Response of the mosquito immune system and symbiotic bacteria to pathogen infection.

Mosquitoes are the deadliest animal in the word, transmitting a variety of insect-borne infectious diseases, such as malaria, dengue fever, yellow fever, and Zika, causing more deaths than any other vector-borne pathogen. Moreover, in the absence of effective drugs and vaccines to prevent and treat insect-borne diseases, mosquito control is particularly important as the primary measure. In recent decades, due to the gradual increase in mosquito resistance, increasing attention has fallen on the mechanisms and effects associated with pathogen infection. This review provides an overview of mosquito innate immune mechanisms in terms of physical and physiological barriers, pattern recognition receptors, signalling pathways, and cellular and humoral immunity, as well as the antipathogenic effects of mosquito symbiotic bacteria. This review contributes to an in-depth understanding of the interaction process between mosquitoes and pathogens and provides a theoretical basis for biological defence strategies against mosquito-borne infectious diseases.

Screening for odorant receptor genes expressed in Aedes aegypti involved in host-seeking, blood-feeding and oviposition behaviors.

BACKGROUND: Aedes aegypti is one of the most important vectors of zoonotic diseases worldwide, and its survival and reproductive processes depend heavily on its olfactory system. In this study, the expression levels of all odorant receptor (OR) genes of Ae. aegypti were explored during different physiological periods to identify olfactory genes that may be associated with mosquito blood-feeding and the search for oviposition sites. METHODS: Four experimental groups, consisting of Ae. aegypti males, pre-blood-feeding females, post-blood-feeding females and post-oviposition females, were established. A total of 114 pairs of primers targeting all messenger RNA encoded by OR genes were designed based on the whole genome of Ae. aegypti. The expression of OR genes was evaluated by real-time fluorescence quantitative PCR for relative quantification and the comparison of differences between groups. RESULTS: A total of 53 differentially expressed OR genes were identified between males and females in Ae. aegypti antennae. Also, eight, eight and 13 differentially expressed OR genes were identified in pre- versus post-blood-feeding females, in pre- versus post-oviposition females and in post-blood-feeding versus post-oviposition females, respectively. In addition, 16 OR genes were significantly differentially expressed in multiple physiological periods of the mosquitoes. CONCLUSIONS: A large number of ORs with significant intergroup differences and high expression levels were screened in this study. Some of these genes are reported for the first time, providing possible targets for the development of mosquito control pathways based on the olfactory system.

Metagenome Sequencing Reveals the Microbiome of Aedes albopictus and Its Possible Relationship With Dengue Virus Susceptibility.

Dengue fever virus (DENV) is a mosquito-borne flavivirus that poses a serious risk to human health. Aedes albopictus is a widely distributed vector of dengue fever in China. Based on the impact of physiological activity, the microbiome in A. albopictus will provide a novel environment-friendly approach to control DENV transmission. We performed metagenomic sequencing on A. albopictus before and after exposure to DENV blood meal to detect microbiome variation of A. albopictus with different susceptibilities to DENV. The dominant phyla in A. albopictus microbiome were Proteobacteria and Ascomycota, and the dominant genera were Aspergillus and Metarhizium. Gammaproteobacteria bacterium, Lactobacillus harbinensis, and Neurospora crassa differed significantly after DENV infection. There were 15 different microorganisms found to be involved in mosquito immunity and metabolism, such as Alphaproteobacteria bacterium, Methyloglobulus morosus, and Shigella sonnei, which might have an impact on the DENV susceptibility of A. albopictus. It was hypothesized that the lack of specific bacteria may lead to increased susceptibility of A. albopictus to DENV. Interventions in the microbiome composition or specific bacteria of A. albopictus may affect the susceptibility to DENV and control the mosquito-borne diseases efficiently.

Screening of olfactory genes related to blood-feeding behaviors in Culex pipiens quinquefasciatus and Culex pipiens molestus by transcriptome analysis.

BACKGROUND: Culex pipiens quinquefasciatus Say (Cx. quinquefasciatus) and Culex pipiens form molestus Forskal (Cx. molestus) in the Culex pipiens complex group show considerable differences in host seeking, blood feeding, mating behavior and in vector competence. Blood-feeding mosquito behaviors are closely related to their olfactory gene expression and olfactory gene repertoire composition. Comparing olfactory genes between these two subspecies with significantly different blood-feeding behaviors can support further research on the molecular mechanism of the Culex pipiens complex olfactory sensory system, providing a new approach for determining candidate attractant or repellent compounds. METHODS: Non-blood-feeding (NBF) and post-blood-feeding (PBF) olfactory system transcriptomes of the two subspecies were sequenced, and the biological functions of their differentially expressed genes were described by bioinformatics analysis. A quantitative polymerase chain reaction (qPCR) was applied to validate the RNA-seq data. The roles of particular olfactory receptors in Cx. quinquefasciatus blood-feeding behaviors were evaluated by RNAi. RESULTS: Five, 7, 24, and 3 Cx. quinquefasciatus-specific OBPs, Cx. molestus-specific OBPs, Cx. quinquefasciatus-specific ORs and Cx. molestus-specific ORs were identified, respectively. The majority of selected ORs were consistent with the predicted transcriptome sequencing results after qRT-PCR validation. OR5 was expressed only in Cx. quinquefasciatus, and OR65 was the only gene upregulated after blood feeding in Cx. molestus. The blood-feeding rates of the OR5 and OR78 dsRNA groups were significantly lower (4.3%±3.1% and 13.3%±11.5%) than those of the enhanced green fluorescence protein (EGFP) group (64.5%±8.7%). CONCLUSION: Most OBPs and ORs were expressed in both subspecies but showed divergence in expression level. OR5 and OR65 might be species-specific expressed genes that regulate the olfactory behaviors of Cx. quinquefasciatus and Cx. molestus, respectively. The RNA interference of OR5 and OR78 could inhibit the blood-feeding behavior of Cx. quinquefasciatus, providing new targets for screening effective repellent compounds to control mosquito-borne diseases effectively and efficiently.

Transcriptome Analysis of Responses to Dengue Virus 2 Infection in Aedes albopictus (Skuse) C6/36 Cells.

Dengue virus (DENV), a member of the Flavivirus genus of the Flaviviridae family, can cause dengue fever (DF) and more serious diseases and thus imposes a heavy burden worldwide. As the main vector of DENV, mosquitoes are a serious hazard. After infection, they induce a complex host-pathogen interaction mechanism. Our goal is to further study the interaction mechanism of viruses in homologous, sensitive, and repeatable C6/36 cell vectors. Transcriptome sequencing (RNA-Seq) technology was applied to the host transcript profiles of C6/36 cells infected with DENV2. Then, bioinformatics analysis was used to identify significant differentially expressed genes and the associated biological processes. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to verify the sequencing data. A total of 1239 DEGs were found by transcriptional analysis of Aedes albopictus C6/36 cells that were infected and uninfected with dengue virus, among which 1133 were upregulated and 106 were downregulated. Further bioinformatics analysis showed that the upregulated DEGs were significantly enriched in signaling pathways such as the MAPK, Hippo, FoxO, Wnt, mTOR, and Notch; metabolic pathways and cellular physiological processes such as autophagy, endocytosis, and apoptosis. Downregulated DEGs were mainly enriched in DNA replication, pyrimidine metabolism, and repair pathways, including BER, NER, and MMR. The qRT-PCR results showed that the concordance between the RNA-Seq and RT-qPCR data was very high (92.3%). The results of this study provide more information about DENV2 infection of C6/36 cells at the transcriptome level, laying a foundation for further research on mosquito vector-virus interactions. These data provide candidate antiviral genes that can be used for further functional verification in the future.

Transcriptome analysis of Aedes aegypti Aag2 cells in response to dengue virus-2 infection.

BACKGROUND: Dengue virus (DENV) is a flavivirus transmitted by mosquitoes that is prevalent in tropical and subtropical countries and has four serotypes (DENV1-4). Aedes aegypti, as the main transmission vector of DENV, exhibits strong infectivity and transmission. With the aim of obtaining a better understanding of the Ae. aegypti-DENV interaction, the transcriptome changes in DENV-2-infected Aag2 cells were studied to describe the immune responses of mosquitoes using the Ae. aegypti Aag2 cell line as a model. METHODS: RNAseq technology was used to sequence the transcripts of the Ae. aegypti Aag2 cell line before and after infection with DENV-2. A bioinformatics analysis was then performed to assess the biological functions of the differentially expressed genes, and the sequencing data were verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: The transcriptome analysis generated 8866 unigenes that were found in both groups, 225 unigenes that were only found in the infection group, and 683 unigenes that only existed in the control group. A total of 1199 differentially expressed genes, including 1014 upregulated and 185 downregulated genes, were identified. The bioinformatics analysis showed that the differentially expressed genes were mainly involved in the longevity regulating pathway, circadian rhythm, DNA replication, and peroxisome, purine, pyrimidine, and drug metabolism. The qRT-PCR verification results showed the same trend, which confirmed that the expression of the differentially expressed genes had changed, and that the transcriptome sequencing data were reliable. CONCLUSIONS: This study investigated the changes in the transcriptome levels in the DENV-2-infected Ae. aegypti Aag2 cell line, which provides a faster and effective method for discovering genes related to Ae. aegypti pathogen susceptibility. The findings provide basic data and directions for further research on the complex mechanism underlying host-pathogen interactions.