Juvenile Hormone-Sensitive Ribosomal Activity Enhances Viral Replication in Aedes aegypti.

Zika virus (ZIKV; Flaviviridae) is a devastating virus transmitted to humans by the mosquito Aedes aegypti. The interaction of the virus with the mosquito vector is poorly known. The double-stranded RNA (dsRNA)-mediated interruption or activation of immunity-related genes in the Toll, IMD, JAK-STAT, and short interfering RNA (siRNA) pathways did not affect ZIKV infection in A. aegypti. Transcriptome-based analysis indicated that most immunity-related genes were upregulated in response to ZIKV infection, including leucine-rich immune protein (LRIM) genes. Further, there was a significant increment in the ZIKV load in LRIM9-, LRIM10A-, and LIRM10B-silenced A. aegypti, suggesting their function in modulating viral infection. Further, gene function enrichment analysis revealed that viral infection increased global ribosomal activity. Silencing of RpL23 and RpL27, two ribosomal large subunit genes, increased mosquito resistance to ZIKV infection. In vitro fat body culture assay revealed that the expression of RpL23 and RpL27 was responsive to the Juvenile hormone (JH) signaling pathway. These two genes were transcriptionally regulated by JH and its receptor methoprene-tolerant (Met) complex. Silencing of Met also inhibited ZIKV infection in A. aegypti. This suggests that ZIKV enhances ribosomal activity through JH regulation to promote infection in mosquitoes. Together, these data reveal A. aegypti immune responses to ZIKV and suggest a control strategy that reduces ZIKV transmission by modulating host factors. IMPORTANCE Most flaviviruses are transmitted between hosts by arthropod vectors such as mosquitoes. Since therapeutics or vaccines are lacking for most mosquito-borne diseases, reducing the mosquito vector competence is an effective way to decrease disease burden. We used high-throughput sequencing technology to study the interaction between mosquito Aedes aegypti and ZIKV. Leucine-rich immune protein (LRIM) genes were involved in the defense in response to viral infection. In addition, RNA interference (RNAi) silencing of RpL23 and RpL27, two JH-regulated ribosomal large subunit genes, suppressed ZIKV infection in A. aegypti. These results suggest a novel control strategy that could block the transmission of ZIKV.

Three novel trehalase genes from Harmonia axyridis (Coleoptera: Coccinellidae): cloning and regulation in response to rapid cold and re-warming.

Trehalose is the main blood sugar in insects. To study the function of trehalase during exposure to low temperatures, three other novel cDNAs of trehalase were cloned from Harmonia axyridis by transcriptome sequencing and rapid amplification of cDNA ends. One of the cloned cDNAs encoded a soluble trehalase, the second trehalase cDNA encoded a transmembrane-like domain, and the third cDNA encoded a membrane-bound protein. Therefore, these cDNAs were, respectively, named HaTreh1-5, HaTreh2-like, and HaTreh2. HaTreh1-5, HaTreh2-like, and HaTreh2 cDNAs encoded proteins containing 586, 553, and 633 amino acids with predicted masses of approximately 69.47, 63.46, and 73.66 kDa, and pIs of 9.20, 5.52, and 6.31, respectively. All three novel trehalases contained signal motifs "PGGINKESYYLDSY", "QWDYPNAWPP", and a highly conserved glycine-rich (GGGGEY) region. The expression levels of HaTreh1-5 and HaTreh2 mRNAs were high during adult stages, whereas HaTreh2-like was expressed in low amounts in the fourth larval stage. The results showed that the activity of membrane-bound trehalases decreased from 25 to 10 °C and from 5 to - 5 °C during cooling. The results also revealed a decreasing trend in expression of the three HaTreh mRNAs during the cooling treatment, and an initial decrease followed by an increase during the process of re-warming.

Aedes aegypti HPX8C modulates immune responses against viral infection.

Mosquitoes act as vectors of numerous pathogens that cause human diseases. Dengue virus (DENV) transmitted by mosquito, Aedes aegypti, is responsible for dengue fever epidemics worldwide with a serious impact on human health. Currently, disease control mainly relies on vector targeted intervention strategies. Therefore, it is imperative to understand the molecular mechanisms underlying the innate immune response of mosquitoes against pathogens. In the present study, the expression profiles of immunity-related genes in the midgut responding to DENV infection by feeding were analyzed by transcriptome and quantitative real-time PCR. The level of Antimicrobial peptides (AMPs) increased seven days post-infection (d.p.i.), which could be induced by the Toll immune pathway. The expression of reactive oxygen species (ROS) genes, including antioxidant genes, such as HPX7, HPX8A, HPX8B, HPX8C were induced at one d.p.i. and peaked again at ten d.p.i. in the midgut. Interestingly, down-regulation of the antioxidant gene HPX8C by RNA interference led to reduction in the virus titer in the mosquito, probably due to the elevated levels of ROS. Application of a ROS inhibitor and scavenger molecules further established the role of oxygen free radicals in the modulation of the immune response to DENV infection. Overall, our comparative transcriptome analyses provide valuable information about the regulation of immunity related genes in the transmission vector in response to DENV infection. It further allows us to identify novel molecular mechanisms underlying the host-virus interaction, which might aid in the development of novel strategies to control mosquito-borne diseases.

Effects of starvation on the carbohydrate metabolism in Harmonia axyridis (Pallas).

Trehalose plays an important role in energy storage, metabolism, and protection from extreme environmental conditions in insects. Trehalose is the main blood sugar in insects, and it can be rapidly used as an energy source in times of need. To elucidate the mechanisms of the starvation response, we observed the effects of starvation on trehalose and glycogen, trehalase activity, and the relative gene expression of genes in the trehalose and glycogen metabolic pathways in the invasive beetle Harmonia axyridis Our results show that trehalose levels and the activities of two types of trehalases decreased significantly in the first 8 h of starvation, while the relative expression of HaTreh1-1 increased. While trehalose remained nearly constant at a relatively high level from 8 to 24 h, glycogen levels decreased significantly from 8 h to 24 h of starvation. Likewise, glycogen phosphorylase (HaGP) expression was significantly higher at 12 to 24 h starvation than the first 8 h, while the expression of glycogen synthase (HaGS) was relatively stable. Furthermore, trehalose decreased significantly from 24 h starvation to 72 h starvation, while trehalase activities and the relative expression of some HaTreh genes generally increased toward the end of the starvation period. The expression of trehalose-6-phosphate synthase (HaTPS) increased significantly, supporting the increase in trehalose synthesis. These results show that trehalose plays a key role in the energy provided during the starvation process through the molecular and biochemical regulation of trehalose and glycogen metabolism.

Transcriptome analysis and identification of induced genes in the response of Harmonia axyridis to cold hardiness.

Harmonia axyridis is an important predatory lady beetle that is a natural enemy of agricultural and forestry pests. In this research, the cold hardiness induced genes and their expression changes in H. axyridis were screened and detected by the way of the transcriptome and qualitative real-time PCR under normal and low temperatures, using high-throughput transcriptome and digital gene-expression-tag technologies. We obtained a 10Gb transcriptome and an 8Mb gene expression tag pool using Illumina deep sequencing technology and RNA-Seq analysis (accession number SRX540102). Of the 46,980 non-redundant unigenes identified, 28,037 (59.7%) were matched to known genes in GenBank, 21,604 (46.0%) in Swiss-Prot, 19,482 (41.5%) in Kyoto Encyclopedia of Genes and Genomes and 13,193 (28.1%) in Gene Ontology databases. Seventy-five percent of the unigene sequences had top matches with gene sequences from Tribolium castaneum. Results indicated that 60 genes regulated the entire cold-acclimation response, and, of these, seven genes were always up-regulated and five genes always down-regulated. Further screening revealed that six cold-resistant genes, E3 ubiquitin-protein ligase, transketolase, trehalase, serine/arginine repetitive matrix protein 2, glycerol kinase and sugar transporter SWEET1-like, play key roles in the response. Expression from a number of the differentially expressed genes was confirmed with quantitative real-time PCR (HaCS_Trans). The paper attempted to identify cold-resistance response genes, and study the potential mechanism by which cold acclimation enhances the insect's cold endurance. Information on these cold-resistance response genes will improve the development of low-temperature storage technology of natural enemy insects for future use in biological control.

Molecular Cloning and Induced Expression of Six Small Heat Shock Proteins Mediating Cold-Hardiness in Harmonia axyridis (Coleoptera: Coccinellidae).

The main function of small heat shock proteins (sHSPs) as molecular chaperones is to protect proteins from denaturation under adverse conditions. Molecular and physiological data were used to examine the sHSPs underlying cold-hardiness in Harmonia axyridis. Complementary DNA sequences were obtained for six H. axyridis sHSPs based on its transcriptome, and the expression of the genes coding for these sHSPs was evaluated by quantitative real-time PCR (qRT-PCR) in several developmental stages, under short-term cooling or heating conditions, and in black and yellow females of experimental and overwintering populations under low-temperature storage. In addition, we measured water content and the super cooling and freezing points (SCP and FP, respectively) of H. axyridis individuals from experimental and overwintering populations. The average water content was not significantly different between adults of both populations, but the SCP and FP of the overwintering population were significantly lower than that of the experimental population. Overall, the six sHSPs genes showed different expression patterns among developmental stages. In the short-term cooling treatment, Hsp16.25 and Hsp21.00 expressions first increased and then decreased, while Hsp10.87 and Hsp21.56 expressions increased during the entire process. Under short-term heating, the expressions of Hsp21.00, Hsp21.62, Hsp10.87, and Hsp16.25 showed an increasing trend, whereas Hsp36.77 first decreased and then increased. Under low-temperature storage conditions, the expression of Hsp36.77 decreased, while the expressions of Hsp21.00 and Hsp21.62 were higher than that of the control group in the experimental population. The expression of Hsp36.77 first increased and then decreased, whereas Hsp21.56 expression was always higher than that of the control group in the overwintering population. Thus, differences in sHSPs gene expression were correlated with the H. axyridis forms, suggesting that the mechanism of cold resistance might differ among them. Although, Hsp36.77, Hsp16.25, Hsp21.00, and Hsp21.62 regulated cold- hardiness, the only significant differences between overwintering and experimental populations were found for Hsp16.25 and Hsp21.00.