A Critical Role for CLSP2 in the Modulation of Antifungal Immune Response in Mosquitoes.

Entomopathogenic fungi represent a promising class of bio-insecticides for mosquito control. Thus, detailed knowledge of the molecular mechanisms governing anti-fungal immune response in mosquitoes is essential. In this study, we show that CLSP2 is a modulator of immune responses during anti-fungal infection in the mosquito Aedes aegypti. With a fungal infection, the expression of the CLSP2 gene is elevated. CLSP2 is cleaved upon challenge with Beauveria bassiana conidia, and the liberated CLSP2 CTL-type domain binds to fungal cell components and B. bassiana conidia. Furthermore, CLPS2 RNA interference silencing significantly increases the resistance to the fungal challenge. RNA-sequencing transcriptome analysis showed that the majority of immune genes were highly upregulated in the CLSP2-depleted mosquitoes infected with the fungus. The up-regulated immune gene cohorts belong to melanization and Toll pathways, but not to the IMD or JAK-STAT. A thioester-containing protein (TEP22), a member of α2-macroglobulin family, has been implicated in the CLSP2-modulated mosquito antifungal defense. Our study has contributed to a greater understanding of immune-modulating mechanisms in mosquitoes.

High throughput profiling of the cotton bollworm Helicoverpa armigera immunotranscriptome during the fungal and bacterial infections.

BACKGROUND: Innate immunity is essential in defending against invading pathogens in invertebrates. The cotton bollworm, Helicoverpa armigera (Hübner) is one of the most destructive lepidopteran pests, which causes enormous economic losses in agricultural production worldwide. The components of the immune system are largely unknown in this insect. The application of entomopathogens is considered as an alternative to the chemical insecticides for its control. However, few studies have focused on the molecular mechanisms of host-pathogen interactions between pest insects and their pathogens. Here, we investigated the immunotranscriptome of H. armigera larvae and examined gene expression changes after pathogen infections. This study provided insights into the potential immunity-related genes and pathways in H. armigera larvae. RESULTS: Here, we adopted a high throughput RNA-seq approach to determine the immunotranscriptome of H. armigera larvae injected with buffer, fungal pathogen Beauveria bassiana, or Gram-negative bacterium Enterobacter cloacae. Based on sequence similarity to those homologs known to participate in immune responses in other insects, we identified immunity-related genes encoding pattern recognition receptors, signal modulators, immune effectors, and nearly all members of the Toll, IMD and JAK/STAT pathways. The RNA-seq data indicated that some immunity-related genes were activated in fungus- and bacterium-challenged fat body while others were suppressed in B. bassiana challenged hemocytes, including the putative IMD and JAK-STAT pathway members. Bacterial infection elevated the expression of recognition and modulator genes in the fat body and signal pathway genes in hemocytes. Although fat body and hemocytes both are important organs involved in the immune response, our transcriptome analysis revealed that more immunity-related genes were induced in the fat body than that hemocytes. Furthermore, quantitative real-time PCR analysis confirmed that, consistent with the RNA-seq data, the transcript abundances of putative PGRP-SA1, Serpin1, Toll-14, and Spz2 genes were elevated in fat body upon B. bassiana infection, while the mRNA levels of defensin, moricin1, and gloverin1 were up-regulated in hemocytes. CONCLUSIONS: In this study, a global survey of the host defense against fungal and bacterial infection was performed on the non-model lepidopteran pest species. The comprehensive sequence resource and expression profiles of the immunity-related genes in H. armigera are acquired. This study provided valuable information for future functional investigations as well as development of specific and effective agents to control this pest.

Reactive oxygen species and antimicrobial peptides are sequentially produced in silkworm midgut in response to bacterial infection.

The silkworm, Bombyx mori, is utilized as a research model in many aspects of biological studies, including genetics, development and immunology. Previous biochemical and genomic studies have elucidated the silkworm immunity in response to infections elicited by bacteria, fungi, microsporidia, and viruses. The intestine serves as the front line in the battle between insects and ingested harmful microorganisms. In this study, we performed RNA sequencing (RNA-seq) of the larval silkworm midgut after oral infection with the Gram-positive bacterium Bacillus bombysepticus and the Gram-negative bacterium Yersinia pseudotuberculosis. This enables us to get a comprehensive understanding of the midgut responses to bacterial infection. We found that B. bombysepticus induced much stronger immune responses than Y. pseudotuberculosis did. Bacterial infection resulted in more energy consumption including carbohydrates and fatty acids. The midgut immune system was characterized by the generation of reactive oxygen species and antimicrobial peptides. The former played a critical role in eliminating invading bacteria during early stage, while the latter executed during late stage. Our results provide an integrated insight into the midgut systematic responses to bacterial infection.

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.

CTL14, a recognition receptor induced in late stage larvae, modulates anti-fungal immunity in cotton bollworm Helicoverpa armigera.

C-type lectin (CTL) is usually considered as pattern recognition receptors in insect innate immunity. Here we found that CTL14 of Helicoverpa armigera was only activated in the fifth instar larvae not in the second instar by entomopathogen Beauveria bassiana infection. Recombinant CTL14 protein was found to form aggregates with zymosan and B. bassiana in vitro. Immunoprecipitation studies demonstrated that CTL14 interacted with serine proteinases (SP), serine proteinase inhibitor (serpin), prophenoloxidases (PPO) and vitellogenin (Vg) in the larval hemolymph. Furthermore, depletion of CTL14 using dsRNA led to dramatic decrease in the expression level of PPO1. Additionally, CTL14 depleted H. armigera decreased the resistance to fungal challenge. Taken together, our study showed the direct involvement of CTL14 in the anti-fungal immunity of H. armigera, which further explained the stronger immune responses in the fifth instar compared to the second instar larvae.