The overuse of chemical insecticides highlights the urgent need for novel vector control strategies. Insect-specific viruses (ISVs), such as the cell-fusing agent virus (CFAV), have shown potential to block arbovirus transmission by inhibiting viral replication in mosquitoes. However, the effects of CFAV beyond its natural host, Aedes aegypti, remain largely unexplored. In this study, we established a CFAV infection model in Aedes albopictus, a major vector for Zika virus (ZIKV), via intrathoracic injection. Stable infection was achieved, with viral loads reaching up to 107 copies per mosquito by day 10 post-injection. Nevertheless, high post-injection mortality (median survival: 3 days) was observed, which we attribute primarily to mechanical injury. No evidence of vertical transmission of CFAV was detected in Ae. albopictus. Co-injection of CFAV and ZIKV did not significantly affect ZIKV replication in this species. In contrast, in Ae. aegypti pre-infected with CFAV followed by oral ZIKV challenge, CFAV significantly reduced ZIKV infection rates in the ovaries at day 4 and viral loads in salivary glands at day 10. These findings demonstrate that while CFAV can productively infect Ae. albopictus, it does not undergo vertical transmission in this species, and has no inhibitory effect on ZIKV under the co-infection conditions tested. This study underscores challenges associated with using single ISVs such as CFAV for arbovirus control and highlights the complex, bidirectional role of multiple ISV co-infections. While exploring multi-ISV combinations may offer a potential strategy to enhance antiviral efficacy, their net effect—whether suppression or enhancement of arboviruses—warrants careful investigation.

Aedes aegypti is a major invasive vector in China, where prolonged pyrethroid use has induced resistance, complicating dengue control. This study evaluated the resistance levels of Ae. aegypti to the pyrethroid beta-cypermethrin in Yunnan and explored the underlying mechanisms to inform control strategies. Mosquitoes were collected from five regions of Yunnan in 2015-2016. Larval bioassays, adult diagnostic dose determination, and adult bioassays were conducted to assess resistance to beta-cypermethrin. Mixed-function oxidase (MFO) amount was measured in larvae, and PCR amplification was used to detect mutations in the voltage-gated sodium channel (VGSC) gene. Correlations between enzyme activity, mutations, and phenotypic resistance were analyzed. Larval bioassays indicated that all five populations exhibited resistance to beta-cypermethrin, with resistance ratios ranging from 11.31 to 41.56. The adult diagnostic dose was determined as 0.74668 g/L, with mortality rates ranging from 8.89% to 58.89%, confirming resistance. A significant negative correlation was found between adult mortality and larval LC50 values. MFO amount was closely correlated with resistance levels. The F1534C mutation was the only VGSC mutation detected, and its frequency showed a significant positive correlation with beta-cypermethrin resistance. Our data were collected in 2015-2016; resistance levels may have changed since then, and no confirmatory bioassays were performed by us after 2020. The findings demonstrate that the F1534C mutation and increased MFO amount contribute to enhanced beta-cypermethrin resistance of Ae. aegypti in Yunnan. By linking phenotype resistance to key molecular and biochemical mechanisms, these findings support the continued monitoring of resistance and provide a basis for the evidence-based optimization of dengue vector control strategies.

Background Aedes aegypti is the major vector for massive mosquito-borne viruses, including Dengue, ZIKV, etc., and threatens human health world-widely. The absence of efficient vaccine for those insect-borne diseases highlights the importance of monitoring of genetic changes of the Ae. aegypti populations. Methods The genetic variation and population structure of Ae. aegypti populations collected from three regions of Yunan province in China during 2016 to 2018 were investigated with 9 microsatellite loci and the mitochondrial coxI gene. Results From 2016 to 2018, the genetic diversity of Ae. aegypti populations in Yunnan province displayed a trend of initial increase followed by a decrease. The degree of inbreeding within the populations gradually shifted from heterozygous to moderately inbred. The clustering results indicated that, compared to the populations collected in 2016, those collected in 2017 and 2018 were genetically closer. Ten coxI haplotypes were detected, haplotypes H06 and H09 were detected nearly in all regions, while the others were only detected in single region or even single year. The population diffusion analysis indicated that the diffusion of Ae. aegypti populations among different locations exhibited a trend of first increasing and then decreasing. Meanwhile, the AMOVA analysis results also revealed that the source of variation in the Yunnan Ae. aegypti populations gradually shifted from mainly inter-individual to mainly inter-regional. The evolutionary scenario inference of the invasion and diffusion of Ae. aegypti indicated that the mosquito invaded and colonized Dehong Prefecture from Southeast Asian, and then diffused to Lincang City, and finally to Xishuangbanna Prefecture. Conclusion The results presented here indicate that the current mosquito-control strategies in Yunnan Province have effectively suppressed Ae. aegypti populations within regions. Rather than focusing primarily on preventing imported cases, greater attention should now be paid to intra-provincial spread of Ae. aegypti , particularly between port and urban areas.

Introduction:The interplay between gut microbiota and host physiological processes has been extensively studied in vertebrates, where it plays a crucial role in regulating appetite, emotion, immunity, and other physiological functions. However, whether a similar regulatory mechanism exists in insects remains unclear, especially regarding the long-distance regulation of olfactory function. This study focused on three Culex subspecies (Culex quinquefasciatus, Culex pipiens pallens, and Culex pipiens molestus) that are closely related but exhibit significant differences in olfaction-dependent ecological habits. By integrating antennal transcriptomic and gut metagenomic data, we systematically analyzed the expression characteristics of olfactory-related genes, the structure of gut microbial communities, and their intrinsic associations. Methods:We integrated antennal transcriptomic and gut metagenomic sequencing to analyze olfactory-related gene expression, gut microbial community structure, and their intrinsic associations in male and female individuals of the three Culex subspecies. Bioinformatics analyses included differential gene screening, functional enrichment, microbial taxonomic annotation, and Spearman correlation analysis. Result:The results showed that a large number of sex-specific and species-specific differentially expressed genes (DEGs) were identified in the antennae of the three Culex subspecies. Among these, 345 DEGs were shared sex-specific genes across species, which were significantly enriched in pathways such as odor binding, signal transduction, and xenobiotic metabolism. At the phylum level, the gut microbial composition was dominated by Proteobacteria, Bacteroidetes, and Firmicutes, showing a conserved structure; at the genus level, 11 dominant genera (including Wolbachia, Elizabethkingia, and Asaia) exhibited distinct species-specific distribution patterns. Diversity analysis revealed that the gut microbial richness of male individuals was significantly higher than that of females, and the β-diversity showed an obvious "sex clustering" pattern.Correlation analysis further indicated that 152 DEGs were significantly correlated with 107 microbial genera. Among them, olfactory-related genes were closely associated with several core genera (e.g., Wolbachia, Asaia, Serratia). Gut microbes may remotely regulate the expression and function of olfactory genes in antennae through metabolites or signaling molecules, thereby influencing mosquito behaviors such as host localization, mating, and oviposition. Discussion:This study reveal the intrinsic association between gut microbes and olfactory function in Culex mosquitoes, providing a new perspective for understanding the "microbe-host" cross-organ regulatory mechanism and laying a theoretical foundation for the development of novel mosquito vector control strategies based on microbial or olfactory interference.

Identifying conserved RNAi targets that remain effective across mosquito species and resistance backgrounds is important for developing broadly applicable mosquito control tools. Here, an engineered Escherichia coli HT115-L4440 system was used to produce β-tubulin double-stranded RNA (dsRNA), and its larvicidal activity was evaluated in susceptible and pyrethroid-resistant strains of Culex quinquefasciatus and Aedes albopictus. Recombinant bacteria produced species-specific β-tubulin dsRNA fragments of approximately 360 bp. Immersion treatment caused high larval mortality in both species. In Cx. quinquefasciatus, mortality reached 85.00% and 89.00% in susceptible and resistant strains, respectively, compared with 8.00% and 10.00% in controls, while β-tubulin transcript abundance showed maximum reductions of 571-fold and 333-fold, respectively. In Ae. albopictus, mortality reached 91.00% and 82.00% in susceptible and resistant strains, compared with 9% and 8% in controls, with maximum reductions in β-tubulin expression of 157-fold and 337-fold, respectively. These comparable effects suggest that β-tubulin dsRNA is not compromised by conventional pyrethroid-resistance mechanisms. Transcriptomic and proteomic analyses showed that β-tubulin knockdown disrupted key cellular processes, including cytoskeletal regulation, metabolism, protein synthesis, intracellular transport, detoxification, oxidative stress, DNA replication, and autophagy. Overall, engineered bacteria-derived β-tubulin dsRNA induced consistent gene silencing and larval mortality in both susceptible and resistant mosquito larvae, supporting β-tubulin as a conserved candidate RNAi target for mosquito control.

Blattella germanica (German cockroaches) are recognized as potential carriers of intestinal pathogens, yet their role in delivering human pathogens to mammals via fecal contamination has not been experimentally evaluated. In this study, we established an experimental model using 100 B. germanica infected with a fluorescently labeled Salmonella enterica serovar Typhi Ty21a strain and assessed their ability to contaminate food and water. Fluorescent signals were detected in both midgut tissues and fecal matter of cockroaches within 6 h postinfection, peaking at 1 day (midgut: 4.52 × 1010 relative fluorescence unit [RFU] and feces: 6.12 × 108 RFU) and persisting up to 7 days. Importantly, cockroach-derived fecal suspensions administered to BALB/c mice resulted in rapid and transient intestinal exposure, with detectable bacterial signals in mouse feces within 30 min (1.13 × 103 to 1.08 × 104 RFU) and in intestinal tissues for up to 12 h; no fluorescent signal was detected thereafter (up to 7 days). Fluorescence quantification showed a positive correlation with viable bacterial counts (CFUs) (R 2 = 0.78, p < 0.05), supporting use of RFU as a proxy for Ty21a sfgfp abundance. Collectively, these findings support the potential role of B. germanica as a mechanical vector that can contaminate food and water and facilitate fecal-oral exposure of mammals to Salmonella under enclosed conditions.

Since July 2025, an outbreak of mosquito-borne chikungunya fever occurred in Foshan City, Guangdong Province, China. This was the second outbreak in China following the one that occurred in Dongguan City, Guangdong Province, in 2010. Moreover, the intensity of this outbreak was significantly greater than that of the previous one. Updates to 23 August, more than 10,000 human cases had been reported. Here, we present the first full genome sequence of the chikungunya virus (CHIKV) derived from field-trapped mosquitoes during the outbreak. Adult Aedes albopictus were BG-trap captured from residences and parklands in three hotspot towns with high density of confirmed human cases. Mosquitoes were morphologically identified and pooled by species, sex and environment types. RNA was extracted, screened by CHIKV RT-qPCR, then positive pools underwent sanger and whole-genome sequencing for complete sequences. Lineage and mutational profiles were inferred by maximum likelihood phylogenetic and comparison against human and mosquito genomes. The distribution of amino acid site mutations in different protein coding regions was also analyzed. Through 11 days of collection using 10 BG-traps, 2,803 mosquitoes were captured. 1569 (55.97%) female Ae. albopictus were divided into 77 pools and 9.09% (7/77) of the pools tested positive for CHIKV. The local Ae. albopictus minimum infection rate (MIR, per 1000 females) was 4.46, while the MIR for residences in Lecong Town was the highest at 9.17. The MIR for parklands was slight higher than for residences (4.60 vs. 4.30). All the 5 Ae. albopictus -derived complete CHIKV genome clustered within ECSA-Indian Ocean lineage genotype, closely related to human-derived genomes on 2025 Reunion Island. Amino-acid mutations E1-A226V/E2-L210Q were detected in the strains, which enhanced adaptability to Ae. albopictus and increased the transmission capacity. Novel mutation observed on E1 and E2 were totally consist to the patient-derived CHIKV in 2025 Reunion Island. It was the first mosquito-derived CHIKV whole-genome during the 2025 Foshan outbreak, filling a critical gap between human case and entomological surveillance. Ae. albopictus was confirmed as the primary vector during the outbreak. The current outbreak CHIKV strain with particular amino-acid mutations had adapted to Ae. albopictus transmission. Compared to previous Chikungunya outbreaks over the past decade, the Foshan outbreak occurred earlier (early July), in a larger urban area (with a population of over 9.5 million), and with abundant breeding sites for the vector mosquito Ae. albopictus . However, the outbreak was quickly brought under control, with daily case numbers consistently decreasing, which is closely linked to the strong vector control measures implemented by the Chinese government in the early stages of the outbreak. Moreover, this event once again underscores the necessity of early monitoring of vector mosquitoes and the importance of implementing highly effective vector intervention measures as soon as possible after an outbreak occurs.

The dengue virus (DENV) is primarily transmitted by Aedes aegypti. Investigating genes associated with mosquito susceptibility to DENV2 offers a theoretical foundation for targeted interventions to regulate or block viral replication and transmission within mosquitoes. Based on the transcriptomic analyses of the midgut and salivary glands from Aedes aegypti infected with DENV2, alongside analyses of Aag2 cell infections, 24 genes potentially related to the regulation of Aedes aegypti infection with DENV2 were selected. By establishing transient transfection and overexpression models of Aedes aegypti Aag2 cells, and mosquito target gene interference models, the difference in viral load before and after treatment was compared, and the effects of DEGs on viral replication were evaluated. After overexpressing 24 DEGs in Aag2 cells, 19 DEGs showed a significant difference in DENV2 RNA copies in the cell supernatant (p < 0.05). In adult mosquitoes, knocking down defensin-A, defensin-A-like, and SMCT1 respectively reduced the DENV2 RNA copies, while knocking down UGT2B1 and ND4 respectively increased the DENV2 RNA copies. In this study, to assess the role of genes related to DENV2 replication, and transient transfection and overexpression models in Aag2 cells and mosquito gene knockdown models were established, and five genes, defensin-A, defensin-A-like, SMCT1, UGT2B1, and ND4, were found to have an impact on the replication of DENV2, providing a reference basis for studying the complex mechanism of mosquito-virus interactions.

Background:Since July 2025, an outbreak of mosquito-borne chikungunya fever has occurred in Foshan City, Guangdong Province, China. This was the second large-scale local outbreak in China after the one that occurred in Dongguan City, Guangdong Province, in 2010. As of 23 August, more than 10,000 human cases had been reported. This study aims to investigate mosquito infection and viral genomic characteristics during the Foshan outbreak.Methods:Adult Aedes albopictus were collected using BioGents Sentinel trap in three hotspot towns (Beijiao, Chencun and Lecong). Mosquitoes were morphologically identified and pooled by species, sex and environment type. Each pool was homogenized, and the homogenate was clarified by centrifugation; and the supernatant was used for viral RNA isolation. The isolated RNA was screened using CHIKV RT-qPCR. Positive pools underwent Sanger sequencing and whole-genome sequencing. The CHIKV lineage and mutational profiles were inferred using maximum likelihood phylogenetic analysis and comparison with human- and mosquito-derived genomes. Results:Over 11 days of trapping, 2803 mosquitoes were captured. 1569 (55.97%) female Ae. albopictus were divided into 77 pools and 9.09% (7/77) of these pools were CHIKV-positive. The minimum infection rate (MIR, per 1000 females) for local Ae. albopictus was 4.46, while the MIR for residences in Lecong Town was the highest at 9.17 per 1000 females. The MIR for parklands was slightly higher than for residences (4.60 vs. 4.30 per 1000 females). Five complete Ae. albopictus-derived CHIKV genome clustered within the East/Central/South African-Indian Ocean lineage genotype, and harbored novel E1 and E2 mutations consistent with those detected in the 2025 Reunion Island human strain. Amino-acid mutations E1-A226V/E2-L210Q were detected, enhancing adaptability to Ae. albopictus and increasing the transmission capacity. Conclusions:This study represents the first mosquito-derived CHIKV whole-genome sequence obtained from the 2025 Foshan outbreak. Ae. albopictus was confirmed as the primary vector, and the presence of adaptive mutations indicated an enhanced transmission potential. Despite the outbreak emerging earlier in the season and affecting a dense urban population, it was effectively controlled through timely and intensive vector interventions. These findings highlighted the critical role of mosquito surveillance in early outbreak preparedness and effective vector management.

Background: Attractive Toxic Sugar Baits (ATSBs) are an innovative vector control strategy based on the “attract-and-kill” principle. The core of ATSBs lies in the preparation of attractive and toxic baits through the mixing and proportioning of luring and active ingredients. Although previous studies have investigated the effects of ATSBs on mosquitoes, significant challenges remain for broader field application. Methods: This study evaluated five fruit juices as ATSBs for mosquitoes, focusing on feeding preferences. Preservative concentrations were assessed by measuring antimicrobial activity over time. Two commercial traps were tested for mosquito entry rates. The optimal insecticide species and concentration were determined based on mortality rates. An optimized ATSBs system was developed and tested under a semi-field cage. Statistical analysis was performed using GraphPad Prism. Results: Within 24 h, apple juice-based ATSBs had the highest attractant index for Culex quinquefasciatus and Anopheles sinensis, while a pear juice-based ATSB was most effective for Aedes albopictus. A 0.1% preservative concentration best maintained juice stability. The LC50 values of dinotefuran-based ATSBs for Cx. quinquefasciatus, Ae. albopictus, and An. sinensis were 1.18 × 10−3, 4.06 × 10−4, and 5.20 × 10−5 g/L, respectively. The Spodoptera frugiperda trap outperformed the Drosophilidae trap. Simulated semi-field cage tests showed 48 h mortality rates of 86.00% for Cx. quinquefasciatus and 95.67% for Ae. albopictus. Conclusion: This study optimized an ATSB system by screening various fruit juices, preservative concentrations, insecticides, and trap devices. The system’s efficacy in mosquito control was evaluated under a semi-field cage. These findings provide a strong foundation for the future application and refinement of ATSB-based mosquito control strategies.

This study aimed to investigate the transcriptomic changes in the midgut and salivary glands of Aedes aegypti mosquitoes infected with Zika virus (ZIKV), in order to explore the molecular mechanisms underlying the interaction between the virus and the mosquito vector. Aedes aegypti from Jiegao (JG) and Mengding (MD) in China were experimentally infected with ZIKV, and the midgut and salivary gland tissues were collected at 2-, 4- and 6 days post-infection (dpi). High-throughput sequencing was performed to analyze the transcriptomic changes between ZIKV-infected and non-infected Ae. aegypti midgut and salivary gland tissues. Bioinformatics tools were employed for further analysis of the transcriptomic data. The expression levels of 8 significantly differentially expressed genes (DEGs) were validated using RT-qPCR. A conjoint analysis of small RNA-seq and mRNA-seq was performed to screen interactional miRNA-mRNA pairs during ZIKV infection. Using the Search Tool for the Retrieval of Interacting Genes, we constructed a protein-protein interaction network of genes and subsequently identified hub genes. The most significant transcriptional changes in Ae. aegypti occurred at 2 dpi. On 2, 4 and 6 dpi, 11 genes showed significant changes in both the midgut and salivary glands of the same mosquito strain, while 25 genes exhibited significant changes in the same tissue between the JG and MD strains. The expression tendencies of 8 DEGs obtained by RNA-Seq were similar to those detected by RT-qPCR. Furthermore, we individually identified 10 hub genes in the midgut and salivary glands. Based on previous miRNA research, we discovered the involvement of 9 miRNAs in the regulation of these hub genes. Our findings demonstrate that Ae. aegypti exhibit distinct transcriptomic changes in response to ZIKV infection. The identification of the hub genes and their regulatory miRNAs provides valuable insights into the molecular mechanisms underlying ZIKV infection in mosquitoes. This study contributes to a better understanding of the pathogen-vector interactions and may aid in the development of targeted strategies for ZIKV control.

BACKGROUND:The attractiveness of mosquitoes to humans varies among individuals, with human volatile organic compounds (VOCs) playing a pivotal role in the mosquitoes' host-seeking behavior. Differences between human volatiles detected by GC-MS can effectively modulate mosquito host selection. METHODS:Participants were enrolled and then assessed for mosquito attraction via an olfactometer. Their skin volatiles were collected with a stir bar as the sorptive extraction and were analyzed with high-resolution gas chromatography-mass spectrometry (SBSE-HRGC-MS). These data were then integrated with principal component analysis (PCA), volcano plot analysis, and partial least squares discriminant analysis (PLS-DA) to identify differential compounds between high and low mosquito attraction groups. Odorants with repellent properties were screened and evaluated using behavioral bioassays to assess their impact on the attractiveness of Aedes aegypti. RESULTS:From the 30 volunteers, 24 participants (12/12 with high/low attractiveness to mosquitoes) were enrolled. In the group with high mosquito attraction, human skin compounds such as N,N-dibutyl formamide (10.8%), decanoic acid (9.2%), and decanal (5.9%) were detected with high components. Conversely, in the low mosquito attraction group, relatively high levels of indole (0.9%), fury hydroxymethyl ketone (2.2%), and 2-hydroxy-3-methyl-2-cyclopentenone (0.8%) were observed. The results of two pathway analyses indicated that most of these compounds are associated with fatty acid metabolism, respectively. Three compounds-2-hydroxy-3-methyl-2-cyclopentenone, furfuryl hydroxymethyl ketone, and 1,2-cyclopentanedione-were identified as prominent candidates, exhibiting significant repellent efficacy in behavioral bioassays. CONCLUSIONS:In this study, the impact of differences among VOCs emitted by human skin on the host-seeking behavior of Ae. aegypti was investigated, providing insights for the development of novel mosquito baits and repellents.

Wolbachia species are symbiotic bacteria that are commonly found in arthropods and nematodes and live inside their cells. In nature, endosymbiont-host interactions and dynamics are complex, often depending on environmental conditions and evolutionary history. Both Wolbachia and mitochondrial DNA are maternally inherited in cells, and after a long period of coexistence, the presence of Wolbachia may have an impact on mitochondrial sequence diversity, thereby confounding mtDNA-based host phylogeny. The universal and typing primers for the wsp gene were used for PCR amplification, the number of positive samples was counted, and the infection pattern was analysed. The mitochondrial DNA diversity of four groups (Wolbachia-infected and uninfected samples, as well as between singly and double infected samples.) was analysed. PACo and ParaFitGlobal tests were used to explore evolutionary associations. The overall prevalence of Wolbachia in the 22 natural populations was 94.2 %, with Type A, Type B and A x B mixed infections detected in Aedes albopictus and coinfection between wAlbA and wAlbB prevalent. The mitochondrial DNA haplotype associated with Wolbachia (Hap1) became the dominant haplotype and was the most abundant and widely distributed in the population. The linkage map showed the predominant haplotype, Hap1, was more closely associated with wAlbA than with wAlbB. Neutral evolution deviated significantly from zero. The diversity of mtDNA COI genes associated with Wolbachia infection was reduced. Wolbachia infection may lead to the selective sweep of mitochondrial DNA in Ae. albopictus.

BACKGROUND:Recently, the effect of artificial light at night (ALAN) on the physiology and behavior of insects has gradually attracted the attention of researchers and has become a new research topic. Aedes albopictus is an important vector that poses a great public health risk. Further studies on the diapause of Ae. albopictus can provide a basis for new vector control, and it is also worth exploring whether the effect of ALAN on the diapause of Ae. albopictus will provide a reference for the prevention and control of infectious diseases mediated by Ae. albopictus. METHODS:In this study, we experimentally studied the diapause characteristics of different geographical strains of Ae. albopictus under the interference of ALAN, explored the effect of ALAN on the diapause of Ae. albopictus and explored the molecular mechanism of ALAN on the diapause process through RNA-seq. RESULTS:As seen from the diapause incidence, Ae. albopictus of the same geographic strain showed a lower diapause incidence when exposed to ALAN. The differentially expressed genes (DEGs) were mainly enriched in signaling and metabolism-related pathways in the parental females and diapause eggs of the ALAN group. CONCLUSIONS:ALAN inhibits Ae. albopictus diapause. In the short photoperiod induced diapause of Ae. albopictus in temperate strain Beijing and subtropical strain Guangzhou, the disturbance of ALAN reduced the egg diapause rate and increased the egg hatching rate of Ae. albopictus, and the disturbance of ALAN also shortened the life cycle of Ae. albopictus eggs after hatching.

GNBPB6, a beta-1,3-glucan-binding protein, was identified in the transcriptome of Aedes aegypti (A. aegypti) with dengue (DENV), Zika (ZIKV), and chikungunya viruses (CHIKV). In this study, we not only clarified that DENV2 and ZIKV regulate the changes in GNBPB6 expression but also identified the relationship of this gene with viral infections. The changes in GNBPB6 expression were quantified and showed a decrease in A. aegypti cells (Aag2 cells) at 2 dpi and 3 dpi and an increase at 4 dpi and 5 dpi (p < 0.05). A significant increase was observed only at 5 dpi after DENV2 infection. Subsequently, a GNBPB6 knockout (KO) cell line was constructed using the CRISPR/Cas9 system, and the DENV2 and ZIKV RNA copies, along with cell densities, were quantified and compared between the KO and wild type (WT) cells at different dpi. The result showed that DENV2 and ZIKV RNA copies were significantly increased in the KO cell line with no significant change in cell growth. Finally, DENV2 copies decreased after GNBPB6 was complemented in the KO. In conclusion, GNBPB6 knockout and complementation in Aag2 cells revealed that GNBPB6 can inhibit the replication of both DENV2 and ZIKV. These results contribute to subsequent research on mosquito–virus interactions.

BACKGROUND:Hainan Island and the Leizhou Peninsula, the southernmost part of mainland China, are areas where Aedes aegypti and Ae. albopictus are sympatric and are also high-incidence areas of dengue outbreaks in China. Many studies have suggested that Aedes endogenous viral components (EVEs) are enriched in piRNA clusters which can silence incoming viral genomes. Investigation the EVEs present in the piRNA clusters associated with viral infection of Aedes mosquitoes in these regions may provide a theoretical basis for novel transmission-blocking vector control strategies. METHODS:In this study, specific primers for endogenous Flaviviridae elements (EFVEs) and endogenous Rhabdoviridae elements (ERVEs) were used to detect the distribution of Zika virus infection associated EVEs in the genomes of individuals of the two Aedes mosquitoes. Genetic diversity of EVEs with a high detection rate was also analyzed. RESULTS:The results showed that many EVEs associated with Zika virus infection were detected in both Aedes species, with the detection rates were 47.68% to 100% in Ae. aegypti and 36.15% to 92.31% in sympatric Ae. albopictus populations. EVEs detection rates in another 17 Ae. albopictus populations ranged from 29.39% to 89.85%. Genetic diversity analyses of the four EVEs (AaFlavi53, AaRha61, AaRha91 and AaRha100) of Ae. aegypti showed that each had high haplotype diversity and low nucleotide diversity. The number of haplotypes in AaFlavi53 was 8, with the dominant haplotype being Hap_1 and the other 7 haplotypes being further mutated from Hap_1 in a lineage direction. In contrast, the haplotype diversity of the other three ERVEs (AaRha61, AaRha91 and AaRha100) was more diverse and richer, with the haplotype numbers were 9, 15 and 19 respectively. In addition, these EVEs all showed inconsistent patterns of both population differentiation and dispersal compared to neutral evolutionary genes such as the Mitochondrial COI gene. CONCLUSION:The EFVEs and ERVEs tested were present at high frequencies in the field Aedes mosquito populations. The haplotype diversity of the EFVE AaFlavi53 was relatively lower and the three ERVEs (AaRha61, AaRha91, AaRha100) were higher. None of the four EVEs could be indicative of the genetic diversity of the Ae. aegypti population. This study provided theoretical support for the use of EVEs to block arbovirus transmission, but further research is needed into the mechanisms by which these EVEs are antiviral to Aedes mosquitoes.

BACKGROUND:Aedes aegypti is a main vector of arboviral diseases, principally dengue, chikungunya, and Zika. Insecticides remain the most effective vector control method. Pyrethroid is the main insecticide currently used, and the long-term use of insecticides can cause mosquitoes to develop knockdown resistance. Studying the mutation sites and genotypes of Ae. aegypti can reveal the mutation characteristics and regional distribution of the kdr gene in an Ae. aegypti population. Testing for a correlation between the mutation rate in various populations and pyrethrin resistance can clarify the resistance mechanism. RESULTS:The bioassay results showed that all 15 populations are resistant. In the study of the kdr gene, three non-synonymous mutations were identified in the DNA of first generation females from the wild Ae. aegypti population: S989P (TCC-CCC), V1016G (GTA-GGA), and F1534C (TTC-TGC). The mortality rate of the various populations was correlated with the mutation rate at the V1016G + F1534C locus, but not the S989P + V1016G locus. CONCLUSION:Aedes aegypti populations in border regions of Yunnan Province are resistant to permethrin and beta-cyfluthrin. The insecticidal effect of beta-cyfluthrin is stronger than that of permethrin. The mutation rate at sites V1016G + F1534C is negatively correlated with the mortality of Ae. aegypti based on bioassays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

E-20-monooxygenase (E20MO) is an enzymatic product of the shade (shd) locus (cytochrome p450, E20MO). Initially discovered in Drosophila, E20MO facilitates the conversion of ecdysone (E) into 20-hydroxyecdysone (20E) and is crucial for oogenesis. Prior research has implicated 20E in growth, development, and insecticide resistance. However, little attention has been given to the association between the E20MO gene and DENV2 infection. The transcriptome of Ae. aegypti cells (Aag2 cells) infected with DENV2 revealed the presence of the E20MO gene. The subsequent quantification of E20MO gene expression levels in Aag2 cells post-DENV infection was carried out. A CRISPR/Cas9 system was utilized to create an E20MO gene knockout cell line (KO), which was then subjected to DENV infection. Analyses of DENV2 copies in KO and wild-type (WT) cells were conducted at different days post-infection (dpi). Plasmids containing E20MO were constructed and transfected into KO cells, with pre- and post-transfection viral copy comparisons. Gene expression levels of E20MO increased after DENV infection. Subsequently, a successful generation of an E20MO gene knockout cell line and the verification of code-shifting mutations at both DNA and RNA levels were achieved. Furthermore, significantly elevated DENV2 RNA copies were observed in the mid-infection phase for the KO cell line. Viral RNA copies were lower in cells transfected with plasmids containing E20MO, compared to KO cells. Through knockout and plasmid complementation experiments in Aag2 cells, the role of E20MO in controlling DENV2 replication was demonstrated. These findings contribute to our understanding of the intricate biological interactions between mosquitoes and arboviruses.

Aedes albopictus, a common mosquito in Zhejiang Province, is a carrier of more than twenty arboviruses. There are dozens or even hundreds of imported cases of dengue fever every year in Zhejiang Province, and there have also been many local outbreaks caused by imported cases of dengue fever. The objectives were to assess the resistance of larvae and adults of several Ae. albopictus strains in Zhejiang Province to commonly used pyrethroid insecticides (beta-cypermethrin, deltamethrin and permethrin), and detect mutations in the sodium channel gene, to further analyse the relationship between phenotypic resistance and the frequency of mutations. The resistance of eight field strains of Ae. albopictus larvae to beta-cypermethrin, deltamethrin and permethrin ranged from 8.17 to 36.06, 12.12-107.3 and 1.55-81.9, respectively, and there was a significant positive correlation of interaction resistance among the three insecticides. The mutation frequencies of I1532T and F1534S in the larvae of Ae. albopictus were 0-6.25 % and 42.19-100.00 %. Moreover, the diagnostic doses of the three pyrethroids for adult Ae. albopictus mosquitoes were 0.2510 g/L, 0.1562 g/L, and 0.9072 g/L. Except for the Zhoushan strain, which was suspected to be resistant to beta-cypermethrin, the other field strains were resistant to the three pyrethroids, and there was a significant positive correlation of cross-resistance among the three insecticides. The mutation frequencies of I1532T and F1534S of adult Ae. albopictus were 0-1.56 % and 62.50-100.00 %. In addition, the LC50 of the larvae and the mortality rate of adult Ae. albopictus after treatment with the three pyrethroids were significantly and positively correlated with the frequency of the F1534S mutation. F1534S mutation occurred earlier than I1532T mutation in both larvae and adult Ae. albopictus. F1534S mutation in the sodium channel gene may be a particular biomolecular detection marker for resistance to pyrethroid insecticides in Ae. albopictus in Zhejiang Province.

Microplastics (MPs) transfer from the environment to living organisms is a nonignorable global problem. As a complete metamorphosis insect, the larvae and adult Culex quinquefasciatus mosquito live in aquatic and terrestrial environments, respectively, where they easily access MPs. However, little is known about mosquitoes' potential role in MPs accumulation throughout ecosystems. Therefore, we conducted a study with different MPs particle sizes (0.1/1/10 mu m) and concentrations (0.5/5/50 mu g/mL) on Cx. quinquefasciatus to address this issue. Once exposed at the young larval stage, MPs could accompany the mosquitoes their entire life. The fluorescence signals of MPs in the larvae were mainly located in the intestines. Its intensity increased (from 3.72 x 10(6 )AU to 5.45 x 10(7) AU) as the concentrations of MPs increases. The fluorescence signals of MPs were also detected in the blood and skin tissues of mice bitten by adult mosquitoes with MPs containing in their bodies. Mosquitos exposed to MPs showed longer larval pupation and eclosion time as well as lower adult body weight. In addition, MPs significantly reduced the lethal effect of pyrethroid insecticides (97.77 % vs. 48.88 %, p < 0.05) with 15.1 % removal of the deltamethrin concentration. After MPs exposure, the relative abundance of the Cx. quinquefasciatus gut microbiome, such as Wolbachia spp., Elizabethkingia spp., and Asaia spp., changed as the MPs size and concentration changes.