pSTAT3 activation of Foxl2 initiates the female pathway underlying temperature-dependent sex determination.

Ovarian development was traditionally recognized as a "default" sexual outcome and therefore received much less scientific attention than testis development. In turtles with temperature-dependent sex determination (TSD), how the female pathway is initiated to induce ovary development remains unknown. In this study, we have found that phosphorylation of the signal transducer and activator of transcription 3 (pSTAT3) and Foxl2 exhibit temperature-dependent sexually dimorphic patterns and tempo-spatial coexpression in early embryos of the red-eared slider turtle (Trachemys scripta elegans). Inhibition of pSTAT3 at a female-producing temperature of 31 °C induces 64.7% female-to-male sex reversal, whereas activation of pSTAT3 at a male-producing temperature of 26 °C triggers 75.6% male-to-female sex reversal. In addition, pSTAT3 directly binds to the locus of the female sex-determining gene Foxl2 and promotes Foxl2 transcription. Overexpression or knockdown of Foxl2 can rescue the sex reversal induced by inhibition or activation of pSTAT3. This study has established a direct genetic link between warm temperature-induced STAT3 phosphorylation and female pathway initiation in a TSD system, highlighting the critical role of pSTAT3 in the cross talk between female and male pathways.

Angiotensin-(1-7) plays an important role in regulating spermatogenesis in Trachemys scripta elegans under salinity stress.

Elevation in water salinity can threaten the spermatogenesis and fertility of freshwater animals. The role of the renin-angiotensin system (RAS) in regulating spermatogenesis has attracted considerable attention. Our previous study found that red-eared sliders (Trachemys scripta elegans), could survive in 10 PSU water for over 1 year. To understand the chronic impact of salinity on testicular spermatogenesis and underlying mechanisms, male T. s. elegans were subjected to treatment with water of 5 PSU and 10 PSU for a year, and spermatogenesis and regulation of the RAS signal pathway was assessed. Results showed induced inflammation in the testes of T. s. elegans in the 10 PSU group, as evidenced by a decrease in the number of testicular germ cells from 1586 to 943. Compared with the control group, the levels of proinflammatory genes, including TNF-α, IL-12A and IL-6 were elevated 3.1, 0.3, and 1.4 times, respectively, in animals exposed to 10 PSU water. Testicular antiapoptotic processes of T. s. elegans might involve the vasoactive peptide angiotensin-(1-7) in the RAS, as its level was significantly increased from 220.2 ng ml-1 in controls to 419.2 ng ml-1 in the 10 PSU group. As expected, specific inhibitor (A-779) for the Ang-(1-7) acceptor effectively prevented the salinity-induced upregulation of genes encoding anti-inflammatory and antiapoptotic factors (TGF-ß1, Bcl-6) in the testis of the 10 PSU animals, whereas it promoted the upregulation of proinflammatory and proapoptotic factors (TNF-α, IL-12A, IL-6, Bax and caspase-3). Our data indicated that Ang-(1-7) attenuates the effect of salinity on inflammation and apoptosis of the testis in T. s. elegans. A new perspective to prevent salinity-induced testis dysfunction is provided.

Roles of estrogen receptors during sexual reversal in Pelodiscus sinensis.

BACKGROUND: The Chinese soft-shelled turtle, Pelodiscus sinensis, exhibits distinct sexual dimorphism, with the males growing faster and larger than the females. During breeding, all-male offspring can be obtained using 17ß-estradiol (E2). However, the molecular mechanisms underlying E2-induced sexual reversal have not yet been elucidated. Previous studies have investigated the molecular sequence and expression characteristics of estrogen receptors (ERs). METHODS AND RESULTS: In this study, primary liver cells and embryos of P. sinensis were treated with ER agonists or inhibitors. Cell incubation experiments revealed that nuclear ERs (nERs) were the main pathway for the transmission of estrogen signals. Our results showed that ERα agonist (ERα-ag) upregulated the expression of Rspo1, whereas ERα inhibitor (ERα-Inh) downregulated its expression. The expression of Dmrt1 was enhanced after ERα-Inh + G-ag treatment, indicating that the regulation of male genes may not act through a single estrogen receptor, but a combination of ERs. In embryos, only the ERα-ag remarkably promoted the expression levels of Rspo1, Wnt4, and ß-catenin, whereas the ERα-Inh had a suppressive effect. Additionally, Dmrt1, Amh, and Sox9 expression levels were downregulated after ERß inhibitor (ERß-Inh) treatment. GPER agonist (G-ag) has a significant promotion effect on Rspo1, Wnt4, and ß-catenin, while the inhibitor G-Inh does not affect male-related genes. CONCLUSIONS: Overall, these results suggest that ERs play different roles during sexual reversal in P. sinensis and ERα may be the main carrier of estrogen-induced sexual reversal in P. sinensis. Further studies need to be performed to analyze the mechanism of ER action.

Isolation, identification and characterization of Aeromonas jandaei from diseased Chinese soft-shell turtles.

Aeromonas jandaei is a gram-negative bacterium commonly found in aquatic environments and can induce illnesses in amphibians, reptiles and aquatic animals. In this study, a strain of bacteria was isolated from the diseased Chinese soft-shell turtle (Pelodiscus sinensis), then named strain JDP-FX. This isolate was identified as A. jandaei after analysis of morphological, physiological and biochemical characteristics, as well as 16S rRNA and gyrB gene sequences. Virulence genetic testing further detected temperature-sensitive protease (eprCAI), type III secretion system (TTSS) (ascv), nuclease (nuc), cytotonic enterotoxin (alt) and serine proteinase (ser) in JDP-FX. Compared with healthy Chinese soft-shell turtle, the serum levels of total protein (TP), albumin (ALB) and globulin (GLB) were significantly decreased in the diseased Chinese soft-shell turtle, while, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were significantly increased. Histopathological observations showed that multiple tissues, including intestinal mucosa, liver and kidney, were severely damaged in the diseased Chinese soft-shell turtle. Moreover, the diseased Chinese soft-shell turtle had significant cell degeneration, necrosis, sloughing and interstitial inflammatory cell infiltration. The pathogenicity of JDP-FX was tested via artificial infection. The median lethal dosage (LD50 ) of the strain was 1.05 × 105 colony forming units (CFU/g) per weight of Chinese soft-shell turtle. Drug susceptibility analysis revealed that JDP-FX was susceptible to ceftazidime, minocycline, cefoperazone, ceftriaxone and piperacillin. In addition, JDP-FX was resistant to doxycycline, florfenicol, sulfonamides, gentamicin, ampicillin and neomycin. Therefore, this study may provide guidance for further research into the diagnosis, prevention and treatment of JDP-FX infection.

Mercury biomagnification in the food chain of a piscivorous turtle species (Testudines: Chelidae: Chelus fimbriata) in the Central Amazon, Brazil.

Due to their natural history and ecological attributes, turtles are excellent organisms for studies of heavy metal contamination. Turtles have a large geographical distribution, occupy different aquatic habitats, and occupy various trophic levels. The present study investigated mercury bioaccumulation in the carnivorous chelonian Chelus fimbriata (Matamata turtle) and Hg biomagnification in relation to its aquatic food chain in the middle Rio Negro, AM-Brazil. Tissue samples of muscle, carapace and claws were collected from 26 C. fimbriata individuals, as well as collections of autotrophic energy sources found in the turtle's aquatic habitat area. The samples were collected in February-March/2014 and analyzed for THg concentrations and carbon (δ13C) and nitrogen (δ15N) stable isotopes. The highest THg levels were found in claws (3780 ng.g-1), carapace (3622 ng.g-1) and muscle (403 ng.g-1), which were found to be significantly different [F(2.73) = 49.02 p < 0.01]. However, THg concentrations in muscle tissue were below the consumption threshold indicated by the WHO and Brazilian Health Ministry. The average δ13C and δ15N values in Matamata samples were -31.7‰ and 11.9‰, respectively. The principal energy source sustaining the food chain of C. fimbriata was found to be terrestrial shrubs, with smaller contributions from emergent aquatic herbaceous plants and algae, while δ15N values showed its trophic position to be two levels above the autotrophic energy sources. There was a positive correlation between THg and turtle size, while a significant relationship was found between THg and δ15N, showing strong biomagnification in the food chain of C. fimbriata: y = 0.21x + 0.46; r2 = 0.45; p < 0.001, for which the slope presented a value of 0.21.

Pharmacokinetic characteristics of florfenicol in green sea turtles (Chelonia mydas) and hawksbill sea turtles (Eretmochelys imbricata) after intramuscular administration.

The pharmacokinetics of florfenicol (FFC) in green sea and hawksbill sea turtles were evaluated following intramuscular (i.m.) administration at two different dosages of 20 or 30 mg/kg body weight (b.w.). This study (longitudinal design) used 5 green sea and 5 hawksbill sea turtles for the two dosages. Blood samples were collected at assigned times up to 168 h. FFC plasma samples were analyzed using validated high-performance liquid chromatography equipped with diode array detection. The pharmacokinetic analysis was performed using a non-compartment approach. The FFC plasma concentrations increased with the dosage. The elimination half-life was similar between the treatment groups (range 19-25 h), as well as the plasma protein binding (range 18.59%-20.65%). According to the surrogate PK/PD parameter (T > MIC, 2 µg/mL), the 20 and 30 mg/kg dosing rates should be effective doses for susceptible bacterial infections in green sea and hawksbill sea turtles.

PHARMACOKINETICS OF TRAMADOL AND O-DESMETHYLTRAMADOL IN GIANT TORTOISES (CHELONOIDIS VANDENBURGHI, CHELONOIDIS VICINA).

The objective of this study was to determine the pharmacokinetics of two orally administered doses of tramadol (1 mg/kg and 5 mg/kg) and its metabolite, O-desmethyltramadol (M1) in giant tortoises (Chelonoidis vandenburghi, Chelonoidis vicina). Eleven giant tortoises (C. vandenburghi, C. vicina) received two randomly assigned, oral doses of tramadol (either 1 mg/kg or 5 mg/kg), with a washout period of 3 wk between each dose. The half-life (t½) of orally administered tramadol at 1 mg/kg and 5 mg/kg was 11.9 ± 4.6 h and 13.2 ± 6.1 h, respectively. After oral administration of tramadol at 1 mg/kg and 5 mg/kg, the maximum concentration (Cmax) was 125 ± 69 ng/ml and 518 ± 411 ng/ml, respectively. There were not enough data points to determine pharmacokinetic (PK) parameters for the M1 metabolite from either dose. Tramadol administered orally to giant tortoises at both doses provided measurable plasma concentrations of tramadol for approximately 48 h with occasional transient sedation. Oral tramadol at 5 mg/kg, on average, achieves concentrations of >100 ng/ml, the reported human therapeutic threshold, for 24 h. Based on the low levels of M1 seen in this study, M1 may not be a major metabolite in this taxon.

Low production of mitochondrial reactive oxygen species after anoxia and reoxygenation in turtle hearts.

Extremely anoxia-tolerant animals, such as freshwater turtles, survive anoxia and reoxygenation without sustaining tissue damage to their hearts. In contrast, for mammals, the ischemia-reperfusion (IR) injury that leads to tissue damage during a heart attack is initiated by a burst of superoxide (O2·-) production from the mitochondrial respiratory chain upon reperfusion of ischemic tissue. Whether turtles avoid oxidative tissue damage because of an absence of mitochondrial superoxide production upon reoxygenation, or because the turtle heart is particularly protected against this damage, is unclear. Here, we investigated whether there was an increase in mitochondrial O2·- production upon the reoxygenation of anoxic red-eared slider turtle hearts in vivo and in vitro. This was done by measuring the production of H2O2, the dismutation product of O2·-, using the mitochondria-targeted mass-spectrometric probe in vivo MitoB, while in parallel assessing changes in the metabolites driving mitochondrial O2·- production, succinate, ATP and ADP levels during anoxia, and H2O2 consumption and production rates of isolated heart mitochondria. We found that there was no excess production of in vivo H2O2 during 1 h of reoxygenation in turtles after 3 h anoxia at room temperature, suggesting that turtle hearts most likely do not suffer oxidative injury after anoxia because their mitochondria produce no excess O2·- upon reoxygenation. Instead, our data support the conclusion that both the low levels of succinate accumulation and the maintenance of ADP levels in the anoxic turtle heart are key factors in preventing the surge of O2·- production upon reoxygenation.

Effects of florfenicol on the antioxidant and immune systems of Chinese soft-shelled turtle (Pelodiscus sinensis).

Florfenicol is a commonly used antibiotic for the treatment of bacterial diseases of the Chinese soft-shelled turtle (Pelodiscus sinensis). The study investigated the effects of florfenicol on the antioxidant and immune system of P. sinensis. Results showed that the total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and catalase (CAT) activities were significantly increased in the 10 mg/kg and 40 mg/kg florfenicol treatment groups compared with the control group. Besides, the malondialdehyde (MDA) content was significantly increased, and the glutathione peroxidase (GSH-Px) activity was significantly decreased with 40 mg/kg florfenicol treatment. In addition, florfenicol has effects on the immune system, 10 mg/kg of florfenicol significantly promoted the activities of acid phosphatase (ACP) and alkaline phosphatase (AKP), whereas 40 mg/kg of florfenicol significantly inhibited their activities. To elucidate the molecular mechanisms, a comparative transcriptome analysis was conducted. A total of 59 differentially expressed genes (DEGs) and 12 significantly enriched KEGG pathways were identified in the 10 mg/kg group; 150 DEGs and 10 significantly enriched KEGG pathways were identified in the 40 mg/kg group. Among them, the complement and coagulation cascade pathways were the most significant which may play an important regulatory role in the immune response. The MADCAM1, STAT3, and IL4I1 genes may be the key genes of florfenicol affecting the immune response. The APOA1, APOA4, SPLA2, FADS1, and FADS2 genes may play a key role in anti-inflammatory and antioxidant effects through redox-related pathways. The study lays the foundation for a deeper understanding of the mechanism of the florfenicol effect on P. sinensis.

Probiotics and Immunostimulant modulate intestinal flora diversity in Reeves' Turtle (Mauremys reevesii) and effects of Clostridium butyricum on its spleen transcriptome.

In this study, we investigated the effects of Clostridium butyricum (group A), Bacillus subtilis (group B), and the immune enhancer algal ß-1,3 glucan (group C) on the intestinal flora of Reeves' turtle Mauremys reevesii and the effects of C. butyricum on the transcriptome of M. reevesii splenic immune tissues. Reeve' turtles were assigned to four groups, each containing three replicates from 18 samples. Juvenile turtles with an initial weight of 106.35 ± 0.03 g were fed a basic diet containing no probiotics (group D), or a basic diet containing C. butyricum TF20201120, B.subtilis, or algal ß-1,3 glucan supplement, respectively. After the turtles had been fed for 60, 90, and 120 d of the experimental period, high-throughput sequencing of the 16S rRNA gene revealed no significant difference in alpha diversity among the four groups at 60 days of feeding (P > 0.05), and at 90 days, the alpha diversity in group A was significantly different (P < 0.05), with an increase of 26.62% in the Shannon index and a decrease of 83.33% in the Simpson index; at 120 d, the alpha diversity (Shannon index) showed a decreasing trend in order for groups A, B, and C, At the phylum level, the abundance of Bacteroidetes, Proteobacteria, and Fusobacteria in group A increased significantly with increasing feeding time (P < 0.05), At the genus level, the abundance of Ruminococcaceae and Anaerotruncus in group A increased significantly compared with that in the other three groups (P < 0.05). Transcriptome analysis showed that 384 genes were differentially expressed in the spleen of M. reevesii, 195 genes were upregulated and 189 genes were downregulated, and C. butyricum TF201120 regulated the hematopoietic cell lineage signaling pathway in the spleen of M. reevesii (P < 0.05). The regulation of several identified immune-related genes was confirmed by qPCR. These results showed that C. butyricum, B. subtilis, and the immune enhancer algal ß-1,3 glucan can improve the intestinal flora of M. reevesii, with C. butyricum TF20201120 being the most effective and significantly enhancing the immunity of M. reevesii.

Non-targeted proteomics reveals altered immune response in geographically distinct populations of green sea turtles (Chelonia mydas).

All seven species of sea turtle are facing increasing pressures from human activities that are impacting their health. Changes in circulating blood proteins of an individual, or all members of a population, can provide an early indicator of adverse health outcomes. Non-targeted measurement of all detectable proteins in a blood sample can indicate physiological changes. In the context of wildlife toxicology, this technique can provide a powerful tool for discovering biomarkers of chemical exposure and effect. This study presents a non-targeted examination of the protein abundance in sea turtle plasma obtained from three geographically distinct foraging populations of green turtles (Chelonia mydas) on the Queensland coast. Relative changes in protein expression between sites were compared, and potential markers of contaminant exposure were investigated. Blood plasma protein profiles were distinct between populations, with 85 out of the 116 identified proteins differentially expressed (p < 0.001). The most strongly dysregulated proteins were predominantly acute phase proteins, suggestive of differing immune status between the populations. The highest upregulation of known markers of immunotoxicity, such as pentraxin fusion and complement factor h, was observed in the Moreton Bay turtles. Forty-five different organohalogens were also measured in green turtle plasma samples as exposure to some organohalogens (e.g., polychlorinated biphenyls) has previously been identified as a cause for immune dysregulation in marine animals. The few detected organohalogens were at very low (pg/mL) concentrations in turtles from all sites, and are unlikely to be the cause of the proteome differences observed. However, the changes in protein expression may be indicative of exposure to other chemicals or environmental stressors. The results of this study provide important information about differences in protein expression between different populations of turtles, and guide future toxicological and health studies on east-Australian green sea turtles.

Enhanced resistance to Ca2+-induced mitochondrial permeability transition in the long-lived red-footed tortoise Chelonoidis carbonaria.

The interaction between supraphysiological cytosolic Ca2+ levels and mitochondrial redox imbalance mediates the mitochondrial permeability transition (MPT). The MPT is involved in cell death, diseases and aging. This study compared the liver mitochondrial Ca2+ retention capacity and oxygen consumption in the long-lived red-footed tortoise (Chelonoidis carbonaria) with those in the rat as a reference standard. Mitochondrial Ca2+ retention capacity, a quantitative measure of MPT sensitivity, was remarkably higher in tortoises than in rats. This difference was minimized in the presence of the MPT inhibitors ADP and cyclosporine A. However, the Ca2+ retention capacities of tortoise and rat liver mitochondria were similar when both MPT inhibitors were present simultaneously. NADH-linked phosphorylating respiration rates of tortoise liver mitochondria represented only 30% of the maximal electron transport system capacity, indicating a limitation imposed by the phosphorylation system. These results suggested underlying differences in putative MPT structural components [e.g. ATP synthase, adenine nucleotide translocase (ANT) and cyclophilin D] between tortoises and rats. Indeed, in tortoise mitochondria, titrations of inhibitors of the oxidative phosphorylation components revealed a higher limitation of ANT. Furthermore, cyclophilin D activity was approximately 70% lower in tortoises than in rats. Investigation of critical properties of mitochondrial redox control that affect MPT demonstrated that tortoise and rat liver mitochondria exhibited similar rates of H2O2 release and glutathione redox status. Overall, our findings suggest that constraints imposed by ANT and cyclophilin D, putative components or regulators of the MPT pore, are associated with the enhanced resistance to Ca2+-induced MPT in tortoises.

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.

Characterization of prolactin (PRL) and PRL receptor (PRLR) in Chinese soft-shelled turtle: Molecular identification, ligand-receptor interaction and tissue distribution.

Prolactin (PRL) plays crucial roles in many physiological and pathological processes through activating its specific membrane-anchored receptor (PRLR). Although this ligand-receptor pair has been extensively studied in mammals, birds and fishes, researches examining their significance is rather scarce in reptiles. Additionally, the interaction mechanism of PRL-PRLR has abortively understood across vertebrates, since two tandem repeated ligand-binding domains of PRLR have been identified in birds and few reptiles. To lay the foundation to clarify their roles and ligand-receptor interaction in reptiles, using Chinese soft-shelled turtle as model, the cDNAs containing open reading frame of PRL and PRLR were cloned. The cloned PRL consisted of 710 bp and encoded a precursor of 228 amino acid (-aa), while PRLR was 2658 bp in length and predicted to generate a 828-aa precursor. Furthermore, the recombinant PRL protein with high-purity was prepared from Escherichia coli (E. coli) strain Rosetta gamiB (DE3) by using cobalt resin. Using the 5 × STAT5-Luciferase reporter system, we found PRLR and PRLR-M2 (the PRLR-mutant lacking membrane-distal ligand-binding domain) could be dose-dependently activated by recombinant PRL, thereby triggering the intracellular JAK2-STAT5 signaling cascade, suggesting PRL-PRLR is functional in Chinese soft-shelled turtle, and the membrane-proximal ligand-binding domain of PRLR is the critical domain involving in PRL-binding. Quantitative real-time PCR revealed that PRL was predominantly and abundantly expressed in pituitary, while PRLR exhibited ubiquitous expression in all of the tissues examined. Collectively, our data indicate the PRL-PRLR pair may function in reptiles including Chinese soft-shelled turtle, in a way similar to that in birds.

Characterisation of UDP-glucuronosyltransferase activity in sea turtle Chelonia mydas.

Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds, promoting their excretion. The complexity of UGT kinetics, and the location of enzyme active site in endoplasmic reticulum lumen, requires an accurate optimisation of enzyme assays.In the present study, we characterised UGT activity in liver microsomes of green turtles (Chelonia mydas), an endangered species. The conditions for measuring UGT activity were standardised through spectrofluorimetric methods, using the substrates 4-methylumbelliferone (4-MU) and uridine diphosphate glucuronic acid (UDPGA) at 30 °C and pH 7.4.The green turtles showed UGT activity at the saturating concentrations of substrates of 250 µM to 4-MU and 7 mM to UDPGA. The alamethicin, Brij®58, bovine serum albumin (BSA), and magnesium increased UGT activity. The assay using alamethicin (22 µg per mg of protein), magnesium (1 mM), and BSA (0.25%) reached the highest Vmax (1203 pmol·min-1mg·protein-1). Lithocholic acid and diclofenac inhibited UGT activity in green turtles.This study is the first report of UGT activity in the liver of green turtles and provides a base for future studies to understand the mechanisms of toxicity by exposure to contaminants in this charismatic species.

Goldfish and crucian carp are natural models of anoxia tolerance in the retina.

Vertebrates need oxygen to survive. The central nervous system has an especially high energy demand, so brain and retinal neurons quickly die in anoxia. But fish of the genus Carassius are exceptionally anoxia-tolerant: the crucian carp (C. carassius) can survive months without oxygen in ice-covered ponds, and the common goldfish (C. auratus) can withstand hours of anoxia at room temperature. These fish previously offered insights into anoxia tolerance in the brain, heart, and liver. Here, we advance Carassius spp. as models to study anoxia tolerance in the retina. Electroretinogram and evoked potential recordings show that crucian carp reversibly downregulate their visual systems in anoxia, probably to save ATP. Notably, Carassius suppress their visual systems nearly twice as much as anoxia-tolerant turtles, Trachemys and Chrysemys spp., which are often promoted as the champions of anoxia tolerance. We summarize what is known about anoxia tolerance in the goldfish and crucian carp retinas, including cellular pathways which may protect retinal neurons from excitotoxic cell death. We compare the Carassius retina with two relevant models: natural anoxia tolerance in the turtle brain, and ischemic preconditioning in the rat retina. All three models include mitochondria as oxygen sensors: mitochondria depolarize due to mitochondrial ATP-dependent K+ channels, possibly to trigger neuroprotective second messenger cascades. The Carassius retina is an accessible and inexpensive model, with over 70 fruitful years of history in vision research. As a model for anoxia tolerance, it may provide new insights into diseases of the eye (like diabetes, macular degeneration, and eye stroke).

The Seasonal and Stage-Specific Expression Patterns of HMGB2 Suggest Its Key Role in Spermatogenesis in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

HMGB2, a member of the high-mobility group (HMG) proteins, was identified as a male-biased gene and plays a crucial role in the germ cells differentiation of mammals. However, its role in spermatogenesis of turtle is still poorly understood. Here, we cloned the Pelodiscus sinensis HMGB2 and analyzed its expression profile in different tissues and in testis at different developmental ages. P. sinensis HMGB2 mRNA was highly expressed in the testis of 3-year-old turtles (P < 0.01), but was hardly detected in ovaries and other somatic tissues. The results of chemical in situ hybridization (CISH) showed that HMGB2 mRNA was specifically expressed in germ cells, where it was mainly distributed in round spermatids and sperm, but not detected in somatic cells, spermatogonia, primary spermatocytes, or secondary spermatocyte. The relative expression of HMGB2 also responded to seasonal changes in testis development in P. sinensis. In different seasons of the year, the relative expression of HMGB2 transcripts in the testis of 1 year and 2 year olds showed an overall upward trend, whereas, in the testis of 3 year old, it peaked in July and then declined in October. Moreover, in April and July, with an increase in ages, the expression of HMGB2 transcripts showed an upward trend. However, in January and October, there was a decline in expression in testis in 3-year-old turtles. These results showed that HMGB2 is closely related to spermatogenesis in P. sinensis.

Starch and fiber intake effects on energy metabolism, growth, and carapacial scute pyramiding of red-footed tortoise hatchlings (Chelonoidis carbonaria).

Tortoise husbandry includes reports of excessive growth and carapace pyramiding, although triggers still remain to be fully elucidated. Juvenile red-footed tortoises (Chelonoidis carbonaria) were fed with two different diets, one high in fiber (HF; 14.2% crude fiber; 39.2% neutral detergent fiber, NDF; dry matter basis, DMB) and one high in starch (HS; 27.7% DMB), to assess effects on energy metabolism, nutrient digestibility, and growth. A total of 20 hatchlings (10 per diet) were used to evaluate: apparent digestibility coefficients (Da) of nutrients and gross energy (GE), passage times at 5 and 11 months of age; resting and post-prandial metabolic rates at 6 and 12 months of age; growth rates; pyramiding; and estimated body composition. Animals fed HS showed higher mass-specific intake of digestible energy (113.9 ± 32.1 kJ kg-1 day-1 vs. 99.6 ± 35.3 kJ kg-1 day-1; P < 0.05), digestible DM (6.1 ± 1.8 g kg-1 day-1 vs. 5.0 ± 1.8 g kg-1 day-1; P < 0.01), shorter transit (3 ± 1 days vs. 4 ± 1 days; P < 0.01) and retention times (8 ± 2 days vs. 10 ± 2 days; P < 0.01), and higher Da of DM, starch, NDF, and GE. Crude protein Da was higher for HF. Rest and post-prandial metabolic rates, and pyramiding degree were not affected by diets. At 13 months, the animals from HS presented wider plastrons and carapaces, and higher carapace width growth rates. In addition, these animals had lower body mineral content (1.88 ± 0.15% vs. 2.15 ± 0.19%; P < 0.01) and bone density (0.13 ± 0.01 g mm-2 vs. 0.15 ± 0.02 g mm-2; P < 0.02). Results provide evidence that highly digestible foods can accelerate shell growth and lower mineralization in this species.

The interaction between Tu-Izumo1 and Tu-JUNO is involved in turtles hybridization.

The specificity of sperm-egg recognition is crucial to species independence, and two proteins (Izumo1 and JUNO) are essential for gamete adhesion/fusion in mammals. However, hybridization, which is very common in turtles, also requires specific recognition of sperm-egg binding proteins. In this study, we discovered that natural selection plays an important role in the codon usage bias of Tu-Izumo1 and Tu-JUNO. Positively selected sites and co-evolutionary analyses between Tu-Izumo1 and Tu-JUNO have been previously reported, and we confirm these results in a larger analysis containing 25 turtle species. We also showed that Tu-JUNO is expressed on the oocyte surface and that Tu-Izumo1 and Tu-JUNO interact with each other directly in different species hybridization combinations. Co-immunization assays revealed that this interaction is evolutionarily conserved in turtles. The results of avidity-based extracellular interaction screening between Tu-Izumo1 and Tu-JUNO for sperm-oocyte binding pairs (both within and across species) likely suggest that the interaction force between Izumo1 and JUNO has a certain correlation in whether the turtles can hybridize. Our results lay a theoretical foundation for the subsequent development of techniques to detect whether different turtle species can interbreed, which would provide the molecular basis for breeding management and species protection of turtles.

Evaluation of dietary zinc on antioxidant-related gene expression, antioxidant capability and immunity of soft-shelled turtles Pelodiscussinensis.

Zinc (Zn) plays a role in the antioxidant capacity and immunity of aquatic animals. A twelve-week feeding experiment was performed to estimate the impact of dietary zinc on antioxidant enzyme-related gene expression, antioxidant enzyme activity and non-specific immune functions of soft-shelled turtles, Pelodiscus sinensis. Six fishmeal-based experimental diets with 32.45% protein were formulated, which contained 35.43, 46.23, 55.38, 66.74, 75.06 and 85.24 mg/kg Zn, respectively. Catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels improved with an elevation in dietary Zn from 35.43 to 55.38 mg/kg and then reduced when dietary Zn was further elevated. The expression levels of Nrf2 and antioxidant-related genes CuZnSOD, MnSOD, CAT, GPX1, GPX2, GPX3 and GPX4 escalated with elevating Zn concentration up to 55.38 mg/kg in diets and then reduced as dietary Zn elevated. The expression levels of Kelch-like ECH-associating protein 1 (keap1) showed a reverse trend with that of Nrf2. The contents of malondialdehyde (MDA) in the 55.38 and 66.74 mg/kg Zn diet-fed groups were the lowest. Alkaline phosphatase activity (AKP), superoxide anion (O2-), lysozyme activity and total antioxidant capacity (T-AOC) improved with an escalation in dietary Zn concentration up to 66.74 mg/kg. Optimal dietary Zn improved antioxidant capability, immunity, and antioxidant enzyme-related gene expression. The dietary Zn demand for soft-shelled turtles were 60.93 and 61.63 mg/kg, based on second regression analysis of SOD and T-AOC activity, respectively.