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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.

Background Chinese soft-shell turtle (Pelodiscus sinensis) is an important commercial species for its high nutritive and medicinal value, 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, which was mainly attributed to food abundance and living space. The diversity of gut microbial community was obviously higher in turtles from paddy field than pond and lake. Significant differences on dominant phyla, genera and species were found among initial samples and subsequent samples from different habitats. Firmicutes was the most abundant phylum in turtle gut sampled from greenhouse at initial and Proteobacteria was the most abundant phylum after cultivated in different habitats. The functional predictions indicated that both habitat type and sampling time had significant effects on metabolic pathways, especially amino acid and carbohydrate metabolism. Conclusion The microbial abundance was different among the habitats and varied within cultural periods. Gut microbes in turtles from paddy field presented high diversity and richness. Rice-turtle coculture was a potential ecological and economic farming mode for its high production and quality of both turtles and rice, which would play important roles in wild turtle protection, food security and paddy field environment improvement.

Grass carp (Ctenopharyngodon idellus) is one of the most important freshwater cultural species in China. However, obesity, fatty liver and digestive tract inflammation commonly exist in commercial grass carp, which may be due to excessive food and energy intake in daily feeding. Therefore, the feeding strategy should adapt to fish appetite, nutrition requirements and welfare in intensive aquaculture. To investigate the effects of feeding frequency on growth, physiology and metabolism-related fgf21 expression to provide a reference for the selection of feeding strategy, healthy and homogeneous juvenile grass carp (45.2 ± 2.5 g) were randomly divided and adapted to four feeding frequencies of 1, 2, 3 or 4 times/day following satiated feeding for 8 weeks. The growth rate increased with increasing feeding frequency. The hepatosomatic and abdominal fat-somatic indexes were higher in the higher feeding frequency groups, while the gut-somatic index was higher in the low feeding frequency groups (P

The Ultra Performance Liquid Chromatography-Triple-Time of Flight Mass Spectrometer (UPLC-Triple-TOF-MS/MS) based platform was developed to investigate the serum metabolic profile associated with the growth performance in Chinese softshell turtle fed with the diet added with Traditional Chinese Medicines (TCMs) feeding additive. 0.0%, 1.0%, 2.0%, 3.0%, 4.0% of TCMs were added to the base ration of the control group (CG), TCMs treatment group1 (TG1), TCMs treatment group2 (TG2), TCMs treatment group3 (TG3) and TCMs treatment group4 (TG4), respectively. Compared with control group (CG), the weight gain and special growth ratio firstly increased and lately decreased with increased dietary TCMs levels. The highest weight gain (5.824 ± 1.205) and specific growth rate (3.887 ± 0.367) were found in the TCMs treatment group 2 (TG2) (P < 0.05), and the lowest feed conversion ratio (1.785±0.015) also appeared in the TG2 group (P < 0.05). In the metabolomics assay, a total of 795 (VIP> 1) out of 3089 variable metabolites were significantly different between the TG2 and CG. 43 potential biomarkers between TG2 and CG groups were finally screened out on the basis of VIP > 1 and P < 0.05. The potential networks and pathways for the model were explored using MetaboAnalyst. A total of 43 potential biomarkers were confirmed involving thirteen different metabolic pathways. Following the diet added with 2% TCMs feeding additive, five metabolic pathways were affected, these included the following: phenylalanine, tyrosine and tryptophan biosynthesis, tyrosine metabolism, cysteine and methionine metabolism and arginine biosynthesis. This study indicated that the dietary supplementation TCMs as feed additives could influence the growth performance of the healthy juvenile turtles, and the optimal dose of supplementing TCMs to juvenile turtleswas 20 g/kg in diet.

According to the characteristics of gas reservoir development and requirement of production management, a new method for analyzing rate decline in fractured well is presented in this paper, which overcomes the disadvantage that conventional rate decline analysis can be used only to determine deliverability and predict gas well performance but not to determine formation parameters. The presented rate decline analysis method of fractured gas well is based on the change of unstable flow rate, and such parameters as permeability, skin factor, average formation pressure and well bore storage factor are determined by matching productivity data. This new method requires no well shuts down and gas well productivity is not impacted, so, the method can completely take the role of gas well shutting down test to obtain gas reservoir performance parameters, In this paper, a mathematical model of transient rate at variable flowing pressure is presented. By means of the model, the interpretation for fractured well rate decline data can be worked out. The daily production log and time are used to interpret formation parameters. Aiming at the difficulty of data interpretation parameter estimation, the genetic algorithm is used to optimize the way, the fractured well production performance data are interpreted to determine formation parameters and the degree of formation damage. The method provides a more economical mean for gas reservoir development monitoring.