Model predictions and laboratory studies have shown that temperature affects the vector competence of Ae. albopictus for transmitting DENV [16, 25]. However, little is known about the underlying mechanisms. In this study, RNA sequencing of the midguts of Ae. albopictus in the CT, MB and MNB groups was conducted, and the results reveal different transcriptional variations in response to DENV infection and temperature. WGCNA was used to identify key gene networks regulated by temperature. Then, we determined hub pathways associated with temperature.
In this research, we collected Ae. albopictus mosquitoes with different infection statuses. To simultaneously collect mosquitoes with and without midgut breakthrough at 23°C, 28°C and 32°C, we determined the optical concentration of DENV-2 to be 8.625 log10 TCID50/mL based on preliminary experimental results, with a time to harvest of 7 days after infection. Under this condition, the MIR, MBR and MNBR of Ae. albopictus were detected. The trend of the results was consistent with previous experimental research and model predictions [16, 25, 26].
The transcriptome of the midgut of Aedes mosquitoes is altered by the DENV infection. Indeed, expression profiles of the Ae. aegypti midgut responding to DENV infection were changed. For example, antimicrobial peptides (AMPs) induced by the Toll immune pathway reportedly increased at 7 days post-infection [27], and Ae. albopictus midgut genes were modulated by DENV exposure in a time- and site-specific manner [28]. In our study, the expression profiles of the Ae. albopictus midgut clustered after the ingestion of a blood meal containing DENV-2. Furthermore, we analyzed Ae. albopictus transcriptomes of the MB and MNB groups at different temperatures and found that temperature affected the midgut transcriptome clustering: low temperature resulted in more DEGs. Similar to our findings, previous research has demonstrated that temperature shapes Ae. aegypti gene expression after ZIKV infection [19].
To better understand the relationship between temperature and infection in Ae. albopictus, we used WGCNA, which is a method for gene co-expression networks [29, 30]. In 2008, the WGCNA software package was developed using R; it is mainly used to analyze large sample expression data, such as gene expression microarray or RNA sequencing data [23]. WGCNA has been proven to be an effective method to detect co-expressed modules and hub genes in tumors, plants, parasites, etc [31-33]. In this study, the highest correlation with temperature was observed for the ME3 module.
The mosquito transcriptome changed in response to DENV, which might be related to the mosquito's antiviral system. In contrast to the innate and adaptive immunity of humans to resist the invasion of pathogens, mosquitoes lack adaptive immunity and mainly rely on innate immunity to suppress virus proliferation. RNA interference (RNAi), Toll, immunodeficiency factor (IMD), Janus kinase pathway signal transduction and activation (JAK-STAT) and other pathways play an important role in the mosquito antiviral immunity [34]. The gene expression profiles of mosquitoes infected with viruses were transformed following temperature change. In a previous study, Ae. aegypti infected with chikungunya virus (CHIKV) were cultured at 18°C, 28°C and 32°C; the Toll, IMD and JAK-STAT pathways were upregulated at 28°C, and high temperature appeared to damage the immune defenses [35]. In our study, the pathways of the ME3 module regulated by temperature included RNA degradation, Toll pathway and IMD pathway.
DENV is an RNA virus, and the RNA degradation pathway is closely related to the proliferation of DENV in mosquitos [36]. Many eukaryotic proteins that interact with the 5'm7GpppN cap affect the metabolism and translation of mRNA [37]. We speculate that m7GpppN-mRNA hydrolase is involved in the replication of DENV in Ae. albopictus. Sm-like (Lsm) proteins participate in pre-mRNA splicing, nuclear RNA processing and miRNA biogenesis and mediate the antiviral immunity through the RNAi pathway [38]. PAT1 is an RNA-binding protein that plays a role in mRNA decay by physically linking de-adenylation with de-capping and by acting as a translation repressor to regulate the gene expression [39]. The Lsm1–7-Pat1 complex binds to cis-acting regulatory sequences of viral positive-strand RNA genomes, which promotes the transcription, translation and replication of the virus [40]. heat shock cognate 70 protein interacts with CHIKV to promote its entry into C6/36 cells [41], and 70-kDa heat shock cognate proteins have been identified as the most critical components in DENV-4 binding and entry into C6/36 cells [42]. Additionally, the CCR4-NOT transcription complex subunit 6-like belongs to the CCR4-NOT complex family, which is involved in the transcription, translation and mRNA decay [43]. The expression level of CCR4-Not complex genes was up-regulated in DENV-infected cells, which is conducive to the proliferation of DENV [44]. In this study, it was highly expressed at 32°C, which suggests that DENV-2 proliferated more quickly at 32°C, which helped DENV-2 break through the midgut barrier.
Regarding Toll pathways, pattern recognition receptors (PRRs) recognize mosquito-borne viruses, which promotes the maturation of Spätzle (Spz). The Spz interaction with Toll-like receptors (TLRs) involves myeloid differentiation gene 88 (MyD88), followed by the activation of the nuclear transcription factor (NF-κB), which induces the release of nuclear antimicrobial peptides (AMPs) and other antiviral molecules [45]. Three proteins are regulated by temperature: modular serine protease, peptidoglycan-recognition protein (PGRP) and MyD88. Modular serine protease is recruited to the lysine-type peptidoglycan recognition complex, which activates the serine protease cascade [46]. It serves as a crucial enzyme to elicit insect immune responses, especially for Toll pathway activation [47]. PGRP is the most important PRR in insects; it can bind to and hydrolyze bacterial peptidoglycan to activate innate immunity [48,49]. MyD88 serves as the key mediator of Toll signaling. The inhibition of MyD88 significantly enhances the replication of DENV-2 in Ae. Aegypti [50]. MyD88 is also involved in the antiviral immunity of Ae. aegypti against Japanese encephalitis virus (JEV) [51].
In this study, functional verification of these genes regulated by temperature was absent. We will design the siRNA and dsRNA of these genes to detect the proliferation ability of DENV-2 at the cell level and analyze the vector competence of Ae. albopictus to transmit DENV-2 at different temperatures.