Abstract:Nitrite has strong toxicity to shrimp, and is one of the main environmental stress factors affecting the growth and survival of shrimp. High nitrite in water is often associated with the phenomenon of shrimp exoskeleton softening (soft skin), and the relevant mechanism needs to be further studied. In order to reveal the mechanism of nitrite resulting in the exoskeleton softening of Penaeus vannamei, this study randomly selected hard-shell shrimp (CH) from three ponds (nitrite concentration: 4.35±1.07 mg/L) as the control group, and from three other ponds (nitrite concentration: 21.05±0.84 mg/L), hard-shell shrimp (NH) and soft-shell shrimp (NS) were selected as experimental groups. The physiological indexes of hepatopancreas were determined and transcriptomic analysis was performed. A total of 404,803,278 raw reads were sequenced on the Illumina Novaseq 6000 platform, and 400,197,920 clean reads were obtained after removing low-quality sequences and splices. An average of 45,457,339, 43,648,589 and 44293,378 clean reads were obtained in groups CH, NS and NH, respectively. The obtained unigenes were annotated in the NR (NCBI non-redundant protein sequences), NT (NCBI nucleotide sequences), PFAM (protein family), KOG (euKaryotic Ortholog Groups), SwissProt (a manually annotated and reviewed protein sequence database), GO (Gene Ontology), and KO (KEGG Orthology) databases. The majority of the genes were annotated in the NR database (92.83%), followed by the EggNOG database (58.58%), the Pfam database (57.39%), the Swiss-Prot database (53.82%), the GO database (52.49%), and the KEGG database (43.22%). Overall, out of 30,746 genes, 28,878 (accounting for 93.92% of the total) were annotated in at least one database. The transcriptomic results showed that a total of 3284 differentially expressed genes (DEGs) were identified across different groups. Among these, the NS vs CH group had the highest number of DEGs, with 2924, including 1375 up-regulated genes and 1549 down-regulated genes. The NH vs CH group had the fewest DEGs, with 479, including 313 up-regulated genes and 166 down-regulated genes. In the NS vs NH group, 837 DEGs were identified, with 588 up-regulated genes and 249 down-regulated genes. According to the KEGG enrichment results, the down-regulated differentially expressed genes in the NS vs NH group were significantly enriched in the retinol metabolism pathway (ko00830) and the cytochrome P450 metabolism of xenobiotics pathway (ko00980). In the NS vs CH group, the most significantly down-regulated pathway was also the cytochrome P450 metabolism of xenobiotics pathway (ko00980), with significant enrichment observed in the pentose and glucuronate interconversions pathway (ko00040), pancreatic secretion pathway (ko04972), and retinol metabolism pathway (ko00830). In the NH vs CH group, the aldosterone-regulated sodium reabsorption pathway (ko04960) and the progesterone-mediated oocyte maturation pathway (ko04914) were significantly enriched. Additionally, the PPAR signaling pathway (ko03320) and the fatty acid degradation pathway (ko00071) were significantly enriched in both the NS vs NH group and the NS vs CH group. The results showed that high concentration of nitrite significantly inhibited the expression levels of glucose-6-phosphate isomerase, trehalose transporter, UDP-n-acetylglucosamine pyrophosphorylase, glutamine synthetase and other key genes related to chitin synthesis. In addition, the expression levels of genes related to chitin degradation (chitinase, chitotriase, neutral α-glucosidase) in NS group were significantly increased. According to the results of GO enrichment analysis, under nitrite stress, hydrolase in NS group, hydrolyzed O-glycosylated compounds (GO: 0004553), hydrolase, acting on glycosylated bond (GO: 0016798) related genes expression were upregulated, which may lead to a decrease in cuticle chitin content. In addition, GO enrichment analysis showed that, compared with CH group, NS differentially expressed genes in pre-ribosomes (GO: 0030684), nucleolus (GO: 0005730), cell modification of amino acid metabolism (GO: 0006575), rRNA metabolism (GO: 0016072), and rRNA processing (GO: 0016072). Significant enrichment, such as long alcohol diphosphate oligosaccharide glycosyltransferase (RPN), translation initiation factor 2 (EIF2), which may lead to stratum corneum disorganization. In addition, the genes related to mineral absorption (calintegrin binding protein, high affinity copper uptake protein, ferritin, etc.) and calreticulin genes were significantly down-regulated in the NS group, which may directly affect exoskeleton sclerosis. KEGG enrichment analysis showed that, compared with the other two groups, the differential expression genes in the NS group were significantly enriched in the PPAR signaling pathway (ko03320) and the fatty acid degradation pathway (ko00071). Specifically, the genes related to lipid metabolism were down-regulated in the NS group, which may lead to the absence of extra energy for exoskeleton hardening. Physiological indexes showed as follows: the activities of chitinase and β-n-acetylglucosaminase in NS group were significantly higher than those in NH group and CH group (P < 0.05), while the trehalase activity in CH group was significantly higher than that in NS group (P < 0.05). The activity of Ca2+-ATPase in CH group was the highest, and significantly higher than that in NH group (P < 0.05). The content of calcium ions in hepatopancreas in NH group was significantly higher than that in NS and CH groups (P < 0.05). These results indicated that high concentration of nitrite in water may affect exoskeleton hardness of P. vannamei through a comprehensive mechanism, including reducing chitin content, damaging cuticle conformation and reducing mineral deposition. This study provides a theoretical basis for further exploring the effect of nitrite on shrimp exoskeleton.
