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Aspartic proteinases are one of the four families of proteinase enzymes that are widely present in living organisms. They are involved in various physiological events, such as protein degradation, development, and host defense. However, the characterization and functional roles of aspartic proteinases remain to be elucidated in crustaceans. Here, we characterized a fragment of cathepsin D-like cDNA from red swamp crayfish, Procambarus clarkii (Pc-cathepsin D-like). The open reading frame of the Pc-cathepsin D-like gene contained 1152 bp, encoding a protein of 383 amino acid residues. We also evaluated the immunological role of the Pc-cathepsin D-like gene in vivo. Spatial distribution analysis revealed that the Pc-cathepsin D-like mRNA was high in the hepatopancreas, followed by the gut, gills, and hemocytes of P. clarkii. The expression levels of the Pc-cathepsin D-like gene increased following challenge with viral (polyinosinic: polycytidylic acid) and bacterial (lipopolysaccharides, peptidoglycan) PAMPs compared with PBS injection. The suppression of the Pc-cathepsin D-like gene by RNA interference significantly increased the expression of immune-associated genes. These results showed that the Pc-cathepsin D-like gene has an essential biological role in innate immune responses because it regulates the expression of immune-associated genes.

The red swamp crayfish, Procambarus clarkii (Girard, 1852), is a paradox in China because it has developed as a popular aquaculture species while becoming a notorious invasive species. We investigated the population genetic diversity and population dynamics of wild Chinese populations of P. clarkii based on proPO fragments and the morphological traits across these populations. The results showed that the populations of P. clarkii exhibit high genetic diversity and significant population structure, inferring that the crayfish was artificially transferred rapidly among different locations for different reasons. Results also revealed distinct phenotypic differentiation, with individuals of some populations having a shorter pleon and wider carapace width, whereas other populations have a small length to width ratio. This study provided comprehensive insights into the population dynamics and morphological variations of P. clarkii, which contribute to understanding the rapid evolution of phenotypic traits after its successful invasion. Results should help establish efficient management strategies for the control of the invasion and at the same time using the genetic information in crayfish aquaculture.

Cathepsins are a group of lysosomal hydrolytic enzymes, broadly distributed in animals, and regulate various physiological processes. However, the immune functions of cathepsins are poorly understood in invertebrates. Therefore, to further provide information about the importance of cathepsins in the innate immune system of crustaceans, cathepsin A from Procambarus clarkii (Pc-cathepsin A) was characterized and its distribution in different tissues was determined. The immunological functions of the Pc-cathepsin A were also evaluated. The Pc-cathepsin A showed high sequence homology to cathepsins of other species, as it contained serine and histidine active sites. Quantitative RT-PCR analysis revealed that the expression of Pc-cathepsin A was highest in the gill, gut, and the hepatopancreas, with variable amounts in the muscle, stomach, heart, and hemocytes. The mRNA expression of Pc-cathepsin A was significantly increased in hepatopancreas challenged with lipopolysaccharide (LPS), peptidoglycan (PGN), and polycytidylic acid (poly I:C). The results of an in vivo analysis revealed that Pc-cathepsin A knockdown by double-stranded RNA in P. clarkii modulated the expression of immune-pathway associated genes in hepatopancreas. Collectively, these results suggest that Pc-cathepsin A modulates innate immune responses by affecting the expression of immune-pathway associated genes, thus revealing a regulatory link between Pc-cathepsin A and immune pathways in P. clarkii, and that Pc-cathepsin A plays an essential biological role in the immune defence against microbial pathogens.

Co-culture modes of rice and aquatic animals as rice-crayfish, rice-fish and rice-turtle have expanded rapidly in paddy rice cultivation regions, due to its ecological efficiency, contributions to food security, food productivity and economic benefits. However, different co-culture strategies have great influences on rice or aquatic animal growth and related ecological benefits. In this study, we investigated the effect of turtle stocking density on the dynamics of paddy soil fertility and microbial community characteristics in a rice-turtle co-culture ecosystem. The Chinese soft-shell turtles (Pelodiscus sinensis) were allocated to experimental paddy fields at four gradient stocking densities ranging from 360 to 540 kg· ha−1. In comparison with conventional rice cultivation with a higher input rate of chemical fertilizer, rice yields were higher in the rice-turtle paddies. In addition, soil pH and organic matter were relatively stable in the rice-turtle mode, whereas both parameters decreased over time in conventional paddies. Stocking density significantly benefited soil fertility, with soil organic matter increasing in response to higher stocking density. Total soil nitrogen concentration was lower in the conventional rice cultivation and low stocking density co-culture groups, compared with the high stocking density groups. Furthermore, the soil microbial communities were also affected by turtle stocking density. Redundancy analysis showed that soil pH, organic matter and available potassium concentration were the major factors influencing the abundance of the dominant microbes. The findings revealed the responses of paddy soil fertility and microbial community characteristics in rice-turtle co-culture modes, which could help optimize the co-culture mode and provide a basis for soil nutrient management in such ecosystems.

Background Chinese soft-shell turtle (Pelodiscus sinensis) is an important commercial species for their high nutritional value and unique taste, but it has been a vulnerable species due to habitat loss. In this study, homologous juvenile turtles were allocated to lake, pond and paddy field to investigate the habitat effects on turtles. Results The growth, morphology and gut microbial communities were monitored during the 4 months cultural period. It showed higher growth rate of turtles in paddy field and pond. The appearance, visceral coefficients, gut morphology and microbial communities in turtles were distinct among different habitats. The microbial community richness on Chao1 was obviously lower in initial turtle guts from greenhouses, whereas it was relative higher in turtle guts sampled from paddy fields than ponds and lake. Significant differences on dominant microbes were found among initial and subsequent samples from different habitats. Firmicutes was the most abundant phylum in the guts of turtles sampled from the greenhouse initially, while Proteobacteria was the most abundant phylum after cultivation in different habitats, followed by Bacteroidetes. The microbial composition were distinct in different habitats at 60d, and the appearance of dominant phyla and genera was more driven by sampling time than habitats at 120d. Both the sampling time and habitats affected the appearance of dominant phyla and genera during the cultivation. The functional predictions indicated that both habitat type and sampling time had significant effects on metabolic pathways, especially amino acid and carbohydrate metabolism. Conclusions The turtles could adapt to natural lakes, artificial ponds and paddy fields. The gut microbial abundance was different among the habitats and sampling time. The species of microbes were significantly more diverse in paddy field specimens than in those from ponds and lakes. Rice-turtle coculture is a potential ecological and economic farming mode that plays important roles in wild turtle protection and food security.