Turtle peptide and its derivative peptide ameliorated DSS-induced ulcerative colitis by inhibiting inflammation and modulating the composition of the gut microbiota.

Ulcerative colitis (UC) is a recurrent intestinal disease with an increasing incidence worldwide that seriously affects the life of patients. Turtle peptide (TP) is a bioactive peptide extracted from turtles that has anti-inflammatory, antioxidant and anti-aging properties. However, studies investigating the effect of TP on the progression of UC are lacking. The aim of this study was to investigate effects and underlying mechanisms of TP and its derivative peptide GPAGPIGPV (GP-9) in alleviating UC in mice. The results showed that 500 mg/kg TP treatment significantly ameliorated colitis symptoms and oxidative stress in UC mice. TP alleviated intestinal barrier damage in UC mice by promoting mucosal repair and increasing the expression of tight junction proteins (ZO1, occludin and claudin-1). TP also modulated the composition of the gut microbiota by increasing the abundance of the beneficial bacteria Anaerotignum, Prevotellaceae_UCG-001, Alistipes, and Lachno-spiraceae_NK4A136_group and decreasing the abundance of the harmful bacteria Prevotella_9 and Parasutterella. Furthermore, we characterized the peptide composition of TP and found that GP-9 ameliorated the symptoms of dextran sodium sulfate (DSS)-induced colitis in mice by inhibiting the TLR4/NF-κB signaling pathway. In conclusion, TP and its derivative peptides ameliorated DSS-induced ulcerative colitis by inhibiting the expression of inflammatory factors and modulating the composition of the intestinal microbiota; this study provides a theoretical basis for the application of TP and its derivative peptides for their anti-inflammatory activity.

Gene expression changes with tumor disease and leech parasitism in the juvenile green sea turtle skin transcriptome.

Emerging infectious diseases are a major threat to biodiversity in the 21st century. Fibropapillomatosis (FP) is an epithelial tumor disease that affects immature and adult marine turtles worldwide, particularly green turtles (Chelonia mydas). We know little about the host factors contributing to FP susceptibility, in part because transcriptomic studies that compare transcript expression in turtles with and without FP are lacking. Here, we performed RNA-Seq on healthy skin tissue from immature C. mydas in the Indian River Lagoon, Florida, USA, comparing turtles (1) with and without FP and (2) with and without leech parasites, a putative vector of FP. We assembled a de novo C. mydas skin transcriptome to identify transcripts with significant differential expression (DE) across FP and leech categories. Significant DE transcripts were found across FP and leech comparisons, including 10 of the same transcripts with DE across both comparisons. Leech-positive individuals significantly upregulated different immune and viral interaction transcripts than did leech-negative individuals, including viral interaction transcripts associated with herpesvirus interactions. This finding strengthens the role of marine leeches as mechanical vectors of Chelonid herpesvirus 5 (ChHV5) which has been implicated as a causative agent of FP. FP-positive turtles upregulated several tumor progression and suppression transcripts relative to FP-negative turtles, which had no significant DE tumor progression transcripts. FP-positive turtles also upregulated significantly more protein interaction transcripts than FP-negative turtles. DE transcripts across leech comparisons showed no functional enrichment, whereas DE transcripts across FP comparisons showed some GO terms were enriched in FP-positive and FP negative turtles. Notably, only FP-negative turtles were enriched for GO terms involved in acquired and inflammatory immune gene regulation. Overall, our DE transcripts included several candidate genes that may play important roles in C. mydas resistance to or recovery from FP, highlighting that transcriptomics provides a promising venue to understand this impactful disease. Continued investigation of C. mydas responses to FP and leech affliction is imperative for species persistence and the conservation of marine ecosystems worldwide due to the essential role of sea turtles in ecosystem function and stability.

Immune and sex-biased gene expression in the threatened Mojave desert tortoise, Gopherus agassizii.

The immune system of ectotherms, particularly non-avian reptiles, remains poorly characterized regarding the genes involved in immune function, and their function in wild populations. We used RNA-Seq to explore the systemic response of Mojave desert tortoise (Gopherus agassizii) gene expression to three levels of Mycoplasma infection to better understand the host response to this bacterial pathogen. We found over an order of magnitude more genes differentially expressed between male and female tortoises (1,037 genes) than differentially expressed among immune groups (40 genes). There were 8 genes differentially expressed among both variables that can be considered sex-biased immune genes in this tortoise. Among experimental immune groups we find enriched GO biological processes for cysteine catabolism, regulation of type 1 interferon production, and regulation of cytokine production involved in immune response. Sex-biased transcription involves iron ion transport, iron ion homeostasis, and regulation of interferon-beta production to be enriched. More detailed work is needed to assess the seasonal response of the candidate genes found here. How seasonal fluctuation of testosterone and corticosterone modulate the immunosuppression of males and their susceptibility to Mycoplasma infection also warrants further investigation, as well as the importance of iron in the immune function and sex-biased differences of this species. Finally, future transcriptional studies should avoid drawing blood from tortoises via subcarapacial venipuncture as the variable aspiration of lymphatic fluid will confound the differential expression of genes.

Comparative studies of the expression of creatine kinase isoforms under immune stress in Pelodiscus sinensis.

The expression and localization of different isoforms of creatine kinase in Pelodiscus sinensis (PSCK) were studied to reveal the role of PSCK isozymes (PSCK-B, PSCK-M, PSCK-S) under bacterial infection-induced immunologic stress. The computational molecular dynamics simulations predicted that PSCK-S would mostly possess a kinase function in a structural aspect when compared to PSCK-B and PSCK-M. The assay of biochemical parameters such as total superoxide dismutase (T-SOD), lactate dehydrogenase (LDH), malondialdehyde (MDA), catalase (CAT), and the content of ATP were measured along with total PSCK activity in different tissue samples under bacterial infection. The expression detections of PSCK isozymes in vitro and in vivo were overall well-matched where PSCK isozymes were expressed differently in P. sinensis tissues. The results showed that PSCK-B mostly contributes to the spleen, followed by the liver and myocardium; PSCK-M mostly contributes to the liver, followed by the myocardium and skeletal muscle, while PSCK-S contributes to the spleen and is uniquely expressed in skeletal muscle. Our study suggests that the various alterations of PSCK isozymes in tissues of P. sinensis are prone to defense the bacterial infection and blocking energetic imbalance before severe pathogenesis turned on in P. sinensis.

Differences in Antibody Responses against Chelonid Alphaherpesvirus 5 (ChHV5) Suggest Differences in Virus Biology in ChHV5-Seropositive Green Turtles from Hawaii and ChHV5-Seropositive Green Turtles from Florida.

Fibropapillomatosis (FP) is a tumor disease associated with a herpesvirus (chelonid herpesvirus 5 [ChHV5]) that affects mainly green turtles globally. Understanding the epidemiology of FP has been hampered by a lack of robust serological assays to monitor exposure to ChHV5. This is due in part to an inability to efficiently culture the virus in vitro for neutralization assays. Here, we expressed two glycoproteins (FUS4 and FUS8) from ChHV5 using baculovirus. These proteins were immobilized on enzyme-linked immunosorbent assay plates in their native form and assayed for reactivity to two types of antibodies, full-length 7S IgY and 5.7S IgY, which has a truncated Fc region. Turtles from Florida were uniformly seropositive to ChHV5 regardless of tumor status. In contrast, in turtles from Hawaii, we detected strong antibody reactivity mainly in tumored animals, with a lower antibody response being seen in nontumored animals, including those from areas where FP is enzootic. Turtles from Hawaii actively shedding ChHV5 were more seropositive than nonshedders. In trying to account for differences in the serological responses to ChHV5 between green turtles from Hawaii and green turtles from Florida, we rejected the cross-reactivity of antibodies to other herpesviruses, differences in viral epitopes, or differences in procedure as likely explanations. Rather, behavioral or other differences between green turtles from Hawaii and green turtles from Florida might have led to the emergence of biologically different viral strains. While the strains from turtles in Florida apparently spread independently of tumors, the transmission of the Hawaiian subtype relies heavily on tumor formation.IMPORTANCE Fibropapillomatosis (FP) is a tumor disease associated with chelonid herpesvirus 5 (ChHV5) that is an important cause of mortality in threatened green turtles globally. FP is expanding in Florida and the Caribbean but declining in Hawaii. We show that Hawaiian turtles mount antibodies to ChHV5 mainly in response to tumors, which are the only sites of viral replication, whereas tumored and nontumored Floridian turtles are uniformly seropositive. Tumor viruses that depend on tumors for replication and spread are rare, with the only example being the retrovirus causing walleye dermal sarcoma in fish. The Hawaiian strain of ChHV5 may be the first DNA virus with such an unusual life history. Our findings, along with the fundamental differences in the life histories between Floridian turtles and Hawaiian turtles, may partly explain the differential dynamics of FP between the two regions.

Effect of seasonal variance on intestinal epithelial barriers and the associated innate immune response of the small intestine of the Chinese soft-shelled turtles.

It is conceivable that pathological conditions can cause intestinal barrier disruption and innate immune dysfunction. However, very limited information has been reported on the effect of seasonal variance on intestinal barriers and innate immunity. The present study was designed to investigate the seasonal variance in intestinal epithelial barriers and the associated innate immune response of turtle intestines during hibernation and nonhibernation periods. Goblet cells (GCs) demonstrated dynamic actions of the mucosal barrier with strong Muc2 protein expression during hibernation. However, weak Muc2 expression during nonhibernation was confirmed by immunohistochemistry, immunofluorescence and immunoblotting. Furthermore, light and transmission electron microscopy revealed that the hypertrophy of GCs resulted in the hypersecretion of mucus granules (MGs) and created a well-developed mucosal layer during hibernation. The absorptive cells (ACs), forming a physical barrier of tight junctions, and desmosomes were firmly anchored during hibernation. Conversely, during nonhibernation, the integrity of tight junctions, adherence junctions and desmosomes was noticeable expanded, causing increased paracellular permeability. As further confirmation, there was strong zonula occluden-1 (ZO-1) and connexins 43 (Cx43) protein expression during hibernation and weak ZO-1 and Cx43 expression during nonhibernation. Moreover, the expression level of the innate immune response proteins Toll-like receptors 2 and 4 (TLR2 and 4) were enhanced during hibernation and were reduced during nonhibernation. These results provide rich information about the seasonal fluctuations that interrupt intestinal epithelial barriers and innate immune response, which might be essential for protection and intestinal homeostasis.

Characterization of a classical 2-cysteine peroxiredoxin1 gene from Chinese soft-shelled turtle Pelodiscus sinensis with its potent antioxidant properties and putative immune response.

Peroxiredoxin family members could function in host defense against oxidative stress, and modulate immune response. In the present study, a 2-cysteine peroxiredoxin gene named PsPrx1 was isolated from Chinese soft-shelled turtle Pelodiscus sinensis. The PsPrx1 cDNA was composed of 1130 bp, consisted of 199 amino acid residues and included a Redoxin and AphC-TSA domain. As detected by qPCR, PsPrx1 was ubiquitously expressed in the examined tissues with the higher levels in liver and spleen. Upon the immune challenge with A. jandaei bacteria and oxidative stress with ammonia pressure, both mRNA and protein expression level in liver could be significantly enhanced. The results of immunohistochemical examinations showed PsPrx1 was mainly distributed at the junction between the hepatic cells. The general functional properties of PsPrx1 were confirmed using purified rPsPrx1 protein. From the results, rPsPrx1 protein was confirmed to exhibit antioxidant activity and antibacterial properties. The potential for scavenging extracellular H2O2 was evidenced by the purified rPsPrx1 protein in vitro system. In the mixed-function oxidase assay, rPsPrx1 also exhibited a dose-dependent inhibition of DNA damage. These results suggest that rPsPrx1 was implicated defense against microbial pathogens and oxidants, and would provide important information to further understand the functional mechanism of Prx1 in P. sinensis immunity.

Diversity, immunoregulatory action and structure-activity relationship of green sea turtle cathelicidins.

Cathelicidins are a family of gene-encoded immune effectors in vertebrate innate immunity. Here, we reported the diversity and biological activity of cathelicidins in green sea turtle, a marine reptile species known for long lifespan and disease resistance. Four novel cathelicidins (Cm-CATH1-4) were identified from green sea turtle. All of them, especially Cm-CATH2, exhibited potent, broad-spectrum and rapid bactericidal and anti-biofilm activities by inducing the disruption of cell membrane integrity. Additionally, Cm-CATH2 effectively induced the macrophages/monocytes and neutrophils trafficking to the infection site, and inhibited the LPS-induced production of inflammatory cytokines, by blocking TLR4/MD2 complex and the downstream signaling pathway activation. In mouse peritonitis and pneumonia models, Cm-CATH2 exhibited evident protection against drug-resistant bacterial infections. Taken together, the diverse structures and functions of Cm-CATHs indicated their pleiotropic role in innate immunity of green sea turtle, and the potent antimicrobial, anti-biofilm and immunomodulatory properties make them ideal candidates for the development of novel anti-infective drugs.

Identification and characterization of the interferon-γ-inducible lysosomal thiol reductase gene in Chinese soft-shelled turtle, Pelodiscus sinensis.

The reduction of disulfide bonds of exogenous antigens is crucial to the MHC-II class antigen processing and presenting pathway and is catalysed by interferon-γ-inducible lysosomal thiol reductase (GILT). In this study, a reptile GILT gene from Chinese soft-shelled turtle, Pelodiscus sinensis (PsGILT), was identified. The full-length cDNA of PsGILT is 1631 nucleotides (nt), including a 5'-untranslated region (UTR) of 3 nt, a 3'-UTR of 860 nt and an open reading frame (ORF) of 768 nt encoding 255 amino acids (aa). The conserved features in known GILTs, such as signal peptide, CXXC motif, GILT signature sequence, N-glycosylation site and conserved cysteines, were all found in the putative PsGILT protein. Genomic analysis revealed that PsGILT kept the "7 exons and 6 introns" structure of vertebrate GILT genes. PsGILT was expressed in all examined organs/tissues and was mainly expressed in spleen and blood. Increased mRNA expression levels of PsIFN-γ and PsGILT in PBLs were observed after induction with LPS, PolyI:C and recombinant IFN-γ (rIFN-γ). We also tested the reductase activity of rGILT in vitro and found that it could reduce intact human IgG into H chains and L chains. These above results implied that PsGILT may play an important role in resisting bacterial and viral infections, like other vertebrate GILTs.

More than Fever: Thermoregulatory Responses to Immunological Stimulation and Consequences of Thermoregulatory Strategy on Innate Immunity in Gopher Tortoises (Gopherus polyphemus).

Organisms possess a range of thermoregulatory strategies that may vary in response to sickness, thereby driving important life-history consequences. Because the immune system is vital to maintaining organism function, understanding the suite of immune responses to infection indicates basic costs and benefits of physiological strategies. Here, we assessed consequences of thermoregulation and seasonality on immune function in both immunologically stimulated and nonstimulated gopher tortoises (Gopherus polyphemus). An ectothermic vertebrate was used as an experimental model because the effects of thermoregulation on immunity remain understudied and are of increasing importance in light of anthropogenic alterations to thermal environments. We found that G. polyphemus increased body temperature (Tb) at 1 h after injection with lipopolysaccharide (LPS) when compared with saline controls (P = 0.04), consistent with behavioral fever. LPS increased plasma bactericidal ability (BA; P = 0.006), reduced plasma iron concentration (P = 0.041), and increased heterophil∶lymphocyte ratios (P < 0.001). In nonstimulated animals, thermoregulatory strategy had a strong effect on innate immunity, which demonstrated that individuals have the ability to facultatively adjust immune function when infection burden is low; this relationship was not present in LPS-injected animals, which suggested that animals stimulated with LPS maximize bactericidal ability independently of temperature. Seasonal acclimation state did not influence responses to LPS, although baseline plasma iron was significantly lower in animals acclimated to winter. These results support that a trade-off exists between immunity and other conflicting physiological interests. Moreover, these results clearly demonstrate the ability of individuals to modulate immune function as a direct result of thermoregulatory decisions.

Lag of Immunity Across Seasonal Acclimation States in Gopher Tortoises (Gopherus Polyphemus).

Disease outbreaks are of increasing importance to ectothermic vertebrates as one of numerous results of global change. Anthropogenic climate change is predicted to increase climatic instability, thereby altering natural thermal environments. In this study, we evaluated the direct effects of rapid temperature change on immunity in Gopher Tortoises (Gopherus polyphemus). Specifically, we tested the lag hypothesis, which predicts significant misalignment of optimal and realized immunity when temperature rapidly changes. We assayed constitutive innate immunity, B-cell humoral responses, and heterophil: lymphocyte ratios in response to rapid temperature changes corresponding to realistic changes in body temperature between winter and summer. We found that during summer, rapid temperature reduction caused a series of changes in immunity, including reduced bactericidal ability (P = 0.002), reduced humoral response (P < 0.0001), and increased heterophil:lymphocyte ratios (P < 0.0001). During winter, we found that a temperature increase provided no benefit to immunity. Specifically, there was no increase in bactericidal ability as was predicted by the lag hypothesis. In winter, humoral responses were significantly reduced as a result of rapid warming (P = 0.011) and the rapid warming caused a significant reduction in heterophil:lymphocyte ratios (P < 0.0001). Independent of temperature, we found a significant acclimation effect of winter relative to summer conditions in humoral response (P < 0.001), which showed an overall increase in this parameter during winter. Our findings demonstrate that rapid temperature change, regardless of its direction, is a constraint on immunity in ectothermic vertebrates.

De novo transcriptome analysis reveals insights into different mechanisms of growth and immunity in a Chinese soft-shelled turtle hybrid and the parental varieties.

The Chinese soft-shelled turtle (Pelodiscus sinensis) is a highly important freshwater aquaculture species in China. The molecular mechanisms underlying changes in immunity and growth in hybrid vigor are not well understood. In the present study, the transcriptomes from significantly different P. sinensis strains (Qingxi black turtle, B and Japanese strain, J) and the resulting hybrid (Zajiao-1, F) were sequenced using an Illumina sequencing platform. Differentially expressed genes (DEGs) between Zajiao-1 and the Qingxi black turtle were enriched mainly in the HTLV-I infection and Hippo signaling pathways, while DEGs between the Zajiao-1 and Japanese strain were enriched mainly in tryptophan metabolism, caner-associated pathways, transcriptional dysregulation in cancer, amebiasis, Fcγ-mediated phagocytosis and the peroxisome pathway. Highly expressed genes involved in the regulation of disorders of the fatty acid biosynthesis, immune and cardiovascular systems in P. sinensis were found among the DEGs. Enrichment categories for gene ontology included cellular processes, metabolic pathways, and the actin cytoskeleton pathway. The reliability of the sequencing data was verified through quantitative real-time polymerase chain reaction analysis of 20 immunity or growth-related genes. These findings offer new insights into heterosis of growth traits and resistance to stresses and potential strategies for selective breeding.

Seasonal Acclimation of Constitutive Immunity in Gopher Tortoises Gopherus polyphemus.

Studies have suggested a role for natural seasonal change to drive patterns of disease, especially within ectothermic vertebrates. In light of recent climate change, it is important to understand baseline disease resistance in a seasonal context to further understand the role that changes in seasonal weather patterns may have in increasing disease frequency. Herein we found support for the seasonal acclimation hypothesis in Gopherus polyphemus (gopher tortoise), which indicated that natural seasonal variation causes differences in baseline immune function across seasonal acclimation states. We found that an innate immune parameter, bactericidal ability (BA), was significantly elevated in the summer (P < 0.00001). Circulating leukocyte profiles varied significantly among seasons, with heterophils and monocytes increased (P = 0.00019 and P = 0.0001, respectively) and lymphocytes decreased (P < 0.00001) during winter. We assayed baseline glucocorticoid concentration (e.g., corticosterone [CORT]) across seasons and sampling conditions to test whether CORT drove the seasonal pattern in immunological acclimation. CORT was significantly lowest during winter and in animals temporarily maintained in seminatural conditions. These changes in CORT occurred independently of the immunological adjustments, suggesting that the seasonal pattern of immunity was not mediated by CORT secretion. The reduction in lymphocytes and BA and also BA during winter suggest that seasonal acclimation is likely a restraint on energetic output when temperature is low and physiological performance is thermally constrained. While these parameters were reduced in winter, the increase in heterophils and monocytes may indicate a compensatory immune adjustment to increase the number of innate phagocytic cells.

Acute cold stress improved the transcription of pro-inflammatory cytokines of Chinese soft-shelled turtle against Aeromonas hydrophila.

Chinese soft-shelled turtle, Pelodiscus sinensis, is widely cultured in East and Southeast Asian countries. It frequently encounters the stress of abrupt temperature changes, which leads to mass death in most cases. However, the mechanism underlying the stress-elicited death remains unknown. We have suspected that the stress impaired the immune function of Chinese soft-shelled turtle, which could result in the mass death, as we noticed that there was a clinical syndrome of infection in dead turtles. To test our hypothesis, we first performed bioinformatic annotation of several pro-inflammatory molecules (IL-1ß, TNFα, IL-6, IL-12ß) of Chinese soft-shelled turtle. Then, we treated the turtles in six groups, injected with Aeromonas hydrophila before acute cold stress (25 °C) and controls, after acute cold stress (15 °C) and controls as well as after the temperature was restored to 25 °C and controls, respectively. Subsequently, real-time PCR for several pro-inflammatory cytokines (IL-1ß, TNFα, IL-6, IL-12ß, IL-8 and IFNγ) was performed to assess the turtle immune function in spleen and intestine, 24 hours after the injection. We found that the mRNA expression levels of the immune molecules were all enhanced after acute cold stress. This change disappeared when the temperature was restored back to 25 °C. Our results suggest that abrupt temperature drop did not suppress the immune function of Chinese soft-shelled turtle in response to germ challenge after abrupt temperature drop. In contrast, it may even increase the expression of various cytokines at least, within a short time after acute cold stress.

Characterisation of the green turtle's leukocyte subpopulations by flow cytometry and evaluation of their phagocytic activity.

Phagocytosis is a fundamental aspect of innate immunity that is conserved across many species making it a potentially useful health-assessment tool for wildlife. In non-mammalian vertebrates, heterophils, monocytes, macrophages, melanomacrophages, and thrombocytes all have phagocytic properties. Recently, B lymphocytes from fish, amphibians, and aquatic turtles have also showed phagocytic capacity. Phagocytes can be studied by flow cytometry; however, the use of this tool is complicated in reptiles partly because nucleated erythrocytes complicate the procedure. We separated green turtle leukocytes by density gradient centrifugation and identified subpopulations by flow cytometry and confocal microscopy. Additionally, we assessed their ability to phagocytize Fluorspheres and Ovoalbumin-Alexa. We found that heterophils and lymphocytes but not monocytes could be easily identified by flow cytometry. While heterophils from adults and juvenile turtles were equally able to phagocytize fluorspheres, adults had significantly more phagocytic ability for OVA-Alexa. Lymphocytes had a mild phagocytic activity with fluorospheres (27-38 %; 39-45 %) and OVA-Alexa (35-46 %; 14-22 %) in juvenile and adult green turtles, respectively. Confocal microscopy confirmed phagocytosis of fluorospheres in both heterophils and lymphocytes. This provides the first evidence that green turtle lymphocytes have phagocytic activity and that this assay could potentially be useful to measure one aspect of innate immunity in this species.

Molecular characterization and expression analysis of VSIG4 from the Asian yellow pond turtle, Mauremys mutica.

V-set and immunoglobulin domain-containing protein 4 (VSIG4), a member of the immunoglobulin superfamily, plays an important role in the immune system. This study isolated and characterized a cDNA encoding VSIG4 (MaVSIG4) from the Asian yellow pond turtle (Mauremys mutica). The MaVSIG4 cDNA is 1840 bp long and contains an open reading frame of 1,182 bp that encodes a polypeptide of 372 amino acids. The genomic sequence of MaVSIG4 spans 7,682 bp, with six exons and five introns. The phylogenetic tree shows that MaVSIG4 is most closely related to Gallus gallus VSIG4. The expression analysis by real-time PCR reveals that MaVSIG4 is ubiquitously expressed in various healthy tissues, with a higher expression level in the liver. After immune stimulation, the expression level of MaVSIG4 sharply decreased in the liver, heart, and kidney at 12 h (P < 0.01). These results provide a basis for further study of the function of MaVSIG4 in the turtle's immune system.

No evidence that estrogens affect the development of the immune system in the red-eared slider turtle, Trachemys scripta.

Exposure to maternally derived substances during development can affect offspring phenotype. In ovo exposure to maternally derived steroids has been shown to influence traits such as growth and behavior in the offspring. The development of the immune system also can be altered by exposure to both androgens and glucocorticoids in a variety of species, but much less is known about the potential for estrogens to influence the development of this system. We examined the effect of estradiol on the development of both innate and adaptive immune components in the red-eared slider turtle (Trachemys scripta). A bacterial killing assay was used to assess innate immunity, a delayed-type hypersensitivity test for cellular immunity, and total immunoglobulin levels to measure the humoral immune response. We found no effect of in ovo estradiol treatment on any of our immune measures despite using doses that are known to influence other phenotypic parameters during development and varying the timing of dosing across development. Our results suggest that maternally derived estradiol does not affect the development of the immune system in T. scripta.

Identification and characterization of the first reptilian CD9, and its expression analysis in response to bacterial infection.

In this study, a CD9 homologue in a reptile, Chinese soft-shelled turtle, has been cloned and identified for the first time. The full-length cDNA of turtle CD9 was 1146bp and contained a 672bp open reading frame (ORF) coding for a protein of 224 amino acids. Four transmembrane domains (TMs) divided the turtle CD9 into several parts: short N-, C-termini, an intracellular loop and two (small and large) extracellular loops (SEL and LEL). A CCG motif, a potential N-linked glycosylation site and 10 cysteine residues were well conserved. The deduced amino acid sequence analysis showed that the turtle CD9 shared 82% identity to duck CD9. Most of the differences were found in the LEL. Phylogenetic analysis showed that the turtle CD9 sequence clustered together with bird CD9 sequence. RT-PCR analysis showed that turtle CD9 was ubiquitously expressed in liver, spleen, kidney, heart, blood and intestine tissues of un-infected turtles. Real-time PCR analysis further indicated that after Aeromonas hydrophila infection, the turtle CD9 mRNA was up-regulated in various tissues at 8h, and significantly up-regulated during 8h to 7d. These results indicated that turtle CD9 may be involved in anti-bacterial immune response.

Molecular characterization and expression profiles in response to bacterial infection of Chinese soft-shelled turtle interleukin-8 (IL-8), the first reptilian chemokine gene.

In this study, an IL-8 homologue has been cloned and identified from a reptile, Chinese soft-shelled turtle for the first time. The full-length cDNA of turtle IL-8 was 1188bp and contained a 312bp open reading frame (ORF) coding for a protein of 104 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in turtle IL-8. The 4924bp genomic DNA of turtle IL-8 contained four exons and three introns. Phylogenetic analysis showed that the amino acid sequence of turtle IL-8 clustered together with birds. RT-PCR analysis showed that turtle IL-8 mRNA was constitutively expressed liver, spleen, kidney, heart, blood and intestine tissues of control turtles. Real-time quantitative PCR analysis further indicated that the turtle IL-8 mRNA expression was apparent in various tissues at 8h and up-regulated significantly during 8h-7d after Aeromonas hydrophila infection. The present studies will help us to understand the evolution of IL-8 molecule and the inflammatory response mechanism in reptiles.

Production of monoclonal antibodies against Chinemys reevesii turtle vitellogenin and their usage for comparison of biochemical and immunological characters of vitellogenins and yolk proteins of freshwater turtles.

Four hybridoma clones (ACV-1, -3, -4, and -5) were established for Chinemys reevesii (Reeves' turtle) vitellogenin (VTG) as a precursor protein of egg yolk and a biomarker of environmental pollution. Binding-inhibition experiments indicated that the epitopes of four mAbs were distinct. No binding of ACV-4 to C. reevesii VTG in the Western blot suggests that the epitope of ACV-4 would be dependent on the three-dimensional structure. ACV-1, -3, and -5 bound to C. reevesii VTG in the Western blot. The signal for ACV-1 and -5 disappeared by reduction of the VTG, suggesting that the construction of the epitopes for ACV-1 and -5 were dependent on the disulfide bridge in the VTG molecule. All four mAbs recognized Trachemys scripta and Mauremys japonica VTGs in the ELISA. The yolk proteins were tested for the binding of the mAbs in the Western blot. ACV-1 being capable to bind to the VTG in the reduced condition did not bind to any protein bands of the yolk. This indicates that ACV-1 recognizes a part of the VTG molecule that is not incorporated in the oocytes. Both ACV-3 and -5 bound to the 32- and 70-kDa yolk proteins. Since a mAb recognizes only one site (epitope) on a protein molecule, the 32-kDa protein originated from the 70-kDa one. An ELISA system using ACV-5 as the capture antibody and ACV-3 as the detecting antibody showed the lower detectable concentration (2 ng/mL) and a wide detectable range to 1000 ng/mL (R2=0.999). The system was used to determine serum VTG levels of juvenile turtles treated with estradiol-17beta or vehicle (corn oil). By the use of the mAbs described in this paper, basic and applied studies for turtle VTGs would be improved.