Transcriptomic Insights into Host Metabolism and Immunity Changes after Parasitization by Leptopilina myrica

Abstract

Parasitoids commonly manipulate their host's metabolism and immunity to facilitate their offspring survival, but the mechanisms remain poorly understood. Here, we deconstructed the manipulation strategy of a newly discovered parasitoid wasp, L. myrica, which parasitizes D. melanogaster. Using RNA-seq, we analyzed transcriptomes of L. myrica-parasitized and non-parasitized Drosophila host larvae. A total of 22.29 Gb and 23.85 Gb of clean reads were obtained from the two samples, respectively, and differential expression analysis identified 445 DEGs. Of them, 304 genes were upregulated and 141 genes were downregulated in parasitized hosts compared with non-parasitized larvae. Based on the functional annotations in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, we found that the genes involved in host nutrition metabolism were significantly upregulated, particularly in carbohydrate, amino acid, and lipid metabolism. We also identified 30 other metabolism-related DEGs, including hexokinase, fatty acid synthase, and UDP-glycosyltransferase (Ugt) genes. We observed that five Bomanin genes (Boms) and six antimicrobial peptides (AMPs) were upregulated. Moreover, a qRT-PCR analysis of 12 randomly selected DEGs confirmed the reproducibility and accuracy of the RNA-seq data. Our results provide a comprehensive transcriptomic analysis of how L. myrica manipulates its host, laying a solid foundation for studies on the regulatory mechanisms employed by parasitoid wasps in their hosts.

Keywords: Leptopilina myrica; immunity; metabolism; parasitoid wasp; transcriptome.

Figure 1

Experimental design and identification of DEGs between the parasitized and non-parasitized groups. (A) Experimental design and comparisons employed in this study. Transcriptomes were generated from Drosophila host larvae at 48 h following parasitization by L. myrica, and non-parasitized individuals at the same developmental stages served as the control. (B) Volcano plot of the 16,941 unigenes; each point in the volcano diagram represents one unigene, and only those with | log2 (FC) | > 1 and a q-value < 0.05 were identified as DEGs. The red points represent the upregulated DEGs, the blue points represent the downregulated DEGs, and the gray points represent the unigenes that are not significant. (C) Number of DEGs identified from the parasitized (P) and non-parasitized (non-P) Drosophila host groups. The orange column represents the upregulated DEGs and the blue column represents the downregulated DEGs.

Figure 2

GO classification of the DEGs between parasitized and non-parasitized Drosophila larvae at 48 h after parasitization. Top 20 enriched GO classifications of annotated upregulated (A) and downregulated (B) DEGs. The distributions are summarized into three main categories: biological processes (BP), molecular functions (MF), and cellular components (CC). The x-axis shows the number of DEGs in each category, and the y-axis shows the different GO terms.

Figure 3

Expression profiles of DEGs involved in carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Each column represents an individual parasitized or non-parasitized larva sample. The color gradient from blue to red represents low to high gene expression levels normalized using Z-score normalization. Abbreviations: non-P, non-parasitized larvae; P, parasitized host; Hex-C, hexokinase C; Ugt37C1, UDP-glycosyltransferase family 37 member C1; Ugt35C1, UDP-glycosyltransferase family 35 member C1; Ect3, ectoderm-expressed 3; Ugt49C1, UDP-glycosyltransferase family 49 member C1; tobi, target of brain insulin; Ugt37C2, UDP-glycosyltransferase family 37 member C2; Akr1B, aldo-keto reductase 1B; Ugt37B1, UDP-glycosyltransferase family 37 member B1; Ugt317A1, UDP-glycosyltransferase family 317 member A1; FASN1, fatty acid synthase 1; ACC, acetyl-CoA carboxylase; PPO3, prophenoloxidase 3; hgo, homogentisate 1,2-dioxygenase; Hpd, 4-hydroxyphenylpyruvate dioxygenase; Faa, fumarylacetoacetase; GstZ2, glutathione S transferase Z2; AsnS, asparagine synthetase; Ddc, dopa decarboxylase; Prat2, phosphoribosylamidotransferase 2; b, black; PPO1, prophenoloxidase 2; Gal, β galactosidase; sro, shroud.

Figure 4

Expression profiles of DEGs involved in immune responses. Each column represents an individual parasitized or non-parasitized larva sample. The color gradient from blue to red represents low to high gene expression levels normalized using Z-score normalization. Abbreviations: non-P, non-parasitized larvae; P, parasitized host; PPO3, prophenoloxidase 3; ItgaPS4, integrin alphaPS4 subunit; BomS3, Bomanin short 3; BomS1, Bomanin short 1; BomS5, Bomanin short 5; BomBc1, Bomanin bicipital 1; BomS2, Bomanin short 2; IM4, immune-induced molecule 4; ItgaPS5, integrin alphaPS5 subunit; IM14, immune-induced molecule 14; Mtk, metchnikowin; IMPPP, Baramicin A2; Dro, drosocin; GstO2, glutathione S transferase O2; CalpA, calpain-A; Itgbn, integrin betanu subunit; AttB, attacin B; Npc2h, Niemann–Pick type C-2h; AttD, attacin D; PPO2, prophenoloxidase 2; Drsl4, drosomycin-like 4; ac, achaete; PGRP-SD, peptidoglycan recognition protein SD; Eig71Ea, ecdysone-induced gene 71Ea; PPO1, prophenoloxidase 1; GILT2, gamma-interferon-inducible lysosomal thiol reductase 2; e, ebony.

Figure 5

Validation of RNA-seq data using qRT-PCR. Log2(FC) in gene expression following 48 h of L. myrica parasitization detected by RNA-seq plotted against the qRT-PCR data. The reference line indicates a linear relationship between the qRT-PCR and RNA-seq results (Pearson correlation coefficient, R = 0.8926; p = 9.350 × 10⁻⁵).