Growth Axis Somatostatin, Growth Hormone Receptor, and Insulin-like Growth Factor-1 Genes Express and Are Affected by the Injection of Exogenous Growth Hormone in Chinemys reevesii.

In this study, to explore the effect of growth hormone changes on the related genes and regulatory roles of the turtle, PCR amplification, real-time fluorescence quantitative analysis, and enzyme cutting technology were used to clone and sequence the somatostatin (SS) gene, growth hormone receptor (GHR), and insulin-like growth factor-1 (IGF-I) sequence of Chinemys reevesii. The effects of human growth hormone on the mRNA expression of growth-axis-related genes SS, GHR, and IGF-1 in different sexes were observed. The study of the SS gene in turtles using real-time fluorescence quantitative PCR showed that the SS gene was mainly expressed in the nervous system and the digestive system, with the highest expression found in the brain, while the GHR gene and the IGF-I gene were expressed in all tissues of Chinemys reevesii. The SS gene was expressed in the brain, pituitary, liver, stomach, and intestine, with the highest expression in the brain and the lowest expression in the liver. Within 4 weeks of the injection of exogenous growth hormone, the expression level of the SS gene in the brain of both sexes first increased and then decreased, showing a parabolic trend, and the expression level of the experimental group was lower than that of the control group. After the injection of growth hormone (GH), the expression of the GHR gene in the liver of both sexes showed a significant increase in the first week, decreasing to the control group level in the second week, and then gradually increasing. Finally, a significant level of difference in the expression of the GHR gene was reached at 3 and 4 weeks. In terms of the IGF-I gene, the changing trend of the expression level in the liver was the same as that of the GHR gene. After the injection of exogenous growth hormone, although the expression of the SS gene increased the inhibition of the secretion of the GHR gene by the Reeves' turtle, exogenous growth hormone could replace the synthesis of GH and GHR, accelerating the growth of the turtle. The experiments showed that the injection of recombinant human growth hormone affects the expression of SS, GHR, and IGF-1 genes, and promotes the growth of the Reeves' turtle.

Role of Clostridium butyricum, Bacillus subtilis, and algae-sourced ß-1,3 glucan on health in grass turtle.

This study investigated the effects of two probiotics namely Clostridium butyricum and Bacillus subtilis, and one prebiotic known as algae-sourced ß-1,3 glucan, on the overall performances of grass turtles (Chinemys reevesii) juveniles. Growth performance, immune responses, enzymatic antioxidant activities, intestinal histomorphology, and disease resistance against the challenge with Aeromonas veronii were assessed. Two hundred and sixteen (216) juvenile turtles with an average initial weight of 106.35 ± 0.03 g were divided into four groups, each containing three replicates with 18 turtles per each replicate, which were fed a basic diet (control group, GD) and a basal diet supplemented with C. butyricum 1.0 × 108 CFU per kg (GA group), or with B. subtilis 1.0 × 108 CFU per kg (GB group) and with algal-sourced ß-1,3-glucan 50 mg per kg (GC group), respectively. After the turtles had been fed for 60 d, 90 d, and 120 d of the experimental period, the growth performance and survival rate (SR), intestinal digestive enzyme, hepatic and intestinal antioxidant capacity, serum biochemical indexes, and immune performance were measured. The results showed that the weight gain rate and SR were significantly enhanced (P < 0.05) after fed probiotics and algae-sourced ß-1,3-glucan in all test times;The pepsin, amylase, acid phosphatase, total antioxidant capacity, triglyceride, alkaline phosphatase, urea nitrogen, cholesterol, total protein, IgA, IgG, IgM at 120 d were significantly enhanced (P<0.05) after fed C. butyricum. The intestinal villi heights, widths, and the thickness of the muscle layer were significantly higher in groups GA, GB, and GC than those reared within the GD control group (P < 0.05). After injecting the challenge by A. veronii the survival rate of grass turtles in the GA group (75%) was significantly higher than the other three groups (P<0.05), while there was no significant difference between the GB and GC groups compared with the control GD group, respectively (P>0.05). Overall, these results indicated that dietary supplementation with probiotics or algae-sourced ß-1,3 glucan, exhibited positive effects on C. reevesii. In particular, C. butyricum, showed the greatest improvements relating to growth, immune response, antioxidant activity, intestinal health, and disease resistance.

Diverse Response Pattern to Anoxia in Three Freshwater Turtle Species.

With increasing water eutrophication and global warming, anoxia and hypoxia are becoming more and more common in water environments. Most vertebrates have a limited tolerance to anoxia of only a few minutes, but some species, such as turtles, can survive for months being exposed to anoxia. Antioxidant defense systems may have a potential role in resisting anoxia stress in freshwater turtles. The three-keeled pond turtle Chinemys reevesii, the snapping turtle Chelydra serpentina and the soft-shelled turtle Pelodiscus sinensis are three popular aquaculture species and share similar habitats in China. While C. reevesii and C. serpentina are hard-shelled turtles with poor skin permeability, P. sinensis is soft-shelled turtle whose skin permeability is good. We examined the antioxidant defense responses in different tissues of the three turtle species under acute anoxia stress for 10 h and subsequently recovered for 24 h in order to reveal the response patterns of the antioxidant defense system of the three turtle species that differed in morphological structure and life history strategy. We found that the antioxidant response patterns to acute anoxia stress were tissue- and species-specific. The soft-shelled turtle was more sensitive to anoxia than the hard-shelled turtles. Under anoxia stress, the three species kept the activities of most antioxidant enzymes stable. C. reevesii and P. sinensis were highly dependent on vitamin C in antioxidant defense, while high activities of structural antioxidant enzymes were found in the tissues of C. serpentina. The above diverse patterns may be related with adaptive evolution of morphological structure and physiological functions of the three turtle species.

Degree of hydrolysis, functional and antioxidant properties of protein hydrolysates from Grass Turtle (Chinemys reevesii) as influenced by enzymatic hydrolysis conditions.

Grass turtle muscle was hydrolyzed with papain enzyme to produce protein hydrolysate (PH) and the degree of hydrolysis (DH) was determined. Under optimal conditions, the highest DH was 19.52% and the yield was recorded as 17.26%. Protein content of the hydrolysates was ranged from 73.35% to 76.63%. Total amino acids were more than 96.77% for each PH. The PH obtained at DH 19.52% achieved excellent solubility and emulsifying activity which were 95.56% and 108.76 m2/g, respectively at pH 6. Foam capacity amounted 100% in PH of DH 19.52% at pH 2, and water-holding capacity was 4.38 g/g. The antioxidant activity showed the strongest hydroxyl radical scavenging activity (95.25%), ABTS (84.88%), DPPH (75.89%), iron chelating (63.25%), and cupper chelating (66.90%) at DH 11.96%, whereas reducing power (0.88) at DH 19.52%. Thus, the findings indicated that utilization of grass turtle muscle protein hydrolysate is a potential alternative protein resource to improve the nutritional and functional properties in food ingredients and product formulations.

The invasive red-eared slider turtle is more successful than the native Chinese three-keeled pond turtle: evidence from the gut microbiota.

BACKGROUND: The mutualistic symbiosis between the gut microbial communities (microbiota) and their host animals has attracted much attention. Many factors potentially affect the gut microbiota, which also varies among host animals. The native Chinese three-keeled pond turtle (Chinemys reevesii) and the invasive red-eared slider turtle (Trachemys scripta elegans) are two common farm-raised species in China, with the latter generally considered a more successful species. However, supporting evidence from the gut microbiota has yet to be collected. METHODS: We collected feces samples from these two turtle species raised in a farm under identical conditions, and analyzed the composition and relative abundance of the gut microbes using bacterial 16S rRNA sequencing on the Roach/454 platform. RESULTS: The gut microbiota was mainly composed of Bacteroidetes and Firmicutes at the phylum level, and Porphyromonadaceae, Bacteroidaceae and Lachnospiraceae at the family level in both species. The relative abundance of the microbes and gene functions in the gut microbiota differed between the two species, whereas alpha or beta diversity did not. Microbes of the families Bacteroidaceae, Clostridiaceae and Lachnospiraceae were comparatively more abundant in C. reevesii, whereas those of the families Porphyromonadaceae and Fusobacteriaceae were comparatively more abundant in T. s. elegans. In both species the gut microbiota had functional roles in enhancing metabolism, genetic information processing and environmental information processing according to the Kyoto Encyclopedia of Genes and Genomes database. The potential to gain mass is greater in T. s. elegans than in C. reevesii, as revealed by the fact that the Firmicutes/Bacteroidetes ratio was lower in the former species. The percentage of human disease-related functional genes was lower in T. s. elegans than in C. reevesii, presumably suggesting an enhanced potential to colonize new habitats in the former species.

A First Insight into the Structural and Functional Comparison of Environmental Microbiota in Freshwater Turtle Chinemys reevesii at Different Growth Stages under Pond and Greenhouse Cultivation.

The microbial community structure of water is an important indicator for evaluating the water quality of the aquaculture environment. In this study, the investigation and comparison of the bacterial communities of pond cultivation (PC) and greenhouse cultivation (GC) between hatchling, juvenile, and adult growth stages of C. reevesii were performed. In addition, the V4 regions of the 16S rRNA gene were sequenced. The Chao1 richness estimator of the PC group was significantly higher than that of the GC group. The beta diversity showed that the microbiotas of the two groups were isolated from each other. The dominant phyla were Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes in the PC group and Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, Chloroflexi, and Actinobacteria in the GC group. Both the numbers and the types of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations differed between the PC and GC groups. The prediction of bacterial phenotype implied that the GC environment is more likely to deteriorate, and turtles are more susceptible to pathogens than those of the PC environment. In addition, a total of nine potential pathogenic bacteria were identified and the correlation of environmental factors analyses showed significant differences of bacterial species between the PC and GC groups, while the potential pathogenic bacteria showed significant correlation with the stocking density, temperature, pH, orthophosphate (PO4-P), and dissolved oxygen (DO) in both the PC and GC groups. Noticeably, this is the first report to describe the different microbiota characteristics of the different cultivation environments in the different growth stages of C. reevesii, which will provide valuable data for water quality adjustment, disease prevention, and the healthy breeding of turtles.

Identification of the cDNA Encoding the Growth Hormone Receptor (GHR) and the Regulation of GHR and IGF-I Gene Expression by Nutritional Status in Reeves' Turtle (Chinemys reevesii).

Chinemys reevesii (Reeves' turtle) is a slow-growing reptile that is distributed widely across China. Prior to this study, the cDNA sequence of the growth hormone receptor (GHR) in the Reeve's turtle, or how periods of starvation might influence the gene expression of GHR and insulin-like growth factor I (IGF-I) in this species, were unknown. Here, we identified the full-length sequence of the cDNA encoding GHR in Reeves' turtle by using RT-PCR and RACE. The full-length GHR cDNA was identified to be 3936 base-pairs in length, with a 1848 base-pair open reading frame (ORF) that encodes a 615 amino acid protein. Analysis showed that GHR mRNA was detectable in a wide range of tissues; the highest and lowest levels of expression were detected in the liver and the gonad, respectively. IGF-I was also expressed in a range of tissues, but not in the gonad; the highest levels of IGF-I expression were detected in the liver. After 4 weeks of fasting, the expression levels of GHR and IGF-I in the liver had decreased significantly; however, these gradually returned to normal after refeeding. We report the first cloned cDNA sequence for the GHR gene in the Reeve's turtle. Our findings provide a foundation from which to investigate the specific function of the GHR in Reeve's turtle, and serve as a reference for studying the effects of different nutrient levels on GHR expression in this species.

Effects of cadmium on the gene transcription of the liver in the freshwater turtle (Chinemys reevesii).

This study investigated the related gene transcription of liver in freshwater turtle Chinemys reevesii exposed to cadmium (Cd). After acclimation, healthy turtles were selected for experiments. They were randomly divided into four experimental groups and each group had 5 animals. The turtles were treated with 0 mg/kg, 7.5 mg/kg, 15 mg/kg, and 30 mg/kg Cd chloride separately by intraperitoneal injection. Liver samples were collected for examination of the transcription of related genes at 2 weeks after Cd exposure. The transcription of mRNA of MT, SOD, CAT, PNKP, and GPX4 genes in turtle liver cells were analyzed. Results showed that Cd promoted MT mRNA transcription in turtle's liver at low dose (7.5 mg/kg) and inhibited MT mRNA transcription in turtle's liver at middle dose (15 mg/kg) and high dose (30 mg/kg). Cd inhibited the transcription of SOD, CAT, and PNKP mRNA in turtle's liver, and the inhibition was obvious at high dose (30 mg/kg). Cd promoted GPX4 mRNA transcription in turtle's liver, especially at low dose (7.5 mg/kg). In conclusion, Cd had different effects on the mRNA transcription of liver cells in the freshwater turtle Chinemys reevesii exposed to Cd.

Identification of Plastrum Testudinis used in traditional medicine with DNA mini-barcodes

ABSTRACT The carapace of the tortoise Chinemys reevesii is an ingredient of "Guijia", a traditional Chinese medicine. However, C. reevesii is difficult to raise in aquaculture and is rare in the wild. Counterfeit tablets are made from carapaces of other species. In addition to C. reevesii, other species including Mauremys sinensis, Indotestudo elongate and Trachemys scripta have been used in Plastrum Testudinis as well. After processing, these carapaces are difficult to identify on the basis of morphological characteristics, which impedes law enforcement. Our study used DNA barcoding technology to identify C. reevesii and its substitutes. We extracted concentrated genomic DNA for PCR amplification. Based on the analysis of 61 full-length COI sequences, we designed four pairs of mini-barcode primers: Tu-A, Tu-B, Tu-C and Tu-D. The Tu-B primers sequenced genomic DNA with a success rate of 76.47%, and the Tu-D primers sequenced genomic DNA with a success rate of 88.24%. The identification efficiency of these two mini-barcodes was 70.59% and 64.71%, and the overall identification efficiency was approximately 76.47%. Similarly, a set of mini barcode systems was generated, which may provide an effective and low-cost method for the identification of authentic tortoise shells.

Effects of cadmium on oxidative stress activities in plasma of freshwater turtle Chinemys reevesii.

Cadmium (Cd) has been recently found in high concentrations in the aquatic environment. This study was designed to examine the effects of Cd on the oxidative stress activities in plasma of freshwater turtle Chinemys reevesii. Experimental turtles were exposed to Cd at the concentration of 15 mg/kg by intraperitoneal injection, and redox status was investigated. Compared to the controls, superoxide dismutase (SOD) and catalase activities in plasma of the treated animals significantly decreased in week 1, week 2, and week 4. However, SOD activities gradually increased from week 4 to week 8. The treated animals had higher content of MDA and lower content of GSH in plasma over the observation period. In conclusion, our results showed that Cd decreased the antioxidant capacity and increased the level of oxidative damage product in plasma, which suggest that Cd causes oxidative stress and damage in the animal under the experimental conditions.

Cadmium toxicokinetics in the freshwater turtle, Chinemys reevesii.

This study was designed to investigate the toxicokinetics of Cadmium (Cd) in Chinemys reevesii. The animals were exposed to 15 mg/kg Cd chloride by intraperitoneal injection, and the Cd absorption, distribution, and excretion in different organs were determined. The results showed that Cd absorption reached its peak in the blood at 3 h after treatment. The accumulation of Cd was the highest in the liver and the second highest in the pancreas. All other tissues also accumulated Cd, such as spleen, kidney, intestine, lung, stomach, heart, brain, muscle. A small amount of Cd was found in the faeces. The urine and bile had low concentrations of Cd. In conclusion, absorbance of Cd reaches its peak at 3 h in blood. The liver and pancreas are the major organs of Cd accumulation, and the major excretion route of Cd is through feaces.

Compensatory growth responses to food restriction in the Chinese three-keeled pond turtle, Chinemys reevesii.

Juvenile Chinese three-keeled pond turtles (Chinemys reevesii) were subjected to one of four different feeding regimens: ad libitum (AL), restricted (R), ad libitum-restricted (AL-R), or restricted-ad libitum (R-AL) for 13 weeks, to assess the compensatory growth (CG) response to food restriction and subsequent re-alimentation. After switching to ad libitum feeding, the turtles in R-AL group ate more food and grew faster than those in other groups. At the end of the trial, R-AL turtles achieved the comparable body weight as AL turtles, indicating that a complete CG response occurred. Cumulative food consumption over the entire period did not differ between R-AL turtles and AL turtles. Experimental treatment affected carcass composition. Carcass lipid content of AL turtles was greater than that of R and AL-R turtles, with R-AL turtles in between. Carcass protein content of R-AL turtles was slightly greater than that of other groups without statistical differences. Stored lipids might be consumed firstly when animals underwent food restriction. Our results reconfirmed the CG of C. reevesii after food restriction. However, it is still difficult to achieve a reduction in the cost of farm-raised turtle production by adopting a restricted-satiation feeding protocol.