A Gut-Specific LITAF-Like Gene in Antheraea pernyi (Lepidoptera: Saturniidae) Involved in the Immune Response to Three Pathogens

Abstract

Antheraea pernyi (Guérin-Méneville 1855) is an important resource for silk, food, and biohealth products; however, exogenous pathogens largely affect the commercial application potential of this species. Since the gut is a key organ for the digestion and absorption of nutrients as well as for immune defense, we used comparative transcriptome analysis to screen for a gut-specific molecular tool for further functional research in A. pernyi. In total, 3,331 differentially expressed genes (DEGs) were identified in the gut compared with all other pooled tissues of A. pernyi, including 1,463 upregulated genes in the gut. Among these, we further focused on a lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF) gene because of its high gut-specific expression and the presence of a highly conserved SIMPLE-like domain, which is related to the immune response to pathogenic infections in many species. The cDNA sequence of ApLITAF was 447-bp long and contained a 243-bp open reading frame encoding an 80-amino acid protein. Immune challenge assays indicated that ApLITAF expression was significantly upregulated in the gut of A. pernyi naturally infected with nucleopolyhedrovirus (NPV) or fed leaves infected with the gram-negative bacterium Escherichia coli (Migula 1895) and the gram-positive bacterium Bacillus subtilis (Ehrenberg 1835). Cell transfection showed that ApLITAF localized to the lysosome. Collectively, these results suggested that ApLITAF played a role in the immune response of A. pernyi and could facilitate the future research and breeding application in this species.

Pupal Diapause Termination and Transcriptional Response of Antheraea pernyi (Lepidoptera: Saturniidae) Triggered by 20-Hydroxyecdysone

“…A total of 3 μg of RNA from each sample was used to construct a sequencing library. Fifteen sequencing libraries were generated using a VAHTS mRNA-seq V2 Library Prep Kit (Vazyme, China) following the manufacturer's recommendations and details (Lei et al, 2021). Paired-end sequencing was carried out on an Illumina HiSeq ™ 2500 platform (Illumina, United States) at Suzhou Transcriptome Biotechnology Co., Ltd. (Suzhou, China).…”

“…qRT-PCR was performed with an SYBR Green qPCR Mix (Toyobo, Japan) using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, United States) to detect the relative expression levels of the genes related to adult development. The qRT-PCR conditions were described in Lei et al (Lei et al, 2021). Each assay was performed in triplicate.…”

“…Each assay was performed in triplicate. The glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) in A. pernyi was used as the control (Lei et al, 2021). Supplementary Table S1 lists all of the primers used for qRT-PCR.…”