Shigui Jiang

Black tiger shrimp (Penaeus monodon) is the largest species of penaeid, being commercially cultured globally, ranking as the second most farmed species in the shrimp industry. However, with the transformation of shrimp aquaculture from semi-intensive to high-density farming, the concentration of ammonia nitrogen in the aquatic environment has increased, severely affecting the growth and survival of shrimps. To increase production efficiency, breeding new strains of shrimp with the trait of tolerance to high ammonia nitrogen is desired in the black tiger shrimp aquaculture. Previous studies have shown that glutamate dehydrogenase (GDH) and aspartate aminotransferase 2 (AST2) play important roles in the metabolism of ammonia nitrogen in crustaceans. In the present study, we conducted synteny analysis of PmGDH and PmAST2 in different species. The interactions of PmGDH with ammonium (NH₄⁺) and PmAST2 with aspartate were analyzed by docking. To develop molecule markers associated with ammonia nitrogen tolerance, SNPs of PmGDH and PmAST2 were identified by direct sequencing, genotyped by the SNaPshot technique, and characterized through genotype-phenotype association analysis by PLINK software version 1.9 in the three geographical populations, two families with different ammonia tolerance, and 120 susceptible and resistant individuals of black tiger shrimp. The results indicate that the GDH and AST2 genes are evolutionarily conserved in vertebrates, except for the black tiger shrimp, which suggests divergence in selective pressure between invertebrates and vertebrates. Notably, PmGDH may interact with NH₄⁺ via the residue Asp178 within loop 1 in the GdhA domain through a hydrogen bonding interaction, and PmAST2 may interact with aspartate via helix 1, sheet 1, loop 1, and loop 2, through both hydrogen bonding interactions and a salt bridge interaction. A total of 12 SNPs were detected in the exons of PmGDH and PmAST2. Among these candidate SNPs, the distributions of PmGDH-1227 and PmAST2-132 showed both significant differences in the genotype and allele association analysis between susceptible and resistant groups. Haplotype association analysis showed that three haplotypes exhibited significantly different distributions between susceptible and resistant groups. In conclusion, PmGDH-1227 and PmAST2-132 were associated with ammonia nitrogen tolerance, and these SNP markers are expected to contribute to marker-assisted selection (MAS) breeding programs to obtain new strains of Penaeus monodon.

High concentrations of ammonia nitrogen could result in the death of aquatic animals and cause a huge economic loss in the aquaculture industry. However, the metabolic responses to acute ammonia nitrogen stress remain largely unknown in Penaeus monodon. In this study, we first investigated the histological change in tissues in Penaeus monodon under 96 h acute ammonia nitrogen stress. The result of the paraffin section showed that acute ammonia nitrogen stress induced severe epithelial detachment and lumen dilatation of the hepatopancreas, swollen and hemocyte infiltration of the gills, and mucosa exfoliation and shortened villi of the intestine in Penaeus monodon, suggesting the impairment of the normal physiological function in these tissues. We next examined the change in the metabolic product in the plasma and the enzyme activity in the hepatopancreas after ammonia nitrogen stress. Upon ammonia stress, both the concentration of ammonia and urea nitrogen significantly increased, while there was no significant increase in the concentration of uric acid, which is consistent with the results that the enzyme activity of glutamine synthetase (GS), glutamate dehydrogenase (GDH), and aspartate transaminase (GOT) became significantly elevated and the enzyme activity of adenosine deaminase (ADA) in the purine metabolism pathway significantly decreased after ammonia stress, suggesting that shrimp could convert excessive ammonia to urea for ammonia detoxification through the ammonia–nitrogen metabolism pathways. Interestingly, we also observed a significant increase in superoxide dismutase (SOD) activity, suggesting a potential role of this antioxidant enzyme in the clearance of reactive oxygen species (ROS) induced via ammonia stress. Moreover, we found that acute ammonia nitrogen stress inhibited the enzyme activity of caspase 3 and caspase 8, suggesting an important role of apoptosis in protecting Penaeus monodon against acute ammonia stress. Overall, our findings revealed that Penaeus monodon may employ metabolic and purine pathways and undergo oxidative stress and apoptosis for ammonia detoxification under ammonia nitrogen stress, thus providing new insight into the metabolic response of shrimp to acute ammonia stress.

This study aimed to evaluate the quality and genetic diversity of farmed Litopenaeus vannamei across three distinct populations from Maoming City (MM), Zhanjiang City (ZJ), and Yangjiang City (YJ) in Guangdong Province. Muscle tissues from L. vannamei were analyzed for phenotypic traits, conventional nutrients, amino acids, and fatty acids, while genetic diversity was assessed using whole genome sequencing techniques. The analysis revealed that the crude protein content in shrimp across the three populations ranged from 20.87 to 21.95 g/100 g, crude fat content ranged from 0.90 to 1.50 g/100 g, essential amino acid content ranged from 5.55 to 5.86 g/100 g, total amino acid content ranged from 14.73 to 15.27 g/100 g, total fatty acid content ranged from 682.73 to 793.97 mg/100 g, total antioxidant capacity (T-AOC) ranged from 2.68 to 2.72 μmol/g, superoxide dismutase (SOD) activity ranged from 1021.97 to 1057.21 U/g, and catalase (CAT) activity ranged from 78.65 to 81.33 μmoL/min. No significant differences were observed in ash and crude fat levels among conventional nutrients, nor in the biochemical indexes T-AOC, CAT, and SOD. Genetic analysis showed that the single nucleotide polymorphism density (SNP/Kb) ranged from 15.323 to 17.461, nucleotide diversity (π) ranged from 2.98 × 10⁻⁵ to 15.84 × 10⁻⁵, polymorphism information content (PIC) ranged from 0.300 to 0.317, heterozygosity (Ho) ranged from 0.033 to 0.048, and inbreeding coefficients (FIS) ranged from 0.834 to 0.887. The genetic differentiation index (FST) values among the three populations ranged from 0.056 to 0.106. This study provides an evaluation of the germplasm resources and genetic diversity of farmed L. vannamei, offering insights for the efficient management and sustainable utilization of this species’ germplasm resources.

Penaeus monodon is one of the world’s most important aquaculture species, with its host-associated microbial community playing a crucial role in its growth, metabolism, immune response, and adaptation. In our study, we utilized Illumina high-throughput sequencing to investigate the composition, structure, and function of the intestinal microbial communities of P. monodon from two different regions in Guangdong. Our results identified 176 phyla across both populations, with Proteobacteria and Firmicutes being predominant. Furthermore, we identified 3095 genera, with Photobacterium, Vibrio, and Aliiroseovarius being the most dominant. Functional gene analysis based on KEGG data indicated that the carbohydrate metabolism and amino acid metabolism were significant at the secondary metabolic pathway level. The eggNOG functional annotation revealed that the genes involved in replication, recombination, and repair are of paramount importance. The CAZy annotation results indicated that Glycoside Hydrolases (GH) have the highest abundance. The Pfam annotation analysis showed that the two most prevalent domains are P-loop NTPase and NADP Rossmann. Our investigation provides a reference for species-level and functional-level analyses of the intestinal microbiota of P. monodon, contributing valuable insights into its microbial ecology.

Salinity is a crucial environmental factor influencing the survival, growth, development, and reproduction of aquatic animals. However, the underlying molecular mechanisms of the shrimp’s response to salinity stress are not yet fully understood. Therefore, we used the Illumina NovaSeq 6000 platform to perform transcriptome sequencing of the hepatopancreas of Litopenaeus vannamei under high-salinity (30 PSU), medium-salinity (10 PSU), and low-salinity (0.5 PSU) conditions. We obtained 63.23 Gb of high-quality data and identified 3589 differentially expressed genes (DEGs), including 1638 upregulated and 1951 downregulated genes. Notably, a comparison between the control group (30 PSU) and the low-salinity group (0.5 PSU) revealed that the BBOX1 and CHE1 genes were significantly upregulated, while the ACOX1, MPV, CYP2L1, GCH, MVK, TREt1, and XDH genes were significantly downregulated. These genes are primarily involved in key metabolic pathways, such as fatty acid oxidation, cholesterol metabolism, and hormone synthesis and metabolism. The key genes involved in fatty acid β-oxidation, such as ACOX1, ACAD, HADH, HSD17B4, PECR, CROT, PIPOX, and CG5009, all showed a downward trend, suggesting that L. vannamei may respond to salt stress by regulating fatty acid oxidative metabolism, optimizing energy utilization, and maintaining cell homeostasis under low-salinity conditions. Functional annotation of gene ontology (GO) and KEGG pathway enrichment analysis highlighted the roles of these significant DEGs in the adaptation of L. vannamei to environments of varying salinity, underscoring the importance of metabolic pathways in their adaptive physiological responses. This study provides a crucial molecular biological basis for understanding the molecular mechanisms and physiological protection strategies of L. vannamei under salinity stress.

Salinization of freshwater ecosystems is a pressing global issue. Changes in salinity can exert severe pressure on aquatic animals and jeopardize their survival. Procambarus clarkii is a valuable freshwater aquaculture species that exhibits some degree of salinity tolerance, making it an excellent research model for freshwater aquaculture species facing salinity stress. In the present study, crayfish were exposed to acute low salt (6 ppt) and high salt (18 ppt) conditions. The organisms were continuously monitored at 6, 24, and 72 h using RNA-Seq to investigate the mechanisms of salt stress resistance. Transcriptome analysis revealed that the crayfish responded to salinity stress with numerous differentially expressed genes, and most of different expression genes was observed in high salinity group for 24h. GO and KEGG enrichment analyses indicated that metabolic pathways were the primary response pathways in crayfish under salinity stress. This suggests that crayfish may use metabolic pathways to compensate for energy loss caused by osmotic stress. Furthermore, gene expression analysis revealed the differential expression of immune and antioxidant-related pathway genes under salinity stress, implying that salinity stress induces immune disorders in crayfish. More genes related to cell proliferation, differentiation, and apoptosis, such as the Foxo, Wnt, Hippo, and Notch signaling pathways, responded to high-salinity stress. This suggests that regulating the cellular replication cycle and accelerating apoptosis may be necessary for crayfish to cope with high-salinity stress. Additionally, we identified 36 solute carrier family (SLC) genes related to ion transport, depicting possible ion exchange mechanisms in crayfish under salinity stress. These findings aimed to establish a foundation for understanding crustacean responses to salinity stress and their osmoregulatory mechanisms.

As the intensive development of aquaculture persists, the demand for fishmeal continues to grow; however, since fishery resources are limited, the price of fishmeal remains high. Therefore, there is an urgent need to develop new sources of protein. They are rich in proteins, fatty acids, amino acids, chitin, vitamins, minerals, and antibacterial substances. Maggot meal-based diet is an ideal source of high-quality animal protein and a new type of protein-based immune enhancer with good application prospects in animal husbandry and aquaculture. In the present study, we investigated the effects of three different diets containing maggot protein on the growth and intestinal microflora of Litopenaeus vannamei. The shrimp were fed either a control feed (no fly maggot protein added), FM feed (compound feed with 30% fresh fly maggot protein added), FF feed (fermented fly maggot protein), or HT feed (high-temperature pelleted fly maggot protein) for eight weeks. The results showed that fresh fly maggot protein in the feed was detrimental to shrimp growth, whereas fermented and high-temperature-pelleted fly maggot protein improved shrimp growth and survival. The effects of different fly maggot protein treatments on the intestinal microbiota of L. vannamei also varied. Fermented fly maggot protein feed and high-temperature-pelleted fly maggot protein feed increased the relative abundance of Ruegeria and Pseudomonas, which increased the abundance of beneficial bacteria and thus inhibited the growth of harmful bacteria. In contrast, fresh fly maggot proteins alter the intestinal microbiome, disrupting symbiotic relationships between bacteria, and causing invasion by Vibrio and antibiotic-resistant bacteria. These results suggest that fresh fly maggot proteins affect the composition of intestinal microorganisms, which is detrimental to the intestinal tract of L. vannamei, whereas fermented fly maggot protein feed affected the growth of L. vannamei positively by improving the composition of intestinal microorganisms.

Air exposure is an important environmental stressor during the transportation and cultivation of Procambarus clarkii. We evaluated the effect of re-submersion for 24 h after dry transportation for 24 h on the histological structure, antioxidant activity, and gene expression of crayfish. The antioxidant parameters of catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and lactate dehydrogenase (LDH), and the relative expression of CAT, SOD, HSP70, and ferritin genes were subsequently measured in the hepatopancreas and gills at both stages. Histopathology found that air exposure led to vacuolation of the hepatopancreas and disorderly arrangement of respiratory epithelial cells (REC) in the gills. The activities of catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and lactic dehydrogenase (LDH) in the hepatopancreas and gills increased with short-term air exposure. The relative expression of genes (CAT, SOD, HSP70, and Ferritin) were induced after short-term air exposure. During re-submersion, MDA content and CAT and SOD activities in the hepatopancreas and gills were restored after 24 h, however, LDH activity and hepatopancreatic tissue damage were not repaired. Our results indicate that air exposure can cause oxidative damage to P. clarkii, and CAT and SOD can be used to determine the response of crayfish exposed to air, in addition to some damage that can be eliminated after re-submersion to a limited degree. This study provides foundational data that re-submersion can improve crayfish performance under hypoxic stress to a certain extent and will lead to the development of more effective transportation strategies and decrease economic losses in the future.

Na⁺/H⁺-exchanger (NHE) assumes a significant part in different particle transport in creatures. A clone of Penaeus monodon NHE cDNA was examined in this study (PmNHE), and its impact on high-concentration ammonia nitrogen stress was researched. The 877-amino acid (aa) protein was encoded by a full-length PmNHE cDNA that was 2788 base pairs (bp) long and had a 2643-bp open reading frame (ORF). The findings show that PmNHE was expressed in all of the P. monodon organs that were tested, including the intestine, muscle, hemolymph, heart, hepatopancreas, stomach, epidermis, gill, testis, and ovary, and the intestine and muscle were found to have the highest levels of PmNHE expression. The expression of PmNHE in the gill tissue of P. monodon was significantly up-regulated under high levels of ammonia nitrogen stress. The expression of PmNHE in the intestine of P. monodon under high-concentration ammonia nitrogen stress was significant. When exposed to high concentrations of ammonia nitrogen stress, P. monodon exhibited shorter survival times than the two control groups. Hence, it is suggested in the present study that PmNHE may have a significant impact on the environment with high levels of ammonia nitrogen.

Mitogen-activated protein kinase kinase 4 (MKK4) is a component of the JNK signaling pathway and plays an important role in immunity and stress resistance. In this study, MKK4 cDNA was cloned, and its bacterial infection and low-salinity challenge responses were researched. The full-length PmMKK4 cDNA was 1582 bp long, with an 858-bp open reading frame (ORF) encoding a 285-amino acid (aa) protein. Results showed that PmMKK-4 was expressed in all examined tissues of P. monodon. The PmMKK4 expression level was found to be lowest in eyestalk ganglion and highest in muscle (approximately 41.25 times than in eyestalk ganglion). Following the infection of Staphylococcus aureus, PmMKK4 was up-regulated in both hepatopancreatic and gill tissues. However, after infection with Vibrio harveyi, PmMKK4 was down-regulated for a period of time in gill tissue, with fluctuating up- and down-regulation in hepatopancreas tissue. Furthermore, after infection with Vibrio anguillarum, gill tissue and hepatopancreas tissue showed a continuous downward trend. The PmMKK4 gene in the gill tissue and hepatopancreas tissue of P. monodon was activated after low-salinity stress. The expression change of PmMKK4 in gill tissue was more significant. The research showed that the PmMKK4 gene plays an important role in both innate immunities after pathogen infection and adaptation in a low-salt environment.