Search

Abstract Background Culex pipiens pallens is a well-known mosquito vector for several diseases. Deltamethrin, a commonly used pyrethroid insecticide, has been frequently applied to manage adult Cx. pipiens pallens. However, mosquitoes can develop resistance to these insecticides as a result of insecticide misuse and, therefore, it is crucial to identify novel methods to control insecticide resistance. The relationship between commensal bacteria and vector resistance has been recently recognized. Bacteriophages (= phages) are effective tools by which to control insect commensal bacteria, but there have as yet been no studies using phages on adult mosquitoes. In this study, we isolated an Aeromonas phage vB AhM-LH that specifically targets resistance-associated symbiotic bacteria in mosquitoes. We investigated the impact of Aeromonas phage vB AhM-LH in an abundance of Aeromonas hydrophila in the gut of Cx. pipiens pallens and its effect on the status of deltamethrin resistance. Methods Phages were isolated on double-layer agar plates and their biological properties analyzed. Phage morphology was observed by transmission electron microscopy (TEM) after negative staining. The phage was then introduced into the mosquito intestines via oral feeding. The inhibitory effect of Aeromonas phage vB AhM-LH on Aeromonas hydrophila in mosquito intestines was assessed through quantitative real-time PCR analysis. Deltamethrin resistance of mosquitoes was assessed using WHO bottle bioassays. Results An Aeromonas phage vB AhM-LH was isolated from sewage and identified as belonging to the Myoviridae family in the order Caudovirales using TEM. Based on biological characteristics analysis and in vitro antibacterial experiments, Aeromonas phage vB AhM-LH was observed to exhibit excellent stability and effective bactericidal activity. Sequencing revealed that the Aeromonas phage vB AhM-LH genome comprises 43,663 bp (51.6% CG content) with 81 predicted open reading frames. No integrase-related gene was detected in the vB AH-LH genome, which marked it as a potential biological antibacterial. Finally, we found that Aeromonas phage vB AhM-LH could significantly reduce deltamethrin resistance in Cx. pipiens pallens, in both the laboratory and field settings, by decreasing the abundance of Aeromonas hydrophila in their midgut. Conclusions Our findings demonstrate that Aeromonas phage vB AhM-LH could effectively modulate commensal bacteria Aeromonas hydrophila in adult mosquitoes, thus representing a promising strategy to mitigate mosquito vector resistance. Graphical Abstract

Abstract 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. Graphical Abstract

Background: Culex pipiens pallens is a well-known mosquito vector for several diseases. Deltamethrin, a commonly used pyrethroid insecticide, has been frequently applied to manage adult Cx. pipiens pallens. However, mosquitoes can develop resistance to these insecticides as a result of insecticide misuse and, therefore, it is crucial to identify novel methods to control insecticide resistance. The relationship between commensal bacteria and vector resistance has been recently recognized. Bacteriophages (= phages) are effective tools by which to control insect commensal bacteria, but there have as yet been no studies using phages on adult mosquitoes. In this study, we isolated an Aeromonas phage vB AhM-LH that specifically targets resistance-associated symbiotic bacteria in mosquitoes. We investigated the impact of Aeromonas phage vB AhM-LH in an abundance of Aeromonas hydrophila in the gut of Cx. pipiens pallens and its effect on the status of deltamethrin resistance. Methods: Phages were isolated on double-layer agar plates and their biological properties analyzed. Phage morphology was observed by transmission electron microscopy (TEM) after negative staining. The phage was then introduced into the mosquito intestines via oral feeding. The inhibitory effect of Aeromonas phage vB AhM-LH on Aeromonas hydrophila in mosquito intestines was assessed through quantitative real-time PCR analysis. Deltamethrin resistance of mosquitoes was assessed using WHO bottle bioassays. Results: An Aeromonas phage vB AhM-LH was isolated from sewage and identified as belonging to the Myoviridae family in the order Caudovirales using TEM. Based on biological characteristics analysis and in vitro antibacterial experiments, Aeromonas phage vB AhM-LH was observed to exhibit excellent stability and effective bactericidal activity. Sequencing revealed that the Aeromonas phage vB AhM-LH genome comprises 43,663 bp (51.6% CG content) with 81 predicted open reading frames. No integrase-related gene was detected in the vB AH-LH genome, which marked it as a potential biological antibacterial. Finally, we found that Aeromonas phage vB AhM-LH could significantly reduce deltamethrin resistance in Cx. pipiens pallens, in both the laboratory and field settings, by decreasing the abundance of Aeromonas hydrophila in their midgut. Conclusions: Our findings demonstrate that Aeromonas phage vB AhM-LH could effectively modulate commensal bacteria Aeromonas hydrophila in adult mosquitoes, thus representing a promising strategy to mitigate mosquito vector resistance. [ABSTRACT FROM AUTHOR]

Copyright of China Tropical Medicine is the property of China Tropical Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Mosquitoes are vectors of various diseases, posing significant health threats worldwide. Chemical pesticides, particularly pyrethroids like deltamethrin, are commonly used for mosquito control, but the emergence of resistant mosquito populations has become a concern. In the deltamethrin‐resistant (DR) strain of Culex pipiens pallens, the highly expressed cytochrome P450 9 J34 (CYP9J34) gene is believed to play a role in resistance, yet the underlying mechanism remains unclear. RESULTS: Quantitative polymerase chain reaction with reverse transcription (qRT–PCR) analysis revealed that the expression of CYP9J34 was 14.6‐fold higher in DR strains than in deltamethrin‐susceptible (DS) strains. The recombinant production of CYP9J34 protein of Cx. pipiens pallens showed that the protein could directly metabolize deltamethrin, yielding the major metabolite 4′‐OH deltamethrin. Through dual luciferase reporter assays and RNA interference, the transcription factor homeobox protein B‐H2‐like (B‐H2) was identified to modulate the expression of the CYP9J34 gene, contributing to mosquito resistance to deltamethrin. CONCLUSIONS: Our findings demonstrate that the CYP9J34 protein could directly degrade deltamethrin, and the transcription factor B‐H2 could regulate CYP9J34 expression, influencing the resistance of mosquitoes to deltamethrin. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Copyright of China Tropical Medicine is the property of China Tropical Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Background Mosquitoes are important vectors for a range of diseases, contributing to high rates of morbidity and mortality in the human population. Culex pipiens pallens is dominant species of Culex mosquito in northern China and a major vector for both West Nile virus and Bancroftian filariasis. Insecticide application were largely applied to control the mosquito-mediated spread of these diseases, contributing to increasing rates of resistance in the mosquito population. The voltage-gated sodium channel (Vgsc) gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). While these kdr mutations are known to be critical to pyrethroid resistance, their evolutionary origins remain poorly understood. Clarifying the origins of these mutations is potential to guide further vector control and disease prevention efforts. Accordingly, the present study was designed to study the evolutionary genetics of kdr mutations and their association with the population structure of Cx. p. pallens in Shandong province, China. Methods Adult Culex females were collected from Shandong province and subjected to morphological identification under a dissection microscope. Genomic DNA were extracted from the collected mosquitoes, the Vgsc gene were amplified via PCR and sequenced to assess kdr allele frequencies, intron polymorphisms, and kdr codon evolution. In addition, population genetic diversity and related population characteristics were assessed by amplifying and sequencing the mitochondrial cytochrome C oxidase I (COI) gene. Results Totally, 263 Cx. p. pallens specimens were used for DNA barcoding and sequencing analyses to assess kdr allele frequencies in nine Culex populations. The kdr codon L1014 in the Vgsc gene identified two non-synonymous mutations (L1014F and L1014S) in the analyzed population. These mutations were present in the eastern hilly area and west plain region of Shandong Province. However, only L1014F mutation was detected in the southern mountainous area and Dongying city of Shandong Province, where the mutation frequency was low. Compared to other cities, population in Qingdao revealed significant genetic differentiation. Spatial kdr mutation patterns are likely attributable to some combination of prolonged insecticide-mediated selection coupled with the genetic isolation of these mosquito populations. Conclusions These data suggest that multiple kdr alleles associated with insecticide resistance are present within the Cx. p. pallens populations of Shandong Province, China. The geographical distributions of kdr mutations in this province are likely that the result of prolonged and extensive insecticide application in agricultural contexts together with frequent mosquito population migrations. In contrast, the low-frequency kdr mutation detected in central Shandong Province populations may originate from the limited selection pressure in this area and the relative genetic isolation. Overall, the study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.

Abstract 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. Graphical Abstract

Introduction: Tahyna virus (TAHV), an arbovirus of the genus Orthobunyavirus, is a cause of human diseases and less studied worldwide. In this study, a new strain of TAHV was isolated from Aedes sp. mosquitoes collected in Panjin city, Liaoning province. However, the competent vector of TAHV in China is still unknown. Methods: The genome of newly isolated TAHV was sequenced and phylogenetic analysis is performed. Aedes albopictus and Culex pipiens pallens were orally infected with artificial virus blood meals (1:1 of virus suspension and mouse blood), the virus was detected in the midgut, ovary, salivary gland and saliva of the mosquitoes. Then, the transmission and dissemination rates, vertical transmission and horizontal transmission of the virus by the mosquitoes were assessed. Methods: The genome of newly isolated TAHV was sequenced and phylogenetic analysis is performed. Aedes albopictus and Culex pipiens pallens were orally infected with artificial virus blood meals (1:1 of virus suspension and mouse blood), the virus was detected in the midgut, ovary, salivary gland and saliva of the mosquitoes. Then, the transmission and dissemination rates, vertical transmission and horizontal transmission of the virus by the mosquitoes were assessed. Methods: The genome of newly isolated TAHV was sequenced and phylogenetic analysis is performed. Aedes albopictus and Culex pipiens pallens were orally infected with artificial virus blood meals (1:1 of virus suspension and mouse blood), the virus was detected in the midgut, ovary, salivary gland and saliva of the mosquitoes. Then, the transmission and dissemination rates, vertical transmission and horizontal transmission of the virus by the mosquitoes were assessed. Results: Phylogenetic analysis revealed that the virus shared high similarity with TAHV and was named the TAHV PJ01 strain. After oral infection with virus blood meals, Ae. albopictus showed positive for the virus in all tested tissues with an extrinsic incubation period of 2 days and a fluctuating increasement of transmission and dissemination rates. Whereas no virus was detected in the saliva of Cx. pipiens pallens. Suckling mice bitten by infectious Ae. albopictus developed obvious neurological symptoms, including inactivity, hind-leg paralysis and difficulty turning over, when the virus titer reached 1.70×105 PFU/mL in the brain. Moreover, TAHV was detected in the eggs, larvae and adults of F1 offspring of Ae. albopictus. Discussion: Ae. albopictus is an efficient vector to transmit TAHV but Cx. pipiens pallens is not. Ae. albopictus is also a reservoir host that transmits the virus vertically, which further increases the risk of outbreaks. This study has important epidemiological implications for the surveillance of pathogenic viruses in China and guiding comprehensive vector control strategies to counteract potential outbreaks in future. [ABSTRACT FROM AUTHOR]

Background: Mosquitoes are important vectors for a range of diseases, contributing to high rates of morbidity and mortality in the human population. Culex pipiens pallens is dominant species of Culex mosquito in northern China and a major vector for both West Nile virus and Bancroftian filariasis. Insecticide application were largely applied to control the mosquito-mediated spread of these diseases, contributing to increasing rates of resistance in the mosquito population. The voltage-gated sodium channel (Vgsc) gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). While these kdr mutations are known to be critical to pyrethroid resistance, their evolutionary origins remain poorly understood. Clarifying the origins of these mutations is potential to guide further vector control and disease prevention efforts. Accordingly, the present study was designed to study the evolutionary genetics of kdr mutations and their association with the population structure of Cx. p. pallens in Shandong province, China. Methods: Adult Culex females were collected from Shandong province and subjected to morphological identification under a dissection microscope. Genomic DNA were extracted from the collected mosquitoes, the Vgsc gene were amplified via PCR and sequenced to assess kdr allele frequencies, intron polymorphisms, and kdr codon evolution. In addition, population genetic diversity and related population characteristics were assessed by amplifying and sequencing the mitochondrial cytochrome C oxidase I (COI) gene. Results: Totally, 263 Cx. p. pallens specimens were used for DNA barcoding and sequencing analyses to assess kdr allele frequencies in nine Culex populations. The kdr codon L1014 in the Vgsc gene identified two non-synonymous mutations (L1014F and L1014S) in the analyzed population. These mutations were present in the eastern hilly area and west plain region of Shandong Province. However, only L1014F mutation was detected in the southern mountainous area and Dongying city of Shandong Province, where the mutation frequency was low. Compared to other cities, population in Qingdao revealed significant genetic differentiation. Spatial kdr mutation patterns are likely attributable to some combination of prolonged insecticide-mediated selection coupled with the genetic isolation of these mosquito populations. Conclusions: These data suggest that multiple kdr alleles associated with insecticide resistance are present within the Cx. p. pallens populations of Shandong Province, China. The geographical distributions of kdr mutations in this province are likely that the result of prolonged and extensive insecticide application in agricultural contexts together with frequent mosquito population migrations. In contrast, the low-frequency kdr mutation detected in central Shandong Province populations may originate from the limited selection pressure in this area and the relative genetic isolation. Overall, the study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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 observed high expression levels in legs and wings. Furthermore, expression of CPR63 in the legs of deltamethrin-resistant (DR) strains was 2.17-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 cuticle of the tarsi in the siCPR63 group than in the siNC (negative control siRNA) group. Transmission electron microscopy (TEM) revealed that the exocuticle and endocuticle thickness of the tarsi were significantly thinner, which contributes the thinner procuticle of tarsi in the siCPR63 group than in the siNC group. Our results suggested that CPR63 might contribute to the resistance phenotype by thickening the cuticle and thereby possibly increasing the tolerance of mosquitoes to deltamethrin.

BACKGROUND: Due to the development of insecticide resistance in mosquitoes, with worldwide mosquito‐borne diseases resurgence in recent years, recent advances in proteome technology have facilitated a proteome‐wide analysis of insecticide resistance‐associated proteins in mosquitoes. Understanding the complexity of the molecular basis of insecticide resistance mechanisms employed by mosquitoes will help in designing the most effective and sustainable mosquito control methods. RESULTS: After 30 generations, insecticide‐selected strains showed elevated resistance levels to the cypermethrin used for selection. Proteome data allowed the detection of 2892 proteins, of which 2885 differentially expressed proteins (DEPs) achieved quantitative significances in four stages (egg, larvae, pupae, adult) of Culex pipiens pallens cypermethrin‐resistant strain as compared to the susceptible strain. Among them, a significant enrichment of proteins, including cuticular proteins, enzymes involved in the detoxification (cytochrome P450, glutathione S‐transferases, esterase, ATP‐binding cassette) and some biological pathways (oxidative phosphorylation, hippo signalling) that are potentially involved in cypermethrin resistance, was observed. Thirty‐one representative DEPs (cytochrome P450, glutathione S‐transferase, cuticle protein) during Cx. pipiens pallens developmental stages were confirmed by a parallel reaction monitoring strategy. CONCLUSIONS: The present study confirmed the power of isobaric tags for relative and absolute quantification for identifying concomitantly quantitative proteome changes associated with cypermethrin in Cx. pipiens pallens. Proteome analysis suggests that proteome modifications can be selected rapidly by cypermethrin, and multiple resistance mechanisms operate simultaneously in cypermethrin‐resistance of Cx. pipiens pallens, Our results interpret that an up‐regulated expression of proteins and enzymes like cytochrome P450, glutathione S‐transferases, esterase etc. has an impact in insecticide resistance. Previously neglected penetration resistance (cuticular proteins) may play an important role in the adaptive response of Cx. pipiens pallens to insecticides. This information may serve as a basis for future work concerning the possible role of these proteins in cypermethrin resistance in mosquito Cx. pipiens pallens. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Mosquitoes within the Culex pipiens complex play a crucial role in human disease transmission. Insecticides, especially pyrethroids, are used to control these vectors. Mosquito legs are the main entry point and barrier for insecticides to gain their neuronal targets. However, the resistance mechanism in mosquito legs is unclear. Herein, we employed transcriptomic analyses and isobaric tags for relative and absolute quantitation techniques to investigate the resistance mechanism, focusing on Cx. pipiens legs. We discovered 2346 differentially expressed genes (DEGs) between deltamethrin-resistant (DR) and deltamethrin-sensitive (DS) mosquito legs, including 41 cytochrome P450 genes. In the same comparison, we identified 228 differentially expressed proteins (DEPs), including six cytochrome P450 proteins. Combined transcriptome and proteome analysis revealed only two upregulated P450 genes, CYP325G4 and CYP6AA9 . The main clusters of DEGs and DEPs were associated with metabolic processes, such as cytochrome P450-mediated metabolism of drugs and xenobiotics. Transcription analysis revealed high CYP325G4 and CYP6AA9 expression in the DR strain at 72 h posteclosion compared with that in the DS strain, particularly in the legs. Mosquitoes knocked down for CYP325G4 were more sensitive to deltamethrin than the controls. CYP325G4 knockdown reduced the expression of several chlorinated hydrocarbon (CHC)-related genes, which altered the cuticle thickness and structure. Conversely, CYP6AA9 knockdown increased CHC gene expression without altering cuticle thickness and structure. P450 activity analysis demonstrated that CYP325G4 and CYP6AA9 contributed to metabolic resistance in the midgut and legs. This study identified CYP325G4 as a novel mosquito deltamethrin resistance factor, being involved in both metabolic and cuticular resistance mechanisms. The previously identified CYP6AA9 was investigated for its involvement in metabolic resistance and potential cuticular resistance in mosquito legs. These findings enhance our comprehension of resistance mechanisms, identifying P450s as promising targets for the future management of mosquito vector resistance, and laying a theoretical groundwork for mosquito resistance management.

The Culex pipiens complex, a common species in urban areas, has not yet caused a disease in the Republic of Korea, although it is abundant across residential areas. This complex is a major vector of West Nile virus in the United States. Additionally, Culex pipiens molestus and Culex pipiens pallens in the Republic of Korea are known to have vector competence. However, it is difficult to morphologically distinguish Culex pipiens complex species in the Republic of Korea. In this study, samples were collected from 48 sites near residential areas in the Republic of Korea, and the distribution of Culex pipiens complex species was firstly analyzed using species‐specific primers. Culex p. molestus and Cx. p. pallens were collected in large numbers in all the study regions except Ulleungdo, where the number of samples collected was five. Of the total 1063 samples, 70.1% were Cx. p. molestus and 29.9% were Cx. p. pallens. As there has been no research on the diversity of these species in the Republic of Korea, the results of this study would provide important baseline data for further studies on the Culex pipiens complex. [ABSTRACT FROM AUTHOR]

BACKGROUND: Mosquito control is still the main prevention and control measure for numerous mosquito‐borne diseases causing millions of deaths each year. New strategies for mosquito control are in demand. Proteases play an important role in mosquito physiology, therefore this study explored the inhibition of a serpin (serine protease inhibitor) in mosquitoes and its effect on reproductive capacity. RESULTS: A factor Xa inhibitor homolog (named Pipiserpin) was amplified and identified in Culex pipiens pallens mosquitoes. We expressed a recombinant Pipiserpin protein in vitro against which a mouse antiserum was generated. We found that female mosquitoes expressed more Pipiserpin protein than male mosquitoes. After mating, female mosquitoes were fed with blood mixed with different amounts of antisera and results showed that consumption of Pipiserpin impeded ovary development and decreased eggs hatching rates compared to that of the pre‐immune serum group. CONCLUSION: We identified a Culex mosquito factor Xa inhibitor, Pipiserpin, which affects female reproductive potential. Our results suggest that Pipiserpin may be a novel target for mosquito population control. The conclusions from our study on Cx. pipiens pallens might serve as a reference for the development of control measures for other mosquitoes as well. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

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 observed high expression levels in legs and wings. Furthermore, expression of CPR63 in the legs of deltamethrin-resistant (DR) strains was 2.17-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 cuticle of the tarsi in the siCPR63 group than in the siNC (negative control siRNA) group. Transmission electron microscopy (TEM) revealed that the exocuticle and endocuticle thickness of the tarsi were significantly thinner, which contributes the thinner procuticle of tarsi in the siCPR63 group than in the siNC group. Our results suggested that CPR63 might contribute to the resistance phenotype by thickening the cuticle and thereby possibly increasing the tolerance of mosquitoes to deltamethrin. [ABSTRACT FROM AUTHOR]

Mosquitoes transmit many damaging vector-borne diseases. Unfortunately, the rise of insecticide resistance has become a major obstacle to mosquito control. A preliminary study showed that a CYP6 cluster is significant for deltamethrin resistance in colonized Culex pipiens pallens. Here, several field strains were collected to explore the association of the cluster in deltamethrin tolerance. We examined the effect of deltamethrin treatment on the cluster expression at a deltamethrin concentration of LC50 in these strains using five time points. As a result, both P450 induction and constitutive overexpression were associated with deltamethrin resistance. Deltamethrin could stimulate different expression sets in the P450 cluster in different strains, predominately correlated with the resistance level of the strain. Our results will offer more insight into working with the characterization of P450s related to insecticide resistance. [ABSTRACT FROM AUTHOR]

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.

Culex pipiens pallens Coquillett, 1898 (Diptera: Culicidae) was the dominant health threat to mosquito species in Beijing, and it is important to unravel the spatial distribution and environmental correlations of Cx. pipiens pallens in Beijing. 3S technology methods and spatial statistics were used to clarify the distribution pattern. Subsequently, linear and spatial regression were performed to detect the environmental factors linked with the density of Cx. pipiens pallens. The same "middle peak" spatial distribution pattern was observed for Cx. pipiens pallens density at the community, subdistrict, and loop area levels in our study area. In addition, there were various correlated environmental factors at the community and subdistrict scales. At the community scale, the summary values of the Modified Normalized Difference Water Index (MNDWI) in 2 km buffer zone (MNDWI_2K) were negatively correlated, and the summary values of Normalized Difference Built-up Index (NDBI) in 800 m buffer zone (NDBI_800) was positively correlated to the Cx. pipiens pallens density. However, the summary values of Normalized Difference Vegetation Index and Nighttime Light Index significantly affected Cx. pipiens pallens density at the subdistrict scale. Our findings provide insight into the spatial distribution pattern of Cx. pipiens pallens density and its associated environmental risk factors at different spatial scales in the Haidian district of Beijing for the first time. The results could be used to predict the Cx. pipiens pallens density as well as the risk of lymphatic filariasis (LF) infection, which would help implement prevention and control measures to prevent future risks of biting and LF transmission in Beijing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

Abstract Background The growth and development of insects is strictly dependent on the precise regulation of chitin synthase (CHS), which is absent in vertebrates and plants. Therefore, CHS represents an attractive target for insecticides. At present, the research on the CHS gene in mosquitoes, especially its biological functions, remains limited. Results The full-length cDNA of the chitin synthase B gene in Culex pipiens pallens (CpCHSB) was prepared and consists of 5158 nucleotides with an open reading frame (ORF) of 4722 nucleotides encoding a protein of 1573 amino acid residues. Among different tissues, CpCHSB gene is mainly expressed in the midgut tissue with the highest expression in adult mosquitoes. Knockdown of CpCHSB in the larval stage significantly lowered the chitin content (16.5%) decreased body size (reduced by 25.6% in the larval stage and by 25.6% in the adult stage), and diminished reproduction (20%). Injecting siCHSB into adult mosquito mainly decreased reproduction (27%). Conclusions CpCHSB plays essential roles in growth and development, by severely reducing larval chitin content, midgut permeability, and reducing the number of female mosquito offspring. These results indicate that CHSB may serve as a potential novel target for exploring biosafe insecticides.

Abstract Background Culex pipiens pallens is the most abundant Culex mosquito species in northern China and is an important vector of bancroftian filariasis and, potentially, West Nile virus. Insecticides, particularly pyrethroids, are widely used for adult mosquito control. Insecticide resistance has become common in several mosquito species, and vector control is the main method currently available to prevent disease transmission. The voltage-gated sodium channel (Vgsc) gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). Methods Culex pipiens pallens larvae were collected from May to November over two decades, from 1992 to 2018, in four cities in Shandong Province, China. The World Health Organization (WHO) standard resistance bioassay was applied to test the resistance levels of Cx. p. pallens larvae to five different insecticides and to test deltamethrin resistance in adults, using the F1 generation. Mutations at Vgsc codon 1014 were also screened in 471 adult samples collected in 2014 to determine the association between kdr mutations and phenotypic resistance. Results Larval resistance against deltamethrin showed an increasing trend from the 1990s until 2018, which was statistically significant in all populations; resistance to cypermethrin increased significantly in mosquitoes from the Zaozhuang population. However, larval resistance to other insecticides remained relatively stable. Larval resistance against deltamethrin was consistent with adult bioassays in 2014, in which all tested populations were highly resistant, with mortality rates ranging from 39.4 to 55.23%. The L1014S and L1014F mutations were both observed in five Cx. p. pallens populations, with L1014F significantly associated with deltamethrin resistance. Conclusions The long-term dataset from Shandong demonstrates major increases in pyrethroid resistance over a 20-year period. The L1014F kdr mutation may be considered a viable molecular marker for monitoring pyrethroid resistance in Cx. p. pallens.

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. [ABSTRACT FROM AUTHOR]

Objective: To investigate the microbiota composition and diversity between autogenous and anautogenous Culex pipiens pallens , so as to provide insights into unraveling the pathogenesis of autogeny in Cx. pipiens pallens ., Methods: Autogenous and anautogenous adult Cx. pipiens pallens samples were collected at 25 ℃, and the hypervariable regions of the microbial 16S ribosomal RNA ( 16S rRNA ) gene was sequenced on the Illumina NovaSeq 6000 sequencing platform. The microbiota abundance and diversity were evaluated using the alpha diversity index, and the difference in the microbiota structure was examined using the beta diversity index. The microbiota with significant differences in the abundance between autogenous and anautogenous adult Cx. pipiens pallens samples was identified using the linear discriminant analysis effect size (LEfSe)., Results: The microbiota in autogenous and anautogenous Cx. pipiens pallens samples belonged to 18 phyla, 28 classes, 70 orders, 113 families, and 170 genera, and the dominant phyla included Proteobacteria, Bacteroidetes, and so on. At the genus level, Wolbachia was a common dominant genus, and the relative abundance was (77.6 ± 11.3)% in autogenous Cx. pipiens pallens samples and (47.5 ± 8.5)% in anautogenous mosquito samples, while Faecalibaculum (0.4% ± 0.1%), Dubosiella (0.5% ± 0.0%) and Massilia (0.5% ± 0.1%) were specific species in autogenous Cx. pipiens pallens samples. Alpha diversity analysis showed that higher Chao1 index and ACE index in autogenous Cx. pipiens pallens samples than in anautogenous samples (both P values > 0.05), and lower Shannon index ( P > 0.05) and Simpson index ( P < 0.05) in autogenous Cx. pipiens pallens samples than in anautogenous samples. LEfSe analysis showed a total of 48 significantly different taxa between autogenous and anautogenous Cx. pipiens pallens samples (all P values < 0.05)., Conclusions: There is a significant difference in the microbiota diversity between autogenous and anautogenous Cx. pipiens pallens .

Abstract Background Investigation of insecticide resistance mechanisms is considered a vital first step towards the creation of effective strategies to control resistant mosquitoes and manage mosquito-borne diseases. Our previous study revealed that NYD-OP7 may be associated with deltamethrin resistance in Culex pipiens pallen. However, the precise function of NYD-OP7 in deltamethrin resistance is still unclear. In this study, we investigated the role of NYD-OP7 in the molecular mechanisms underlying pyrethroid resistance. Results Knockdown of NYD-OP7 not only increased the susceptibility of the mosquitoes to deltamethrin in vivo but also simultaneously repressed both expression and enzyme activity of its downstream effector molecule, phospholipase C (PLC) and expression of several insecticide resistance-related P450 genes. Knockdown of PLC also sensitized the mosquitoes to deltamethrin and reduced the expression of the P450 genes. Conclusions Our results revealed that NYD-OP7 and its downstream effector PLC contribute to deltamethrin resistance by regulating the expression of P450s in Cx. pipiens pallens.

China used to be one of the most heavily endemic countries for lymphatic filariasis (LF) in the world. Bancroftian filariasis, which is caused by the filarioid nematode Wuchereria bancrofti, is the only filariasis in Shandong Province. A total of 864 endemic counties (cities) in 16 provinces/autonomous regions/municipalities with a total population of 330 million people were at risk of infection. Shandong Province was a highly LFendemic area in the 1950s, the epidemiological investigation of LF conducted in 1957 indicated that the disease was endemic in 74 counties and the highest microfilaria rate was up to 26%. There were*5 million people in the province infected with LF; among which almost 2.5 million people had lymphedema, elephantiasis, or hydrocele. Through vigorous prevention and scientific research, Shandong Province was the first to propose to treat LF with fortified diethylcarbamazine salt, more than 25 million people in the province had taken the salt, and more than 3 million people took intermittent medication. After more than 50 years of unremitting efforts, Shandong Province basically eliminated LF in 1983 and became the first province in China that successfully eradicated LF in 2004, which has played an important role in accelerating the elimination of filariasis in the country in 2007 and has made tremendous contributions to social and economic development of China. Since 1980, Shandong Province has carried out extensive international cooperation as the WHO Collaborating Center for Lymphatic Filariasis. This article is intended to share the experience in eliminating LF to other parts of the world and improve public health capacity in regions such as Africa and Oceania where the disease is still endemic for interest. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

The wide application of pyrethroids has led to the rapid development of insecticide resistance in mosquitoes, leading to a rise in mosquito-borne diseases. We previously identified five differentially expressed lipase family genes upon evaluating the transcriptomes of deltamethrin-resistant and deltamethrin-susceptible strains of Culex pipiens pallens. Herein, the gene expression levels were verified by quantitative real-time PCR, and two lipase family genes, lipase A and pancreatic triacylglycerol lipase A, were chosen for further investigations. Using cell viability assays and Centers for Disease Control and Prevention bottle bioassays, lipase A was found to increase the resistance of mosquitoes against deltamethrin both in vitro and in vivo. Our findings indicate that lipase A is involved in conferring deltamethrin resistance in Cx. pipiens pallens. [ABSTRACT FROM AUTHOR]

The 60S ribosomal protein L22 (GenBank accession no. EF990190) was cloned from Culex pipiens pallens. An open reading frame (ORF) of 447 bps was found to encode a putative 148 amino acids protein which shares 90% and 80% identity with RPL22 genes from Aedes aegypti and Anopheles gambiae respectively. Real-time quantitative PCR analysis demonstrated that the transcription level of RPL22 in deltamethrin-resistant strain was 2.57 folds higher than in deltamethrin-susceptible strain of Cx. pipiens pallens. Overexpression of RPL22 in C6/36 cells showed that the deltamethrin-resistance was decreased in C6/36-RPL22 cell compared to the control. The mRNA level of cytochrome P450 6A1 (CYP6A1, GenBank accession no. FJ423553) showed that CYP6A1 was down-regulated in the C6/36 transfected with RPL22 (C6/36-RPL22) cells, suggesting that CYP6A1 was repressed by RPL22. Our study provides the first evidence that RPL22 may play some role in the regulation of deltamethrin-resistance in Cx. pipiens pallens.

One mosquito opsin gene, NYD-OP7, has been cloned from Culex pipiens pallens. An open reading frame (ORF) of 1116 bp was found to encode a putative 371 amino acids protein which exhibits high identity with opsins from Aedes and Anopheles mosquitoes. Transcript expression of NYD-OP7 was determined by real-time PCR in all life stages of deltamethrin-susceptible and -resistant strains of the Culex mosquito. The results demonstrated that this gene is expressed at all developmental stages, and it is expressed predominantly at the pupae and adult stages. Meanwhile, in pupae and adults, NYD-OP7 is overexpressed in deltamethrin-resistant strain than in -susceptible strain. Importantly, stable expression of NYD-OP7 in the mosquito C6/36 cells can confer moderate deltamethrin resistance. Our study provides the first direct evidence that increased expression of an opsin gene may play some role in the development of deltamethrin resistance in Cx. pipiens pallens. © 2006 Elsevier Inc. All rights reserved.

Abstract Background Severe fever with thrombocytopenia syndrome (SFTS) is a newly identified emerging infectious disease, which is caused by a novel bunyavirus (termed SFTSV) in Asia. Although mosquitoes have not been identified as the primary vectors, as revealed by epidemiological surveys, their role in transmitting this SFTSV as a suspicious vector has not been validated. Findings In this study, we conducted experimental infections of mosquitoes with SFTSV to examine the role of mosquitoes in the transmission of the virus. We did not detect viral replication in Culex pipiens pallens, Aedes aegyptis and Anopheles sinensis as revealed by qRT-PCR assay. In addition, we failed to isolate SFTSV from the Vero cells cultured with suspensions of SFTSV-infected mosquitoes. Conclusion The results of the present study demonstrate little possibility that mosquitoes act as vectors for the emerging pathogen SFTSV.

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.

Background: The growth and development of insects is strictly dependent on the precise regulation of chitin synthase (CHS), which is absent in vertebrates and plants. Therefore, CHS represents an attractive target for insecticides. At present, the research on the CHS gene in mosquitoes, especially its biological functions, remains limited. Results: The full-length cDNA of the chitin synthase B gene in Culex pipiens pallens (CpCHSB) was prepared and consists of 5158 nucleotides with an open reading frame (ORF) of 4722 nucleotides encoding a protein of 1573 amino acid residues. Among different tissues, CpCHSB gene is mainly expressed in the midgut tissue with the highest expression in adult mosquitoes. Knockdown of CpCHSB in the larval stage significantly lowered the chitin content (16.5%) decreased body size (reduced by 25.6% in the larval stage and by 25.6% in the adult stage), and diminished reproduction (20%). Injecting siCHSB into adult mosquito mainly decreased reproduction (27%). Conclusions: CpCHSB plays essential roles in growth and development, by severely reducing larval chitin content, midgut permeability, and reducing the number of female mosquito offspring. These results indicate that CHSB may serve as a potential novel target for exploring biosafe insecticides. [ABSTRACT FROM AUTHOR]

Field collected populations of two mosquito species, Culex pipiens pallens and Aedes albopictus, were tested for susceptibility to five pyrethroid insecticides using a topical application bioassay. The laboratory strain of Cx. pipiens pallens (KS‐CP) demonstrated the most and the lowest susceptibility to deltamethrin and d‐cis/trans prallethrine with LD50 values of 0.00022 μg/♀ and 0.00178 μg/♀, respectively, while Ae. albopictus (KS‐AL) showed the greatest and the lowest susceptibility to phthalethrin and deltamethrin with LD50 values of 0.00015 μg/♀ and 0.00085 μg/♀, respectively. Cx. pipiens pallens (JF‐CP) collected at a nearby rice field in Jeonju showed the greatest susceptibility to deltamethrin (LD50 value of 0.0473 μg/♀) and the resistance ratios (RRs) and compared to the laboratory strain were 215 fold greater. JF‐CP demonstrated the lowest susceptibility to d‐cis/trans phenothrin (0.7697 μg/♀) with RRs 711.0 fold greater than the KS‐CP. An Ae. albopictus (JF‐AL) collected at a nearby forest in Jeonju showed the highest susceptibility to deltamethrin (0.00203 μg/♀) with RRs 2.4 fold greater than the KS‐AL. JF‐AL demonstrated the lowest susceptibility to d‐cis/trans prallethrine (LD50 value 0.00646 μg/♀) with RRs 9.4 fold greater than the KS‐AL. JF‐AL demonstrated the greatest resistance to phthalethrin with RRs 39.3 fold greater than the KS‐AL. Resistance ratios of Cx. pipiens pallens between field and laboratory strains ranged from 215.0 to 711.0 and Ae. albopictus ranged from 2.4 to 39.3. These results indicate that the choice of effective insecticides to selected species of mosquito populations will result in more effective control against field strains of mosquito vectors and decrease environmental pollution. [ABSTRACT FROM AUTHOR]

Pyrethroid insecticides have been effective and powerful for controlling mosquitoes. However, abuse of these insecticides increases the number of resistant mosquitoes. In this study, Culex pipiens pallens and Aedes koreicus were collected from an artificial reservoir in the vicinity of a populated area in Korea, which is also a migratory bird catchment area. To monitor resistance to pyrethroid insecticides in mosquitoes, genomic DNA from the collected mosquitoes was sequenced for the kdr mutation in the voltage‐gated sodium channel (VGSC) gene. As a result, three samples with homozygous resistance (17.6%) and one with heterozygous resistance (5.9%) were found among 17 Cx. pipiens pallens specimens. One of the samples had a unique sequence at the amplified VGSC region. Of the 15 Ae. koreicus, no insecticide resistant individuals were found. In Korea, this is the first report of kdr genetic traits in Ae. koreicus and Cx. pipiens pallens and of a unique VGSC allele in Cx. pipiens pallens. Further investigation is needed to monitor the kdr resistance of these species in Korea and to determine how the unique sequence found in Cx. pipiens pallens is related to insecticide resistance. [ABSTRACT FROM AUTHOR]

Culex pipiens pallens is an important vector that transmits Bancroftian filariasis, Japanese encephalitis and other diseases that pose a serious threat to human health. Extensive and improper use of insecticides has caused insecticide resistance in mosquitoes, which has become an important obstacle to the control of mosquito-borne diseases. It is crucial to investigate the underlying mechanism of insecticide resistance. The aims of this study were to identify genes involved in insecticide resistance based on the resistance phenotype, gene expression profile and single-nucleotide polymorphisms (SNPs) and to screen for major genes controlling insecticide resistance. Using a combination of SNP and transcriptome data, gene expression quantitative trait loci (eQTLs) were studied in deltamethrin-resistant mosquitoes. The most differentially expressed pathway in the resistant group was identified, and a regulatory network was built using these SNPs and the differentially expressed genes (DEGs) in this pathway. The major candidate genes involved in the control of insecticide resistance were analyzed by qPCR, siRNA microinjection and CDC bottle bioassays. A total of 85 DEGs that encoded putative detoxification enzymes (including 61 P450s) were identified in this pathway. The resistance regulatory network was built using SNPs, and these metabolic genes, and a major gene, CYP9AL1, were identified. The functional role of CYP9AL1 in insecticide resistance was confirmed by siRNA microinjection and CDC bottle bioassays. Using the eQTL approach, we identified important genes in pyrethroid resistance that may aid in understanding the mechanism underlying insecticide resistance and in targeting new measures for resistance monitoring and management. [ABSTRACT FROM AUTHOR]

The emergence and rapid spread of insecticide resistance in several mosquito species has become a significant obstacle in management of mosquito‐borne diseases, including deltamethrin resistance in Culex pipiens pallens. Previous study identified a major deltamethrin resistance quantitative trait locus (DR‐6) that alone explained 62% of the genetic variance. In this study, the marker L4B1.102 and L4B1.175 associated with the DR‐6 were characterized. We searched for potential candidate genes in the flank region of two markers in the genome sequence and showed that a cluster of CYP6 cytochrome P450 genes (CYP6BB4, CYP6BB3, CYP6CC2, CYP6P14, CYP6BZ2, CYP6AA9, CYP6AA8, CYP6AA7) was in the vicinity of DR‐6. Significant differences in the expression of these P450s in the larval and adult stages were identified in the resistant strains compared with the susceptible strain. For CYP6AA9 and CYP6BB4, the correlation analysis showed a highly positive correlation between relative gene expression quantification and the resistance level in different strains. Knockdown of CYP6BB4 increased the sensitivity of mosquitoes to deltamethrin. We identified that the deltamethrin resistance was in a cluster of CYP6 genes in C. pipiens pallens, and CYP6BB4 may play a significant role in the development of deltamethrin resistance. [ABSTRACT FROM AUTHOR]

Background & objectives: Under certain ecological circumstances, pathogens are able to rapidly adapt to new vectors. The great capacity of Plasmodium spp. to adapt to new anopheline mosquito vectors on different continents and the continuous ecological changes attributed to humans might promote their adaptation to culicine vectors, which are known to infect humans. Based on our current knowledge, it is difficult to predict whether such adaptations will occur. This study was aimed to determine the infection susceptibility of Anopheles sinensis, Culex tritaeniorhynchus and Cx. pipiens pallens to Plasmodium vivax in Shandong Province of China. Methods: The susceptibility of the three predominant species of mosquitoes -An. sinensis, Cx. tritaeniorhynchus and Cx. pipiens pallens in Shandong Province was compared with a direct membrane feeding assay with 15 batches of Shandong strain mono-infected gametocyte-containing blood collected from Plasmodium vivax-infected patients. Infectivity was measured by dissecting the midguts and salivary glands of the mosquitoes. The presence of oocysts and sporozoites was determined by microscopy at 6 and 22 days post-blood feeding. Results: From the 15 batches of mosquitoes that were fed infected blood, oocysts and sporozoites were detected only in 7th, 13th and 15th batches of infection for An. sinensis, and no oocysts or sporozoites were detected in Cx. tritaeniorhynchus or Cx. pipiens pallens. The positive rate of An. sinensis infection was 21.2, 13 and 36.3% in the three batches of mosquitoes, with an average infection rate of 23.5%. Interpretation & conclusion: The susceptibility of An. sinensis to P. vivax was very high in Shandong Province. Cx. tritaeniorhynchus and Cx. pipiens pallens failed to exhibit susceptibility to P. vivax.

Abstract Background Malaria infection in mosquitoes is traditionally detected by microscopic examination for Plasmodium oocysts and sporozoites. Although PCR is now widely used, the presence of parasite DNA in a mosquito does not prove that sporogony is achieved. Thus, detection of sporozoites by microscopy is still required to definitively identify vector mosquitoes. The aim of this study was to confirm sporogony of avian Plasmodium spp. in Culex pipiens pallens and C. inatomii caught from the wild. Findings Mosquitoes collected at two sites in Japan were dissected and examined by microscopy for Plasmodium oocysts and sporozoites. DNA was extracted from the midgut and salivary gland of infected mosquitoes, and the infecting Plasmodium species was identified by sequencing 478 bp of cytochrome b. Oocysts, or both oocysts and sporozoites, were found in 3.94 and 0.46 % of C. p. pallens and C. inatomii, respectively. Four (CXPIP09, GRW4, GRW11 and SGS1) and three cytochrome b lineages (CXINA01, CXINA02 and CXQUI01) were confirmed to achieve sporogony in C. p. pallens and C. inatomii, respectively. One mosquito each of C. p. pallens and C. inatomii was co-infected with two different Plasmodium lineages. Conclusions These findings demonstrate that C. p. pallens and C. inatomii are natural vectors of four and three lineages of avian Plasmodium spp., respectively. The data indicate that a systematic procedure combining microscopy and PCR is a feasible and reliable approach to identify natural vectors of wildlife malaria.

Abstract Background Most mosquito species need to obtain sugar from host plants. Little is known about the chemical cues that Culex pipiens pallens use during their orientation to nectar host plants. In this study, we investigated the behavioural responses of female Cx. pipiens pallens to common floral scent compounds and their blends. Methods Behavioural responses of female Cx. pipiens pallens to 18 individual compounds at different concentrations were determined in the olfactometer bioassays. A synthetic blend composed of behaviourally active compounds was formulated, and its attractiveness to mosquitoes was tested. Several most attractive compounds constituted a reduced blend, and its attractiveness was tested against the solvent and the full blend, respectively. Mosquito response in the olfactometer was analyzed by comparing the percentages of mosquitoes caught in the two arms by χ2 test (observed versus expected). Results Fifteen of the 18 compounds were attractive to female Cx. pipiens pallens in the dose-dependent bioassays, with the exception of β-pinene, acetophenone and nonanal. (68.00 ± 2.49) % mosquitoes responded to the full blend composed of these 15 compounds on their optimal doses when tested against the solvent, with the preference index at 46.11 ± 3.57. Six individual compounds whose preference indices were over 40 constituted the reduced blend, and it attracted (68.00 ± 1.33) % mosquitoes when tested against the solvent while its preference index was 42.00 ± 3.54. When tested against the full blend simultaneously in the olfactometer, the reduced blend could attract (45.00 ± 2.69) % of released mosquitoes, which was as attractive as the full blend. Conclusions Our results demonstrate that female Cx. pipiens pallens is differentially attracted by a variety of compounds at different concentrations. Alteration of the concentration strongly affects the attractiveness of the synthetic blend. Several floral scent volatiles might be the universal olfactory cues for various mosquito species to locate their nectar host plants, which could be potentially used in trapping devices for surveillance and control of them.

Abstract Background Mosquito control based on chemical insecticides is considered as an important element in the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of pyrethroid resistance in important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. To date, the mechanisms of pyrethroid resistance are still unclear. Recent advances in proteomic techniques can facilitate to identify pyrethroid resistance-associated proteins at a large-scale for improving our understanding of resistance mechanisms, and more importantly, for seeking some genetic markers used for monitoring and predicting the development of resistance. Methods We performed a quantitative proteomic analysis between a deltamethrin-susceptible strain and a deltamethrin-resistant strain of laboratory population of Culex pipiens pallens using isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Gene Ontology (GO) analysis was used to find the relative processes that these differentially expressed proteins were involved in. One differentially expressed protein was chosen to confirm by Western blot in the laboratory and field populations of Cx. pipiens pallens. Results We identified 30 differentially expressed proteins assigned into 10 different categories, including oxidoreductase activity, transporter activity, catalytic activity, structural constituent of cuticle and hypothetical proteins. GO analysis revealed that 25 proteins were sub-categorized into 35 hierarchically-structured GO classifications. Western blot results showed that CYP6AA9 as one of the up-regulated proteins was confirmed to be overexpressed in the deltamethrin-resistant strains compared with the deltamethrin-susceptible strains both in the laboratory and field populations. Conclusions This is the first study to use modern proteomic tools for identifying pyrethroid resistance-related proteins in Cx. pipiens. The present study brought to light many proteins that were not previously thought to be associated with pyrethroid resistance, which further expands our understanding of pyrethroid resistance mechanisms. CYP6AA9 was overexpressed in the deltamethrin-resistant strains, indicating that CYP6AA9 may be involved in pyrethroid resistance and may be used as a potential genetic marker to monitor and predict the pyrethroid resistance level of field populations.

The larvicidal activity of the crude petroleum ether, ethyl acetate, acetone, chloroform and ethanol extracts of Amorpha fruticosa seeds was individually assayed for toxicity against the early fourth-instar larva of the mosquito, Culex pipiens pallens after 24 h exposure. Of the tested extracts, the ethanol one exhibited the highest larvicidal activity (LC50 = 22.69 mg/L). Amorphigenin (8'-hydroxyrotenone), a rotenoid compound which exhibits a strong larvicidal activity with LC50 and LC90 values of 4.29 and 11.27 mg/L, respectively, was isolated from the ethanol extract by column chromatograpy. Its structure was elucidated by 1H-NMR, UV and IR spectral data. Furthermore, investigation of amorphigenin’s effects on mitochondrial complex I activity and protein synthesis in C. pipiens pallens larvae reveals that amorphigenin decreases mitochondrial complex I activities to 65.73% at 10.45 μmol/L, compared to the control, when NADH were used as the substrate. Meanwhile, amorphigenin at 10.45 μmol/L also caused a 1.98-fold decrease in protein content, compared to the control larvae treated with acetone only.

Abstract: To identify larvicidal compounds from the ethanolic extracts of Curcuma longa root, the active compounds were isolated using activity‐guided fractionation with column chromatography and identified based on nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. The dipping method was used to determine the larvicidal activities of each compound against 4th‐instar larvae of Culex pipiens pallens. Two compounds were isolated and identified, ar‐turmerone and 8‐hydroxyl‐ar‐turmerone. The two compounds exhibited larvicidal activities against the 4th‐instar larvae of C. pipiens pallens after 24 hr of treatment with LC50 values of 138.86 and 257.68 ppm, respectively. The larvicidal activities of ar‐turmerone and 8‐hydroxyl‐ar‐turmerone against C. pipiens pallens are reported herein for the first time. The elucidation of the structure of these phytochemicals and their insecticidal activities are important for assessing the potential of this plant as a botanical insecticide. [ABSTRACT FROM AUTHOR]

Mosquito adults usually need to ingest sugar from nectar host plants to sustain their metabolic needs. Mosquitoes could be differentially attracted by various flowering plant species, and the volatiles were thought to be important factors attributed to the differential attractiveness. However, whether mosquitoes' preference for host plants correlates with their nutritional rewards from sugar sources remains unclear. In the present study, the preference of newly emerged Culex pipiens pallens to three kinds of flowering plants ( Ligustrum quihoui, Abelia chinensis, and Nerium indicum) was determined in the olfactometer. Besides, when the newly emerged mosquitoes were provided with these flowering plants as sugar sources, the content of their metabolic reserves (glycogen, lipid, and protein) was determined. The results revealed that Cx. pipiens pallens could be differentially attracted by the odors emitted by the inflorescences of the tested flowering plants, and the nutritional rewards of mosquitoes were significantly affected by feeding on different inflorescences. The present study demonstrated that feeding on nectar host plants with differential attraction could affect the energy reserves of Cx. pipiens pallens. [ABSTRACT FROM AUTHOR]

The attraction of Culex pipiens pallens Coquillett to various potential sugar sources in their habitats remains unknown. In the present study, we evaluated the attraction of Cx. pipiens pallens to the volatiles of different kinds of flowers, fruits, and seed pods by multiple choice tests under laboratory conditions. Nine of the 21 tested fresh tissues of flowers were attractive to females and six of them were attractive to males. The volatiles of six flowers ( Ligustrum quihoui , Abelia chinensis , Ophiopogon japonicus , Phaseolus vulgaris , Osmanthus fragrans , and Nerium indicum ) were attractive to both female and male Cx. pipiens pallens , and the flowers of Torenia fournieri and Lavandula angustifolia were potentially repellent to female and male mosquitoes, respectively. Besides, the volatiles of three fruits and seed pods ( Broussonetia papyrifera , Amygdalus persica , and Cucumis melo ) were attractive to both female and male Cx. pipiens pallens while the seed pods of Ficus carica and Pyrus bretschneideri were only attractive to females and males, respectively. This study provides valuable information on developing novel control methods against Cx. pipiens pallens in China. [ABSTRACT FROM AUTHOR]

Culex pipiens pallensare the most common mosquito’s vector in Asia. In order to protect the people from diseases, the anti-mosquito population is necessary that uses safe and new bio-pesticides such as bacteria-AgNPs. In our report, we used two kinds of bacteria to synthesize silver nanoparticles to examine the toxic effect on the larvae and pupae ofCx. pipiens pallensand also used as antimicrobial activity. The biosynthesis of AgNPs and its characterization was carried out by UV-Vis spectrophotometry, FTIR, TEM, SEM, and EDX. The larvicidal and pupicidal assays revealed that the lethal concentration LC50values ofBacillus amyloliquefaciens-AgNPs were 0.72 ppm (I), 0.73 ppm (II), 0.69 ppm (III), 1.16 ppm (IV), and 4.18 (Pupae), while LC50values ofBacillus subtilis-AgNPs were 0.60 ppm (I), 0.62 ppm (II), 0.21 ppm (III), 0.28 ppm (IV), and 3.46 ppm (Pupae) after 72 h of exposure. Antibacterial activity test of AgNPs reveals better results against rice pathogenic bacteria than bacteria alone. Thus, bacteria-mediated silver nanoparticles have a rapid effect on vector mosquito and microbial pathogen suggesting savings of energy and resources. Hence, bacteria-AgNPs may be used in the future as an effective weapon to control vector mosquito and harmful bacteria. [ABSTRACT FROM AUTHOR]

Piwi-interacting RNAs (piRNAs) are a novel class of noncoding single-strand RNAs. They play an important role in the germ cell maintenance, brain development, epigenetic regulation of cancer, and antiviral function. However, little is known about the relationship between the piRNAs and insecticide resistance in mosquitoes. In this study, we reported that piRNA-3878 was related with pyrethroid resistance in Culex pipiens pallens. The expression level of piRNA-3878 was lower in both laboratory and field-collected deltamethrin-resistant (DR) strains. Overexpression of piRNA-3878 increased the susceptibility of the DR strain, while inhibiting the expression of piRNA-3878 in DS strain made the mosquitoes more resistant to deltamethrin. Furthermore, we identified that CpCYP307B1 was the target of piRNA-3878. The mosquito mortality rate was increased after downregulating the expression of CpCYP307B1. These findings revealed that piRNA-3878 could target CpCYP307B1 to regulate pyrethroid resistance. [ABSTRACT FROM AUTHOR]