Comparative Transcriptome Analysis of Litopenaeus vannamei Reveals That Triosephosphate Isomerase-Like Genes Play an Important Role During Decapod Iridescent Virus 1 Infection

Abstract

Decapod iridescent virus 1 (DIV1) results in severe economic losses in shrimp aquaculture. However, little is known about the physiological effect of DIV1 infection on the host. In this study, we found that the lethal dose 50 of DIV1-infected Litopenaeus vannamei after 48, 72, 96, and 156 h were 4.86 × 10⁶, 5.07 × 10⁵, 2.13 × 10⁵, and 2.38 × 10⁴ copies/μg DNA, respectively. In order to investigate the mechanisms of DIV1 infection, a comparative transcriptome analysis of hemocytes from L. vannamei, infected or not with DIV1, was conducted. The BUSCO analysis showed that the transcriptome was with high completeness (complete single-copy BUSCOs: 57.3%, complete duplicated BUSCOs: 41.1%, fragmentation: 0.8%, missing: 0.8%). A total of 168,854 unigenes were assembled, with an average length of 601 bp. Based on homology searches, Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), and cluster of orthologous groups of proteins (KOG) analysis, 62,270 (36.88%) unigenes were annotated. Among them, 1,112 differentially expressed genes (DEGs) were identified, of which 889 genes were up-regulated and 223 genes were down-regulated after DIV1 infection. These genes were mainly annotated to the major metabolic processes such as fructose and mannose metabolism, carbon metabolism, and inositol phosphate metabolism. Among these metabolic pathways, the triosephosphate isomerase (TPI) family was the most eye-catching DEG as it participates in several metabolic processes. Three types of TPI, LvTPI-like, LvTPI-Blike, and LvTPI-Blike1, were obtained for gene silencing by RNA interference. The results showed that LvTPI-like and LvTPI-Blike1 silencing caused a high mortality rate among L. vannamei. However, LvTPI-like and LvTPI-Blike silencing reduced DIV1 replication in DIV1-infected L. vannamei. All the results indicated that TPI-like genes play an important role during DIV1 infection, which provides valuable insight into the infection mechanism of DIV1 in shrimp and may aid in preventing viral diseases in shrimp culture.

Keywords: DIV1; Litopenaeus vannamei; RNA interference; transcriptome analysis; triosephosphate isomerase.

Figure 1

Clinical symptoms and virus detection of Litopenaeus vannamei. (A,B) Clinical symptoms of DIV1-infected L. vannamei. (C) Virus detection of infected L. vannamei used for the DIV1 inoculation in LD₅₀ test. Marker: DL2000 molecular mass marker; lane 1: PCR amplified products of WSSV detection; lane 2: PCR-amplified products of IHHNV detection; lane 3: PCR amplified products of DIV1detection. (D) DIV1 detection of L. vannamei in LD₅₀ test using nested PCR method. Marker: DL2000 molecular mass marker; lanes 1 and 3: PCR amplified products of DIV1 detection in healthy L. vannamei; lanes 2 and 4: PCR-amplified products of DIV1 detection in dead L. vannamei.

Figure 2

Cumulative survival rates of Litopenaeus vannamei injected by DIV1 (A) and genome copies of DIV1 in infected L. vannamei (B). (A) Six groups of healthy L. vannamei were intramuscularly injected at the third abdominal segment with 50 μl of DIV1 supernatants at five concentrations and phosphate-buffered saline as a control. (B) The DIV1 copies were investigated in the hemocyte, hepatopancreas, intestine, gill, and muscle of L. vannamei infected by DIV1 at the concentration of LD50 after 48 h of infection. Dissimilar letters show a significant difference (p < 0.05).

Figure 3

Transcriptome sequence length distribution (A) and assembly quality analysis (B).

Figure 4

Functional enrichment of unigenes from Litopenaeus vannamei. (A) KOG classification of unigenes. Each bar represents the number of unigenes classified into each of the 26 KOG functional categories. (B) Gene Ontology (GO) classification of unigenes. Three major GO categories were enriched: biological process, cellular component, and molecular function. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) classification of unigenes. The unigenes were assigned to six special KEGG pathways, including organismal systems, metabolism, human diseases, genetic information processing, environmental information processing, and cellular processes.

Figure 5

Volcano diagram of differentially expressed genes (DEGs) in Litopenaeus vannamei with and without DIV1 infection. The x-axis indicates the fold change, and the y-axis indicates the statistical significance of the differences. Red dots represent the significantly up-regulated DEGs, while green dots represent the significantly down-regulated DEGs (FDR < 0.05 and |log2 ratio| ≥ 1). The gray dots represent the DEGs which are not significantly different.

Figure 6

Analysis of GO term functional enrichment of differentially expressed genes between DIV1-infected and control groups. The x-axis indicates the Gene Ontology processes, and the y-axis indicates the number of unigenes in a process.

Figure 7

Top 20 of pathway enrichment. The x-axis indicates the ratio of the number of genes in the pathway of the DEGs and all genes. The y-axis indicates the pathway.

Figure 8

Comparison of the expression profiles of six selected genes as determined by Illumina sequencing and qRT-PCR.

Figure 9

Function of LvTPI-likes during DIV1 infection. (A) qPCR analysis of the silencing efficiencies of LvTPI-likes. (a) LvTPI-like, (b) LvTPI-Blike, and (c) LvTPI-Blike1. EF1α was used as the internal control. (B) Cumulative survival rates of Litopenaeus vannamei injected by LvTPI-likes dsRNA. (C) Cumulative survival rates of LvTPI-likes-RNAi L. vannamei during DIV1 infection. Error bars represent ± SD of three replicates. Data were analyzed with the GraphPad Prism software using the log-rank (Mantel–Cox) method. All data are given in terms of means ± standard error (SE). Asterisks indicate significant differences. *P < 0.05 and **P < 0.01 (n = 3).

Figure 10

Detection of DIV1 copy numbers in hemocyte (A), hepatopancreas (B), intestine (C), gill (D), and muscle (E) of Litopenaeus vannamei following treatment with dsLvTPI-like and dsLvTPI-Blike. Data are shown as mean ± SD of three animals. Dissimilar letters show a significant difference (p < 0.05).