Comparative Analysis of the Growth, Physiological Responses, and Gene Expression of Chinese Soft-Shelled Turtles Cultured in Different Modes.

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important freshwater aquaculture turtle due to its taste and nutritional and medicinal value. More ecological culturing modes, such as rice-turtle co-culture, should be developed to meet the ecological benefit demand. We compared growth, physiological parameters, and transcriptome data to detect the physiological responses and regulatory mechanisms of pond-cultured turtles as compared to co-cultured turtles. The co-cultured turtles grew slower than pond-cultured turtles. The gonadosomatic index of co-cultured male turtles was lower than that of pond-cultured male turtles, and both the mesenteric fat index and limb fat index were lower in co-cultured turtles than in pond-cultured turtles (p < 0.05). The blood GLU of the co-cultured turtles was significantly lower than the GLU of the pond-cultured turtles (p < 0.05), while the values of CRE, UA, BUN, AKP, ACP, GOT, and CAT were higher in the co-cultured turtles than in the pond-cultured turtles (p < 0.05). In total, 246 and 598 differentially expressed genes (DEGs) were identified in the brain and gut from turtles cultured in the two different modes, respectively. More DEGs were related to environmental information processing, metabolism, and human diseases. In the brain, the top enriched pathways of DEGs included the longevity regulating pathway, glycerolipid metabolism, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, and PI3K-Akt signaling pathway, while in the gut, the top enriched pathways of DEGs included the cell cycle, DNA replication, cellular senescence, and p53 signaling pathway. The turtles acclimated to the different culturing conditions by adjusting their growth, physiological, and biochemical characteristics and related gene expression during a short culture period.

Molecular characterization and functional analysis of the lysosomal cathepsin D-like gene in red swamp crayfish, Procambarus clarkii.

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.

Gut microbiota of homologous Chinese soft-shell turtles (Pelodiscus sinensis) in different habitats.

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.

Chemicals affect color preference in rare minnow (Gobiocypris rarus).

Behavioral response of fish has been shown that was sensitive to chemicals in water. Herein, larval and adult rare minnows of Gobiocypris rarus were studied for their innate color preference and response to a concentration gradient of chemicals (cadmium ion [Cd2+], tricaine methanesulfonate [MS222], and p-chloroaniline). The results showed that both larval and adult rare minnows preferred blue and green over yellow and red in water with no chemicals added. Larval color preference changed significantly under concentrations of Cd2+ ≥ 0.4 mg/L, MS222 ≥ 3 mg/L, and p-chloroaniline ≥ 10 mg/L; for adults, color preference changed significantly when Cd2+ ≥ 3 mg/L, MS222 ≥ 34 mg/L, and p-chloroaniline ≥ 38 mg/L. In addition, the color preference priorities of both larvae and adults also changed at high concentrations of chemicals. The present study provides useful information on how changes in rare minnow behavior could be used as an early indicator of water pollution.

Identification and immunoregulatory role of cathepsin A in the red swamp crayfish, Procambarus clarkii.

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.

Comparative Developmental Toxicity and Stress Protein Responses of Dimethyl Sulfoxide to Rare Minnow and Zebrafish Embryos/Larvae.

Dimethyl sulfoxide (DMSO), a widely used carrier solvent, can be toxic to test organisms and has species-specific sensitivity. In this study, the developmental toxicity and stress protein responses of DMSO to rare minnow (Gobiocypris rarus) and zebrafish (Danio rerio) with two tests were compared in the early life stage. In the first test, fertilized eggs were exposed to 0%, 0.0001%, 0.001%, 0.01%, 0.1%, 1.0%, 1.5%, and 2.0% v/v of DMSO until 3 days post hatching. In the second test, larvae from 0 to 8 d were exposed to 2% DMSO until 4 days. Our results showed that DMSO was toxic to rare minnow and zebrafish on multiple indexes, and the no-observed-effect concentrations of DMSO in both species were 1.0% and 0.001% for developmental toxicity analysis and stress protein analysis, respectively. Furthermore, rare minnow larvae were more sensitive than zebrafish to DMSO for spinal malformation. The sensitive period for induction of spinal malformation by DMSO was 0-7 d after hatch (dah) for rare minnow and 0-4 dah for zebrafish. Together, these results will provide support to the use of DMSO in ecotoxicological studies using rare minnow and will contribute to a better understanding of the toxicity of DMSO.

Effects of Total Hardness and Calcium:Magnesium Ratio of Water during Early Stages of Rare Minnows (Gobiocypris rarus).

The ionic composition of water is important for all fish. In the present study, the effects of total hardness and Ca(2+):Mg(2+) ratio on early life stages of rare minnows (Gobiocypris rarus), a promising laboratory fish in China, were evaluated. Paired parent fish were transferred to spawning aquaria (16 L) containing water at different total hardness and Ca:Mg ratios, and their offspring were further cultured at 25 ± 1 °C and 12:12-h light:dark photoperiod. Fertilization rates were not affected by total hardness to 480 mg L(-1) CaCO3, but egg size decreased with increasing total hardness. Ca:Mg ratios less than 1:20 or greater than 8:1 had adverse influences on hatching, feeding, development, larval growth, and survival. Embryos and larvae incubated in Mg(2+)- and Ca(2+)-deficient waters exhibited high malformation rates and high mortality. Our results demonstrate that rare minnows can adapt to a wide range of total hardness and Ca:Mg ratios, although an imbalance between Ca(2+) and Mg(2+) in water is toxic to this species. To increase the comparability and usefulness of test results, we recommend the use of reconstituted or drinking water of defined total hardness and Ca:Mg ratio for the culture and toxicity testing of rare minnows.

Growth of rare minnows (Gobiocypris rarus) fed different amounts of dietary protein and lipids.

The rare minnow (Gobiocypris rarus) is a laboratory fish that is commonly used for toxicology research, but there is currently no standard lab diet for this model organism. Certain studies with rare minnows require specialized diets, so there is a need to better understand how manipulating nutrients affects the development and growth of this fish. We conducted two separate dose-response experiments to determine the effect of different levels of dietary protein or dietary lipids on the growth of juvenile rare minnows over 60 d. We measured growth rates and food intake over two periods of time: the first 20 or 30 d of diet consumption and the entire 60 d of each experiment. We found that different levels of dietary protein or dietary lipids produced significantly different growth rates during both the early phase and the entire duration of the study. Among experimental protein-variable diets, those with intermediate levels of dietary protein (around 35.2%) produced the highest growth rate. Among experimental lipid-variable diets, those with intermediate levels of dietary lipids (around 7.6%) produced the highest growth rate. Over all periods of both experiments, however, the control diet of bloodworms generally produced the highest growth rate that matched or exceeded that of any experimental diet. These results can guide investigators when using rare minnows in research, particularly when using custom and standardized diets.

Short-term toxicity of ammonia, nitrite, and nitrate to early life stages of the rare minnow (Gobiocypris rarus).

Nitrogenous pollutants including ammonia, nitrite, and nitrate are a widespread concern in natural waters and aquaculture. In the present study, the toxicity of ammonia, nitrite, and nitrate to rare minnow (Gobiocypris rarus) in the early life stage were evaluated by 2 short-term toxicity tests. In the short-term toxicity test, conducted on embryo and sac-fry stages, 30 fertilized eggs with 3 replicates were randomly exposed to varying levels of ammonia, nitrite, and nitrate until 3 d posthatch (dph). In the 7-d larval subchronic toxicity test, 30 newly hatched larvae with 3 replicates were randomly exposed to varying levels of ammonia, nitrite, and nitrate until 7 dph. The results showed that the 7-d larval subchronic toxicity test was more sensitive than the short-term toxicity test on embryo and sac-fry stages. Both toxicity tests revealed that ammonia was most toxic to rare minnows, followed by nitrite and nitrate. High levels of ammonia, nitrite, and nitrate decreased growth, retarded development, and increased mortality. The no-observed-effect concentrations of ammonia, nitrite, and nitrate for larval growth were 2.49 mg L(-1) , 13.33 mg L(-1) , and 19.95 mg L(-1) nitrogen, respectively. The present study's results demonstrate that nitrogenous pollutants pose a threat to wild populations of rare minnows and provide useful information for establishing water quality criteria for this laboratory fish. Environ Toxicol Chem 2016;35:1422-1427. © 2015 SETAC.

Effects of temperature and feeding frequency on ingestion and growth for rare minnow.

Water temperature and feeding frequency are two important components in feeding strategy that directly affect the growth and physiology of fishes. The rare minnow (Gobiocypris rarus) has been cultured for decades in the laboratory as an experimental fish and is widely used in environmental science research. An 8-week factorial feeding experiment was conducted on juvenile rare minnows to investigate the interaction between water temperature (ambient, 20, 24, 28°C) and feeding frequency (one, two, three meals per day) on growth performance, feed utilization, gut evacuation and adaptability to variable environmental conditions. Groups fed three times a day at 28°C attained the maximum final body weight, followed by those fed two times a day at 24°C. There was an obvious curvilinear relationship between specific growth rate and temperature. Increased temperature significantly promoted food consumption and growth rate, but there were no benefits on growth by feeding multiple times at temperatures of 18°C or lower. Temperature and feeding frequency also affected gut evacuation rates: high temperature and frequency lead to fast evacuation. These results show that the optimal temperature and feeding frequency for rare minnow is 24°C and two meals a day for maximal growth, feeding efficiency, and daily management. The findings also suggest that the rare minnow has the ability to rapidly adapt to changing culture conditions by adjusting physiological activities in the short term.