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.

Orchard grass allergy in an African spur-thighed tortoise (Centrochelys sulcata) confirmed via intradermal allergen testing and provocation testing.

The humoral immune system of reptiles is not well-studied. To the best of the authors' knowledge, this case report describes the first case of a type I allergic conjunctivitis associated with orchard grass in an African spur-thighed tortoise (Centrochelys sulcata) supported by intradermal allergen testing and provocative testing. Further studies are needed to better characterize allergic reactions in reptiles.

Le système immunitaire humoral des reptiles n'est pas bien étudié. A la connaissance des auteurs, cet article décrit le premier cas de conjonctivite associé au dactyle pelotonné chez une tortue sillonée (Centrochelys sulcata) confirmé par tests allergéniques intradermiques et test de provocation. Des études supplémentaires sont nécessaires pour mieux caractériser les réactions allergiques chez les reptiles.

El sistema inmunológico humoral de los reptiles no está bien estudiado. A entender de los autores, este artículo describe el primer caso de una conjuntivitis alérgica tipo I asociada con dáctilo (Dactylis glomerata) en una tortuga africana (Centrochelys sulcata) basado en pruebas de alérgenos intradérmicos y pruebas de provocación. Se necesitan más estudios para caracterizar mejor las reacciones alérgicas en reptiles.

O sistema imune humoral de répteis ainda não é bem estudado. De acordo com os conhecimentos dos autores, este relato descreve o primeiro caso de conjuntivite alérgica do tipo I associada a grama (Dactilys glomerata) em uma tartaruga-grega africana (Centrochelys sulcata) baseado em teste alérgico intradérmico e teste de provocação. Mais estudos são necessários para melhor se caracterizar as reações alérgicas em répteis.

Global characterization and expression analysis of interferon regulatory factors in response to Aeromonas hydrophila challenge in Chinese soft-shelled turtle (Pelodiscus sinensis).

Interferon regulatory factors (IRFs) were originally identified as transcriptional regulators of type I interferon (IFN) expression. Recent studies have widely identified the roles of IRFs as central mediators in immune defence against pathogen infection. However, the functional roles and expression profiles of IRFs are still unclear in Chinese soft-shelled turtle (Pelodiscus sinensis). In this study, eight members of the PsIRF family were identified in P. sinensis through a genome-wide search. These PsIRF genes contained the conserved domains of this group of proteins, including the N-terminal DNA-binding domain and C-terminal IRF-associated domain. Phylogenetic analyses among IRF homologs showed that the PsIRFs shared the closest phylogenetic relationships with IRFs of other turtle species. Further molecular evolutionary analyses revealed evolutionary conservation of the PsIRF genes. Moreover, expression profiling demonstrated that eight PsIRF genes exhibited constitutive expression in different tissues of P. sinensis. Several genes, such as PsIRF1, PsIRF2 and PsIRF4, showed predominant expression in the spleen and were significantly upregulated upon Aeromonas hydrophila infection. Remarkably, PsIRF1, PsIRF2 and PsIRF4 exhibited rapid increases in their protein expression levels post-infection and were mainly expressed in the splenic red pulp according to immunohistochemistry analysis. These results provide rich resources for further exploration of the roles of PsIRFs in immune regulation in P. sinensis and other turtles.

Genome-wide identification of Toll-like receptors in the Chinese soft-shelled turtle Pelodiscus sinensis and expression analysis responding to Aeromonas hydrophila infection.

Toll-like receptors (TLRs) recognizing specific pathogen-associated molecular patterns play crucial roles in immune defence against pathogen invasion. Although recent advances in many species have reported the characterization and functional roles of TLRs in innate immunity, systematic knowledge of TLRs is still lacking in the Chinese soft-shelled turtle Pelodiscus sinensis. In this study, a genome-wide search was performed and identified 15 candidate PsTLR family genes in P. sinensis. Protein structure analysis revealed the conserved domain arrangements for these PsTLR proteins. Phylogenetic analysis indicated the evolutionary conservation of TLRs among various species. Additionally, a putative interaction network among PsTLR proteins was proposed and several functional partner proteins involved in TLR signalling pathway were predicted in P. sinensis. Expression profiling showed that these PsTLRs exhibited constitutive expression patterns in different tissues of P. sinensis. Moreover, several genes were highly expressed in the major immune organ spleen. Remarkably, the mRNA levels of PsTLR2-1, PsTLR4 and several TLR signalling molecules were significantly up-regulated in the spleen after Aeromonas hydrophila infection, indicating that PsTLRs and these genes responded to bacterial stress. These results provide rich information for the functional exploration of PsTLRs and will facilitate uncovering the molecular mechanisms underlying immune regulation in P. sinensis.

Identification, characterization and expression analysis of MAVS in Pelodiscus sinensis after challenge with Poly I:C.

Pelodiscus sinensis, which is one of the important reptile species in the aquaculture industry in China, frequently suffers from serious infectious diseases caused by viruses. However, there is a lack of biological knowledge about its antiviral innate immunity. In this study, we identified and characterized the open reading frame (ORF) of PsMAVS cDNA in P. sinensis. It consisted of 2691 nucleotides encoding a protein of 896 amino acid residues, which were composed of an N-terminal CARD, a central proline-rich domain and a C-terminal TM domain. Based on the amino acid sequence, phylogenetic analyses revealed a closer relationship of PsMAVS with those of Chelonia. qRT-PCR analysis indicated that PsMAVS was ubiquitously expressed in all of the examined healthy tissues with different expression levels; it was expressed at high levels in spleen, muscle and heart and at moderate levels in kidney, liver, intestine, intestinum crissum and oesophagus. PsMAVS was detected in embryos at 10 days post hatching, and it gradually upregulated with the embryonic development stage. Its expression levels in the examined tissues were all upregulated significantly after challenge with Poly I:C. The PsMAVS protein was detected in the intestinal tissues from both the challenge and the control groups, and it was distributed widely in the cytoplasm of the intestinal cells, suggesting PsMAVS plays multiple roles in the complicated mechanisms of immune defence against virus invasion in P. sinensis.

Seasonal changes in baseline corticosterone, association with innate immunity, and effects of confinement in free-ranging Eastern Box Turtles, Terrapene carolina carolina.

Seasonal changes in glucocorticoids and their influence on the immune system are widespread in vertebrates, but whether this occurs in lesser studied taxa like turtles is unclear. The purpose of this study was to test for seasonal changes in baseline corticosterone (CORT), innate immunity, body condition, and metabolic factors (triglycerides, uric acid) in free-ranging Eastern Box Turtles (Terrapene carolina carolina), a species which is in decline across its range. In addition, the effect of handling and confinement on CORT levels was measured. In both years of the seasonal study, baseline CORT concentrations were significantly higher in fall than in spring, but summer CORT levels differed between the years. The annual variation in CORT levels may be related to weather conditions or associated with opportunistic mating. CORT levels of turtles confined for one hour were higher than turtles bled as quickly as possible, and unlike the seasonal study, females had higher baseline and post-stressor CORT levels than males. Baseline CORT levels were positively correlated with hemolysis titer, indicating a possible immunoenhancing effect of CORT. Triglycerides were positively correlated with body condition and were higher in females than males. The higher triglyceride level in females was likely associated with the energetic demands of reproduction and nesting. Males had a lower body condition in the fall than in both spring and summer, while females exhibited no seasonal differences in body condition. Uric acid and innate immunity measures failed to exhibit significant seasonal or sex related differences. Overall our findings of substantial annual, seasonal, and sexual variation in turtle physiology are consistent with the findings reported for other vertebrates and indicate the importance of incorporating these sources of variation into the design of future studies.

Humoral Immune Responses to Select Marine Bacteria in Loggerhead Sea Turtles Caretta caretta and Kemp's Ridley Sea Turtles Lepidochelys kempii from the Southeastern United States.

Serum from Kemp's ridley sea turtles Lepidochelys kempii and loggerhead sea turtles Caretta caretta was collected during summer in 2011, 2012, and 2013. Serum immunoglobulin Y (IgY) recognition of lysate proteins from nine bacterial species and whole bacterium-specific IgY titers to these pathogens were quantified. Serum and purified IgY recognized proteins of all bacteria, with protein recognition for some species being more pronounced than others. Circulating IgY titers against Vibrio vulnificus, V. anguillarum, Erysipelothrix rhusiopathiae, and Brevundimonas vesicularis changed over the years in Kemp's ridley sea turtles, while IgY titers against V. vulnificus, Escherichia coli, V. parahaemolyticus, B. vesicularis, and Mycobacterium marinum were different in loggerhead sea turtles. Serum lysozyme activity was constant for loggerhead sea turtles over the 3 years, while activity in Kemp's ridley sea turtles was lower in 2011 and 2012 than in 2013. Blood packed cell volume, glucose, and serum protein levels were comparable to those of healthy sea turtles in previous studies; therefore, this study provides baseline information on antibody responses in healthy wild sea turtles.

The effect of environmental temperature on reptilian peripheral blood B cell functions.

Recent studies have identified phagocytic B cells in a variety of species, yet little is understood about their function and how it is influenced by natural environmental variation, such as temperature. Phagocytic B-cells are present in red-eared slider turtles, Trachemys scripta, and the wide range of temperatures experienced by these ectotherms may have an effect on immunity, including B cell antibody secretion and phagocytosis. We examined the impact of environmental temperature on B cell function in vitro using phagocytic and ELISpot assays conducted at biologically relevant temperatures. We found a significant effect of temperature on antibody secretion, with maximal antibody secretion occurring at intermediate temperatures (estimated maximum of 28.8°C). There was no effect of temperature on phagocytosis. We also noted a difference in the efficiency of phagocytosis in this assay between B cells and non-B cells. Interestingly, in our in vitro assay, phagocytic B cells engulfed more foreign fluorescent beads per cell than phagocytes lacking surface immunoglobulin. This work sheds light on our understanding of phagocytic B cells and the importance of environmental temperature on the behavior of reptilian immune cells, which may have relevance for organismal fitness.

Green Turtles (Chelonia mydas) Have Novel Asymmetrical Antibodies.

Igs in vertebrates comprise equally sized H and L chains, with exceptions such as H chain-only Abs in camels or natural Ag receptors in sharks. In Reptilia, Igs are known as IgYs. Using immunoassays with isotype-specific mAbs, in this study we show that green turtles (Chelonia mydas) have a 5.7S 120-kDa IgY comprising two equally sized H/L chains with truncated Fc and a 7S 200-kDa IgY comprised of two differently sized H chains bound to L chains and apparently often noncovalently associated with an antigenically related 90-kDa moiety. Both the 200- and 90-kDa 7S molecules are made in response to specific Ag, although the 90-kDa molecule appears more prominent after chronic Ag stimulation. Despite no molecular evidence of a hinge, electron microscopy reveals marked flexibility of Fab arms of 7S and 5.7S IgY. Both IgY can be captured with protein G or melon gel, but less so with protein A. Thus, turtle IgY share some characteristics with mammalian IgG. However, the asymmetrical structure of some turtle Ig and the discovery of an Ig class indicative of chronic antigenic stimulation represent striking advances in our understanding of immunology.

Prevalence and genetic diversity of blood parasite mixed infections in Spanish terrapins, Mauremys leprosa.

Blood parasites such as haemogregarines and haemosporidians have been identified in almost all groups of vertebrates and may cause serious damages to their hosts. However, very little is known about biodiversity of these parasites and their effects on some groups of reptiles such as terrapins. Moreover, the information on virulence from blood parasites mixed infection is largely unknown in reptiles. With this aim, we investigated for the first time the prevalence and genetic diversity of blood parasites from one genus of haemoparasitic aplicomplexan (Hepatozoon) in two populations of Spanish terrapins (Mauremys leprosa), a semi-aquatic turtle from southwestern Europe with a vulnerable conservation status. We also examined the association between mixed blood parasite infection and indicators of health of terrapins (body condition, haematocrit values and immune response). Blood parasite infection with Hepatozoon spp was detected in 46·4% of 140 examined terrapins. The prevalence of blood parasites infection differed between populations. We found two different lineages of blood parasite, which have not been found in previous studies. Of the turtles with infection, 5·7% harboured mixed infection by the two lineages. There was no difference in body condition between uninfected, single-infected and mixed-infected turtles, but mixed-infected individuals had the lowest values of haematocrit, thus revealing the negative effects of blood parasite mixed infections. Immune response varied among terrapins with different infection status, where mixed infected individuals had higher immune response than uninfected or single-infected terrapins.

Effects of selenium exposure on the hematology, innate immunity, and metabolic rate of yellow-bellied sliders (Trachemys scripta scripta).

Selenium (Se) is a naturally occurring essential element that can be toxic to vertebrates at high concentrations. Despite studies that have documented that wild reptile species can accumulate copious amounts of Se, little is known regarding specific toxicologic effects of Se. In this study, 70 juvenile yellow-bellied sliders (Trachemys scripta scripta) were exposed to one of three seleno-L-methionine (SetMet) treatments (control, n = 24; 15 mg/kg, n = 23; and 30 mg/kg, n = 23) via weekly oral gavage for 5 weeks. At the conclusion of the experiment, kidney, liver, muscle, and blood samples were collected for quantitative Se analysis. Turtles in the SeMet treatment groups accumulated significantly higher amounts of Se in all tissue types relative to controls (all p < 0.001). Turtles in the 30 mg/kg SeMet group also accumulated significantly higher amounts of Se compared to the 15 mg/kg group (all p < 0.001). Although toxicity thresholds for reptiles have not been established, Se concentrations in liver tissue from both SeMet treatment groups exceeded reported avian toxicity thresholds for liver tissue. Neither oxygen consumption nor innate bactericidal capacity were impacted by SeMet exposure. However, turtles in the 30 mg/kg SeMet group exhibited anemia, which has been reported in other vertebrates exposed to Se. Furthermore, juvenile T. s. scripta in the 30 mg/kg SeMet group experienced 17% mortality compared to 0% in the 15 mg/kg treatment and control groups. To our knowledge, this study is the first to report dose-dependent Se-associated anemia and mortality in a chelonian species.

Evaluation of differentially expressed immune-related genes in intestine of Pelodiscus sinensis after intragastric challenge with lipopolysaccharide based on transcriptome analysis.

Pelodiscus sinensis is the most common turtle species that has been raised in East and Southeast Asia. However, there are still limited studies about the immune defense mechanisms in its small intestine until now. In the present research, histological analysis and transcriptome analysis was performed on the small intestine of P. sinensis after intragastric challenge with LPS to explore its mechanisms of immune responses to pathogens. The result showed the number of intraepithelial lymphocytes (IELs) and goblet cells (GCs) in its intestine increased significantly at 48 h post-challenge with LPS by intragastrical route, indicating clearly the intestinal immune response was induced. Compared with the control, a total of 748 differentially expressed genes (DEGs) were identified, including 361 up-regulated genes and 387 down-regulated genes. Based on the Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG), 48 immune-related DEGs were identified, which were classified into 82 GO terms and 14 pathways. Finally, 18 DEGs, which were randomly selected, were confirmed by quantitative real-time PCR (qRT-PCR). Our results provide valuable information for further analysis of the immune defense mechanisms against pathogens in the small intestine of P. sinensis.

EXPERIMENTAL CHALLENGE STUDY OF FV3-LIKE RANAVIRUS INFECTION IN PREVIOUSLY FV3-LIKE RANAVIRUS INFECTED EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) TO ASSESS INFECTION AND SURVIVAL.

The Maryland Zoo in Baltimore experienced an outbreak of Frog virus-3 (FV3)-like ranavirus during the summer of 2011, during which 14 of 27 (52%) of its captive eastern box turtles (Terrapene carolina carolina) survived. To assess survival, immunity, and viral shedding, an experimental challenge study was performed in which the surviving, previously infected turtles were reinfected with the outbreak strain of FV3-like ranavirus. Seven turtles were inoculated with virus intramuscularly and four control turtles received saline intramuscularly. The turtles were monitored for 8 wk with blood and oral swabs collected for quantitative polymerase chain reaction (qPCR). During that time, one of seven (14%) inoculated turtles and none of the controls (0%) died; there was no significant difference in survival. Clinical signs of the inoculated turtles, except for the turtle that died, were mild compared to the original outbreak. Quantitative PCR for FV3-like ranavirus on blood and oral swabs was positive for all inoculated turtles and negative for all controls. The turtle that died had intracytoplasmic inclusion bodies in multiple organs. Three inoculated and two control turtles were euthanized at the end of the study. No inclusion bodies were present in any of the organs. Quantitative PCR detected FV3-like ranavirus in the spleen of a control turtle, which suggested persistence of the virus. The surviving five turtles were qPCR-negative for FV3-like ranavirus from blood and oral swabs after brumation. Quantitative PCR for Terrapene herpesvirus 1 found no association between ranavirus infection and herpesvirus loads. In conclusion, previously infected eastern box turtles can be reinfected with the same strain of FV3-like ranavirus and show mild to no clinical signs but can shed the virus from the oral cavity.

Honest sexual signaling in turtles: experimental evidence of a trade-off between immune response and coloration in red-eared sliders Trachemys scripta elegans.

Sexual signals can be evolutionarily stable if they are honest and condition dependent or costly to the signaler. One possible cost is the existence of a trade-off between maintaining the immune system and the elaboration of ornaments. This hypothesis has been experimentally tested in some groups of animals but not in others such as turtles. We experimentally challenged the immune system of female red-eared sliders Trachemys scripta elegans, with a bacterial antigen (lipopolysaccharide (LPS)) without pathogenic effects to explore whether the immune activation affected visual colorful ornaments of the head. The LPS injection altered the reflectance patterns of color ornaments. In comparison to the control animals, the yellow chin stripes of injected animals exhibited (1) reduced brightness, (2) lower long wavelength (>470 nm) reflectance, and (3) lower values for carotenoid chroma. The postorbital patches of injected individuals also showed reduced very long wavelength (>570 nm) reflectance but did not change in carotenoid chroma. Thus, experimental turtles showed darker and less "yellowish" chin stripes and less "reddish" postorbital patches at the end of the experiment, whereas control turtles did not change their coloration. This is the first experimental evidence supporting the existence of a trade-off between the immune system and the expression of visual ornaments in turtles. We suggest that this trade-off may allow turtles to honestly signal individual quality via characteristics of coloration, which may have an important role in intersexual selection processes.

Extensive diversification of IgD-, IgY-, and truncated IgY(δFc)-encoding genes in the red-eared turtle (Trachemys scripta elegans).

IgY(ΔFc), containing only CH1 and CH2 domains, is expressed in the serum of some birds and reptiles, such as ducks and turtles. The duck IgY(ΔFc) is produced by the same υ gene that expresses the intact IgY form (CH1-4) using different transcriptional termination sites. In this study, we show that intact IgY and IgY(ΔFc) are encoded by distinct genes in the red-eared turtle (Trachemys scripta elegans). At least eight IgY and five IgY(ΔFc) transcripts were found in a single turtle. Together with Southern blotting, our data suggest that multiple genes encoding both IgY forms are present in the turtle genome. Both of the IgY forms were detected in the serum using rabbit polyclonal Abs. In addition, we show that multiple copies of the turtle δ gene are present in the genome and that alternative splicing is extensively involved in the generation of both the secretory and membrane-bound forms of the IgD H chain transcripts. Although a single µ gene was identified, the α gene was not identified in this species.

Integrated time-series biochemical, transcriptomic, and metabolomic analyses reveal key metabolites and signaling pathways in the liver of the Chinese soft-shelled turtle (Pelodiscus sinensis) against Aeromonas hydrophila infection.

Introduction: Aeromonas hydrophila, a bacterium widely distributed in the natural environment, causes multiple diseases in various animals. Exploring the mechanism of the host defense against A. hydrophila can help develop efficient strategies against Aeromonas infection. Methods: Herein, we investigated the temporal influence of A. hydrophila on the Chinese soft-shelled turtle, an economically important species, at the biochemical, transcriptomic, and metabolomic levels. Plasma parameters were detected with the test kits. Transcriptome and metabolome were respectively applied to screen the differentially expressed genes and metabolites. Results: The contents or activities of these plasma parameters were significantly increased at 24 hpi and declined at 96 hpi, indicating that 24 and 96 hpi were two important time points during infection. Totals of 3121 and 274 differentially expressed genes (DEGs) from the transcriptome while 74 and 91 differentially abundant metabolites (DAMs) from the metabolome were detected at 24 and 96 hpi. The top DEGs at 24 hpi included Ccl2, Ccl3, Ccl4, Il1ß, Il6, Il7, Il15, Tnf, and Tnfr1 while Zap70, Cd3g, Cd8a, Itk, Pik3r3, Cd247, Malt1, and Cd4 were the most abundant at 96 hpi. The predominant DAMs included O-phospho-L-serine, γ-Aminobutyric acid, orotate, L-tyrosine, and L-tryptophan at 24 hpi, as well as L-glutamic acid, L-arginine, glutathione, glutathione disulfide, and citric acid at 96 hpi. Discussion: The combined analysis of DEGs and DAMs revealed that tryptophan metabolism, nicotinate and nicotinamide metabolism, as well as starch and sucrose metabolism, were the most important signaling pathways at the early infective stage while tyrosine metabolism, pyrimidine metabolism, as well as alanine, aspartate and glutamate metabolism were the most crucial pathways at the later stage. In general, our results indicated that the Chinese soft-shelled turtle displays stage-specific physiological responses to resist A. hydrophila infection.

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.

Immunoglobulin genes of the turtles.

The availability of reptile genomes for the use of the scientific community is an exceptional opportunity to study the evolution of immunoglobulin genes. The genome of Chrysemys picta bellii and Pelodiscus sinensis is the first one that has been reported for turtles. The scanning for immunoglobulin genes resulted in the presence of a complex locus for the immunoglobulin heavy chain (IGH). This IGH locus in both turtles contains genes for 13 isotypes in C. picta bellii and 17 in P. sinensis. These correspond with one immunoglobulin M, one immunoglobulin D, several immunoglobulins Y (six in C. picta bellii and eight in P. sinensis), and several immunoglobulins that are similar to immunoglobulin D2 (five in C. picta belli and seven in P. sinensis) that was previously described in Eublepharis macularius. It is worthy to note that IGHD2 are placed in an inverted transcriptional orientation and present sequences for two immunoglobulin domains that are similar to bird IgA domains. Furthermore, its phylogenetic analysis allows us to consider about the presence of IGHA gene in a primitive reptile, so we would be dealing with the memory of the gene that originated from the bird IGHA. In summary, we provide a clear picture of the immunoglobulins present in a turtle, whose analysis supports the idea that turtles emerged from the evolutionary line from the differentiation of birds and the presence of the IGHA gene present in a common ancestor.

Humoral immune responses are maintained with age in a long-lived ectotherm, the red-eared slider turtle.

Aging is typically associated with a decrease in immune function. However, aging does not affect each branch of the immune system equally. Because of these varying effects of age on immune responses, aging could affect taxa differently based on how the particular taxon employs its resources towards different components of immune defense. An example of this is found in the humoral immune system. Specific responses tend to decrease with age while non-specific, natural antibody responses increase with age. Compared with mammals, reptiles of all ages have a slower and less robust humoral immune system. Therefore, they may invest more in non-specific responses and thus avoid the negative consequences of age on the immune system. We examined how the humoral immune system of reptiles is affected by aging and investigated the roles of non-specific, natural antibody responses and specific responses by examining several characteristics of antibodies against lipopolysaccharide (LPS) in the red-eared slider turtle. We found very little evidence of immunosenescence in the humoral immune system of the red-eared slider turtle, Trachemys scripta, which supports the idea that non-specific, natural antibody responses are an important line of defense in reptiles. Overall, this demonstrates that a taxon's immune strategy can influence how the immune system is affected by age.