were conserved in the three mosquito species. The C. p. quinquefasciatus transcriptomes during development showed that the ABCG and ABCC genes were mainly highly expressed at the egg and pupal stages. The pigment-transport relative brown, white, and scarlet, as well as the ABCF subfamily, were highly expressed at the egg stage. The highly expressed genes in larvae included three ABCA3 genes. The majority of the highly expressed genes in adults were ABCG1/4 genes. These results provided insights into the evolution of the ABC transporter family in mosquitoes.

Identification of genes involved in pyrethroid-, propoxur-, and dichlorvos- insecticides resistance in the mosquitoes, Culex pipiens complex (Diptera: Culicidae).

Culex pipiens pallens and Cx. p. quinquefasciatus are important vectors of many diseases, such as West Nile fever and lymphatic filariasis. The widespread use of insecticides to control these disease vectors and other insect pests has led to insecticide resistance becoming common in these species. In this study, high throughout Illumina sequencing was used to identify hundreds of Cx. p. pallens and Cx. p. quinquefasciatus genes that were differentially expressed in response to insecticide exposure. The identification of these genes is a vital first step for more detailed investigation of the molecular mechanisms involved in insecticide resistance in Culex mosquitoes.

Development of multifunctional metabolic synergists to suppress the evolution of resistance against pyrethroids in insects that blood feed on humans.

BACKGROUND: Pyrethroids are the insecticides of choice when exposure to humans is likely, such as occurs in vector and public-health-related control programs. Unfortunately, the pyrethroids share a common resistance mechanism with dichlorodiphenyltrichloroethane (DDT), knockdown resistance (kdr), and prior extensive use of DDT has predisposed the pyrethroids to cross-resistance via kdr. Given the widespread occurrence of kdr, the use of synergists with pyrethroids is considered to be prudent to guard against the selection of multiply resistant insects. RESULTS: 3-Phenoxybenzyl hexanoate (PBH) was synthesized as a multifunctional pyrethroid synergist that, besides being a surrogate substrate for sequestration/hydrolytic carboxylesterases, now also functions as a substrate for oxidative xenobiotic metabolism. The addition of PBH to permethrin-treated females of the ISOP450 strain of Culex pipiens quinquefasciatus resulted in a threefold increase in synergism, as judged by the synergistic ratio. Similarly, PBH synergized the action of deltamethrin sixfold on females of the common bed bug, Cimex lectularius, and was 2.8-fold more synergistic than piperonyl butoxide (PBO). CONCLUSIONS: PBH synergized the action of both type I and type II pyrethroids in a mosquito vector (Cx. p. quinquefasciatus) and in a public-health pest, C. lectularius, respectively, indicating a broad spectrum of action on blood-feeding insects. PBH appears to have residual properties similar to permethrin and is itself non-toxic, unlike PBO, and therefore should be compatible with existing pyrethroid formulations used for insecticide-treated nets and home/residential sprays.

Effects of West Nile virus dose and extrinsic incubation temperature on temporal progression of vector competence in Culex pipiens quinquefasciatus.

Culex pipiens quinquefasciatus were fed blood containing either 7.0 +/- 0.1 logs plaque-forming units (pfu)/ml (high dose) or 5.9 +/- 0.1 logs pfu/ml (low dose) of West Nile virus and held at extrinsic incubation temperatures (EIT) of 28 degrees C or 25 degrees C. Approximately 20 mosquitoes per dose were collected after incubation periods (IP) of 4, 6, 8, and 12 days postinfection (dpi). Infection rates were influenced by EIT and virus dose but not by IP. Body titer was significantly higher for mosquitoes fed the high dose and held at 28 degrees C at the later IPs (6, 8, and 12 dpi). However, leg titer was significantly higher for mosquitoes at the later IPs but did not differ between EITs or doses. Because infection rates varied with EIT and dose, there is likely a midgut infection barrier influenced by these factors that is not influenced by IP. Dissemination rates were influenced by all 3 factors consistent with the presence of a midgut escape barrier. Dissemination rate, body titer, and leg titer were dependent on IP, indicating the need to investigate multiple time points in vector competence studies to elucidate critical events in infection and dissemination.

Fumigating insecticidal activity of 5 essential oils against Culex pipiens quinquefasciatus / 第二军医大学学报

Objective:To observe the fumigating insecticidal activity of 5 essential oils(asteraceae oil,rutaceae oil,mentha piperita oil,carvacryl oil and citronella oil) against Culex pipiens quinquefasciatus.Methods: Fumigating insecticidal activity was investigated by airtight fumigation in conical flask.Results: After different fumigating time,5 essential oils showed different toxity against Culex pipien quinqucfasciatus.Of 5 essential oils,rutaceae oil was the most toxic, with the LC 50 values being 0.013(0.5 h),0.055(4 h) and 0.058(24 h); asteraceae oil was the lowest toxic one, with the LC 50 values being 0.948(0.5 h),0.427(4 h) and 1.711(24 h).When LC 95 values of the 5 essential oils were used to treat Culex pipiens quinquefasciatus,the shortest fumigating time appeared in carvacryl oil(6.087 min) and the longest in citrinella oil(21.143 min).Conclusion: All the 5 essential oils have considerable insecticidal effects against Culex pipiens quinquefasciatus.Rutaceae oil and carvacryl oil are better than the others,which provides basic informations for the related field experiments.

Pathogenicity Tests on Nine Mosquito Species and Several Non-target Organisms with Strelkovimermis spiculatus (Nemata Mermithidae).

Nine species of mosquitoes and several species of non-target aquatic organisms were tested for susceptibility to the mernaithid nematode, Strelkovimermis spiculatus. All species of Anopheles, Aedes, Culex, and Toxorhynchites exposed to S. spiculatus were susceptible. Of the nine mosquito species tested, C. pipiens quinquefasciatus had the greatest tolerance to initial invasion and the highest percent infection of those that survived. High levels of infection were also achieved with Aedes taeniorhynchus and A. albopictus, but these mosquitoes were significantly less tolerant to parasitism than C. pipiens quinquefasciatus. Strelkovimermis spiculatus did not infect or develop in any of the non-target hosts tested.