The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp.

Haemogregarina species are apicomplexan blood parasites infecting vertebrates such as fish, lizards, and turtles. Due to the high morphological similarity of the erythrocytic stages infecting host species, it has always been a challenge to identify the true diversity of these parasites. Therefore, taxonomic studies are presently based on the combination of morphological and molecular data. In Tunisia, two species of Haemogregarina have been reported within the freshwater turtle Mauremys leprosa (Geoemydidae) for more than 40 years. Since M. leprosa occurs in the same aquatic environments as Emys orbicularis (Emydidae) in Tunisia, our objectives were to assess parasite diversity and specificity on the basis of both morphological and molecular approaches. The turtles were surveyed and sampled across six aquatic areas of Tunisia. Among the 39 specimens of M. leprosa and seven of E. orbicularis that were trapped and investigated, the presence of haemogregarines was detected in the blood of turtles only at sites where leeches were observed. Three 18S variants were identified, which corresponded to three distinct Haemogregarina species, among which one was identified as Haemogregarina stepanowi. The two other species that were detected are likely new to science. Because we show the occurrence of more than one blood parasite species within a single host specimen, our study provides the first report of coinfection with molecularly distinct Haemogregarina spp.

Insights into Emydid Turtle Cytogenetics: The European Pond Turtle as a Model Species.

Our knowledge of Testudines evolution is limited by the lack of modern cytogenetic data. Compared to other reptiles, there is little information even on chromosome banding, let alone molecular cytogenetic data. Here, we provide detailed information on the karyotype of the European pond turtle Emys orbicularis, a model Emydidae, employing both chromosome banding and molecular cytogenetics. We provide a high-resolution G-banded karyotype and a map of rDNA genes and telomeric sequences using fluorescence in situ hybridization. We test hypotheses of sex-determining mechanisms in Emys by comparative genomic hybridization to determine if Emys has a cryptic sex-specific region. Our results provide valuable data to guide future efforts on genome sequencing and anchoring in Emydidae and for understanding karyotype evolution in Testudines.

Trace Element Concentrations in European Pond Turtles (Emys orbicularis) from Brenne Natural Park, France.

We assessed trace elements concentration in European pond turtle (Emys orbicularis) from Brenne Natural Park (France). We sampled road-killed turtles (N = 46) to measure the concentrations of 4 non-essential (Ag, Cd, Hg, and Pb) and 10 essential (As, Co, Cr, Cu, Fe, Mn, Ni, Se, V, and Zn) elements in muscle, skin, liver and claws. Body size or sex did not influence the concentrations of most elements; except for Hg (liver, skin and claws) and Zn (muscle) which increased with body size. We found relatively high concentrations of Hg and Zn, possibly linked to fish farming. This result deserves future investigations to evaluate possible ecotoxicological effects on E. orbicularis.

Skin and skeletal system lesions of european pond turtles (Emys orbicularis) from natural habitats.

Water pollution is known to play an important role in the pathogenesis of plastron, carapace and skin diseases of turtles. In this study, a total of 150 European pond turtles (Emys orbicularis) of different age and both sexes, originating from natural habitats in Serbia, were examined for morphological changes of the skin, plastron, carapace and skeletal system. The turtles were taken out from their natural habitats in Lake Ludas, Lake Palic and Lake Tresetiste. After artificial hibernation, they were subjected to detailed examination, sampled and treated, and finally returned into their natural habitat. Biopsies from the skin and shell were subjected to histopathological examination and microbiological analysis. X-ray scanning was also performed to detect changes in the skeletal system. Macroscopic changes of the skin, most frequently degenerative, inflammatory or neoplastic diseases, were diagnosed in 49.33% of the turtles examined. Dermatitis of different origin and form was the most prominent histopathological finding (28.00%). In the plastron, inflammatory and degenerative processes were frequently found. Osteopathy and mechanical injuries were the dominant findings. Macroscopic changes of the plastron, carapace and skeletal system were diagnosed in 67.33% of the turtles examined. Using X-ray scanning, generalised osteopathy, anomalies and malformations of different aetiology were also diagnosed on the tail and legs. Microbiological examinations showed the presence of a variety of bacterial and fungal agents, either primary pathogens or potential polluters, which invaded the skin and shell, or were present in cloacal swab samples. Bacterial infection was diagnosed in 76.66% of the turtles, first of all in those with skin and shell necrosis. Mycoses were diagnosed in 33.33% of the animals.

The turtles from the middle Paleolithic site of Gruta Nova da Columbeira (Bombarral, Portugal): Update through an archaeozoological perspective.

Twenty-five years after the preliminary systematic study of the turtle remains (Agrionemys [=Testudo] hermanni and Emys or Mauremys) recovered from Gruta Nova da Columbeira site (Bombarral, Portugal), the results of its review from systematic and archaeozoological perspectives are presented here. Tortoise remains studies from pre-Upper Paleolithic sites worldwide have provided relevant data confirming its role as a dietary supply for hominid populations and informing about their ability to adapt to local environmental resources. The Iberian Peninsula record in general, and specifically, that from Portugal, have yielded substantial evidence to this highly debated topic. In this sense, turtle remains recovered in Gruta Nova da Columbeira site, discovered in the 1960s and the main ensemble chronologically ascribed to the MIS-5 (87.1 ± 6.3 ka BP), offer new information to this debate. Its detailed restudy, has allowed us the identification, justification, and figuration of remains attributed to two Iberian turtle taxa, Chersine hermanni and Emys orbicularis. Therefore, this update on the data concerning the turtle record from Gruta Nova da Columbeira provides new justified taxonomic evidence regarding the Iberian turtle taxa distribution during the Upper Pleistocene. The previously suggested hypothesis about the tortoise human consumption on the site is here evaluated through the development of an archaeozoological and taphonomical analysis, as well as considering the potential documentation of anthropic alterations (e.g., burning, cutmarks, percussion marks). In this sense, this hypothesis is confirmed. In addition, the presence of carnivore activity evidence indicates the engagement of other agents in the deposit formation.

Molecular Screening of Haemogregarine Hemoparasites (Apicomplexa: Adeleorina: Haemogregarinidae) in Populations of Native and Introduced Pond Turtles in Eastern Europe.

Haemogregarines (Apicomplexa: Adeleorina) are the most common and widespread reptilian blood parasites. Haemogregarina stepanowi was the first haemogregarine described from a reptile, the European pond turtle Emys orbicularis, and initial assessments indicated it was widespread across different pond turtle host species across much of Europe, the Middle East and North Africa. However, recent molecular assessments have indicated the presence of multiple genetically distinct forms in North Africa and the Iberian Peninsula, and extensive mixed infections which may be associated with a negative impact on the hosts. Here, we screened two native species, E. orbicularis and Mauremys rivulata, and the introduced Trachemys scripta from Serbia and North Macedonia for haemogregarines by amplifying and sequencing part of the 18S rRNA gene of these parasites, and used a standard DNA barcoding approach to identify leeches, the final host, attached to pond turtles. Our results again demonstrate the occurrence of considerable diversity of parasites in the analysed pond turtle species, and that T. scripta are likely infected by local haemogregarine parasites, and not those that are found in its native range. Leeches were identified as Placobdella costata, part of a lineage from Northern Europe. Mixed infections within pond turtles were again common. Current haemogregarine taxonomy does not reflect the genetic diversity identified, and a full taxonomic reassessment is needed.

Ludwig Heinrich Bojanus and the anatomy of the European pond turtle: Facts, fiction, and future.

Detailed anatomical knowledge is a prerequisite for studying a variety of biological questions. This holds true particularly for the evolutionary realm, in which fossils play an important role. Putting paleontological data in perspective consequently requires a profound knowledge of the corresponding structures of the extant representatives of a given lineage as well. Numerous morphological studies have contributed to our understanding of chelonian anatomy over the past centuries, but one of the earliest ones-whose completion celebrated its bicentenary with this symposium-still stands out among all of them: Anatome Testudinis Europaeae, published by Ludwig Heinrich Bojanus (1776-1827) in two installments in 1819 and 1821. Numerous myths around this publication have evolved ever since. The intent of this article is to set the record straight for some of them, and, based on personal inspection of multiple copies, to discuss currently unrecognized aspects about the existence of exceptional colored variant editions. This ends in a proposal for how this work could be used even more effectively in its upcoming third century of existence. Last but not least and despite the controversies related to its bibliographical aspects, one fact remains an unchallenged reality: Anatome Testudinis Europaeae, even 200 years after its first publication, still ranks very high among the most detailed and accurate anatomical works on a single chelonian-and for that matter reptilian-species ever produced and has lost absolutely none of its value for modern-day science.

Impact of abiotic factors and road networks on the freshwater turtle Emys orbicularis (Linnaeus, 1758) (Reptilia: Emydidae) in northern Iran.

Emys orbicularis is one of the freshwater turtles occurring in Europe, Middle East and Western Asia, but also occur in Caucasus region as Georgia, Armenia, Azerbaijan and Southern Caspian Sea region (northern Iran provinces as Guilan, Mazandaran, Golestan). In this study, seventeen localities in three Iranian provinces were investigated to study the response of this species to abiotic factors (water quality parameters) and road mortality. To test for differences in occurrence of E. orbicularis between sites that could be explained by variation in abiotic factors, one-way ANOVA test and Tukey's HSD post hoc tests were conducted. These showed significant differences in conductivity (CON) and Total Dissolved Solids (TDS) between locations in the three provinces of Golestan, Mazandaran and Gilan [CON: (F (2, 13) = 9.824, p = 0.003); TDS: (F (2, 13) = 13.957, p = 0.001)]. Differences in four other abiotic variables-water temperature, pH, salinity and dissolved oxygen (DO)-were not significant between provinces, although there were differences. These findings suggested that the species can occur in ponds with the following features: temperature 21-28 °C; pH 7-8; salinity > 750 mg/L; DO > 30%. Road mortality might be another factor that could contribute to the decline of this turtle's population in northern Iran, noting that the roads were created by human within the species' habitat and increased the mortality rate. Roads should be monitored in order to evaluate their effect on freshwater turtle mortality. Overall, our results demonstrated the advantage of using analysis of abiotic variables as a valid approach for defining species-responses to abiotic factors in E. orbicularis and recommended monitoring roads to find high-risk mortality of this species and create under road tunnels for moving turtles without any risk to reduce high rate of mortality.

Fatal spirorchiidosis in European pond turtles (Emys orbicularis) in Switzerland.

Infections with intravascular digenean trematodes of the Spirorchiidae family (spirorchiidoses) are of great conservation concern both in marine and freshwater turtles due to their pathogenic potential. Between 2014 and 2021, Spirorchis sp. infections associated with granulomatous inflammation and sudden death were detected in European pond turtles (Emys orbicularis) from three conservation breeding facilities in Switzerland. Blood fluke eggs associated with lesions were found in the intestine, spleen, testis, skeletal musculature, heart, kidneys, stomach, pancreas, liver, lung, and meninges from nine pond turtles submitted for necropsy and in the intestinal content from five of these animals. Two novel polymerase chain reactions (PCRs) targeting the 28S ribosomal RNA gene and the ITS2 region and subsequent sequencing revealed 100% nucleotide identity with a Spirorchis sp. previously isolated from an Escambia map turtle (Graptemys ernsti) in the USA. Our findings suggest a spill-over event secondary to direct or indirect contact with invasive North American turtle species in Switzerland. We describe the clinical, haematological, ultrasonographical, endoscopical, parasitological, pathological, and molecular findings associated with spirorchiid blood fluke infections of the Spirorchis genus in E. orbicularis, as well as the biosecurity measures that were developed to prevent the spread of this parasite among breeding and highly endangered free-ranging E. orbicularis populations in Switzerland.

Effects of microplastic exposure on the blood biochemical parameters in the pond turtle (Emys orbicularis).

The accumulation of microplastics (MPs) is a growing problem in aquatic ecosystems. Despite increased research on MPs in the last decade, their potential threat to freshwater ecosystems remains an open question. In the present study, the negative impacts of MPs were investigated on blood biochemical parameters in the European pond turtle (Emys orbicularis). Pond turtles were distributed into three experimental groups (n = 9 for each group) and were fed diets containing 250, 500, and 1000 mg MPs (PE100 polyethylene) per kg of food for 30 days, and a control group fed with a standard uncontaminated diet. The results indicated that exposure to 500 and 1000 mg kg-1 MPs caused a significant increase in the activities of alanine and aspartate aminotransferases, and in the levels of cholesterol, glucose, creatinine, urea, and calcium (Ca+2) compared with the control group. On the contrary, the activity of gamma-glutamyl transferase and the levels of total protein, albumin, total immunoglobulins, and phosphorus were significantly reduced in E. orbicularis exposed to 500 and 1000 mg kg-1 MPs when compared with the controls. In all the MP-exposed groups, the activity of lactate dehydrogenase and globulin and magnesium (Mg+2) levels were significantly reduced; while creatine phosphokinase and alkaline phosphatase activities were increased with respect to the control turtles. A significant decrease in triglyceride levels was reported in E. orbicularis exposed to 1000 mg kg-1 MPs. MPs intake induced notable alterations in blood biochemical parameters of E. orbicularis. These results suggest that changes in the blood biochemical parameters could be an appropriate bio-indicator to evidence the existence of tissue damage in E. orbicularis.

The first case of Spiroxys contortus in European pond turtle (Emys orbicularis) in the wild in Poland.

During standard parasitological dissection of 4 individuals of European pond turtle (E. orbicularis) (Linneaus, 1758) one nematode was found. In a morphological examination the parasite was identified as Spiroxys contortus (Rudolphi, 1819) and confirmed by DNA analysis based on sequencing of the small subunit ribosomal RNA gene. The partial 18S rDNA gene was deposited to NCBI GenBank with the accession number MN629917. This is the first molecular evidence of S. contortus in E. orbicularis from wild from Poland. The analyzed sample genotype sequence shows 100% similarity to the reference specimen from Argentina.

Cytochrome c oxydase I phylogenetic analysis of Haemogregarina parasites (Apicomplexa, Coccidia, Eucoccidiorida, Haemogregarinidae) confirms the presence of three distinct species within the freshwater turtles of Tunisia.

Species of Haemogregarina are apicomplexan blood parasites that use vertebrates as intermediate hosts. Due to limited interspecific morphological characters within the genus during the last decade, 18S rRNA gene sequences were widely used for species identification. As coinfection patterns were recently reported from nuclear molecular data for two sympatric freshwater turtles Mauremys leprosa and Emys orbicularis from Tunisia, our objectives were to design COI specific primers to confirm the presence of three distinct species in both host species. Blood samples were collected from 22 turtles, from which DNAs were extracted and used as templates for amplification. Following different rounds of PCR and nested PCR, we designed specific Haemogregarina COI primers that allowed the sequencing of nine distinct haplotypes. Phylogenetic Bayesian analysis revealed the occurrence of three well-differentiated sublineages that clustered together into a single clade. Based on pairwise genetic distances (p-distance), we confirmed the occurrence of three distinct but phylogenetically closely related species coinfecting M. leprosa and E. orbicularis in the same aquatic environments. Our results demonstrate that the use of fast evolving genes within Haemogregarina will help to investigate the parasite diversity within both intermediate vertebrate and definitive invertebrate hosts, and to assess the evolution, historical biogeography and specificity of haemogregarines.

Virulence and Antimicrobial Resistance Pattern of Aeromonas spp. Colonizing European Pond Turtles Emys orbicularis and Their Natural Environment. First Study from Poland.

The aim of the study was to isolate and identify species belonging to the Aeromonas genus and evaluate the antimicrobial resistance and virulence patterns of isolates colonizing European pond turtles (Emys orbicularis) from natural environment of Eastern Poland. In total, 74 turtles and 15 samples of water from their natural environment were examined. More than 40 strains were isolated and identified: A. bestiarum (n = 1), A. hydrophila (n = 13), A. allosaccharophila (n = 2), A. salmonicida (n = 3), and A. veronii (n = 23). The highest incidence of resistance was noted for ampicillin (100%) and sulfamethoxazole (62.0%), followed by erythromycin and colistin (both 40.5%). Moreover, eight strains were intermediately resistant to meropenem (19%). Most Aeromonas isolates were found to possess more than one virulence gene among fla, aer, hlyA, act, ela, alt, and ast. We showed that the population of free-living European pond turtles was highly colonized by Aeromonas spp. Such strains may be an infectious agent not only for the population of turtles but also for other species of animals inhabiting their natural environment. Moreover, the undesirable properties of water quality caused by the presence of drug-resistant aeromonads could have a negative impact on human health.

Optimal body mass-length ratio during hibernation for Emys orbicularis (Linnaeus, 1758) - European Pond Turtle.

The aim of this study was to determine optimal body mass/length ratios of Emys orbicularis before hibernation. We obtained 213 measurements of wild-caught and captive-bred turtles during 9 years (2011-2019) and determined regression curves that show intervals for optimal or suboptimal body mass/length ratios before winter dormancy. Using this data we designed an online calculator that facilitates the process of establishing if a turtle has an optimal body mass/length ratio before hibernation. We also gathered 45 measurements at the end of the hibernation period which showed an average body mass loss of 5.32%. The present study offers practical means of assessing if an Emys orbicularis individual is fit for hibernation.

Freshwater turtles reveal personality traits in their antipredatory behaviour.

Animals respond to predation risk with antipredatory behaviours that may disclose the presence of different personality traits among individuals in a population, and how populations may differ for the expression of those traits. Variation among individuals is a necessary condition for the evolution through natural selection and inter-individual behavioural differences may be selected in different environmental situations. We tested whether individuals of two freshwater turtle species, Emys orbicularis and Mauremys leprosa, show consistent risk-taking behaviour when exposed to the presence of a potential predator. In an outdoor experimental setting, we simulated a predatory attack by overturning a turtle onto its carapace and recording three different behavioural response variables. Our results showed clear differences between species. The appearance time was highly consistent over the trials for both species, but was shorter for E. orbicularis and very long for M. leprosa. Waiting time was consistent only for females of E. orbicularis while M. leprosa did not show repeatability and had significantly longer waiting times. The number of failed righting attempts, likely being partially dependent on turtle individual performance, was highly consistent during the experiment for both species. The hiding behaviour in both species of freshwater turtles was consistent over time and, therefore, could be regarded as a personality trait. However, the differences in waiting times consistency suggested possible differences in defensive strategies between sexes and species.

Demonstrating the value and importance of combining DNA barcodes and discriminant morphological characters for polystome taxonomy (Platyhelminthes, Monogenea).

Polystomes are monogenean parasites that infest mainly semi aquatic vertebrates, such as amphibians and chelonians. Owing to the lack of discriminative morphological characters and because polystomes are considered to be strictly host- and site-specific, host identity is often used as an additional character for parasite identification. Recent genetic studies, however, showed that polystomes infecting freshwater turtles in outdoor turtle enclosures and natural environments, were not strictly host-specific. Therefore, we proposed a new procedure for turtle polystome taxonomy based on the combination of Cytochrome c Oxydase I sequences and two discriminant morphological characters, namely the number of genital spines and the testis shape. We tested the validity of this procedure with Polystomoides oris, which was collected from the pharyngeal cavity of the American painted turtle Chrysemys picta and two undescribed species, both collected from the pharyngeal cavity of the American slider Trachemys scripta and two other European turtles, namely the European pond turtle Emys orbicularis and the Mediterranean turtle Mauremys leprosa. A Principal Component Analysis based on both morphological characters allowed the separation of all specimens in three morphological groups, which matched well with the molecular data. As a result, we describe two new polystome species, i.e., Polystomoides soredensis n. sp. and Polystomoides scriptanus n. sp.

Endohelminths of European pond turtle Emys orbicularis in Southwest Iran.

Very little is known about parasitic diseases of European pond turtles (Emys orbicularis) in Iran. The objective of this study is to examine parasitic fauna of European pond turtles collected from Fars province, southwest Iran. Carcasses of turtles (n = 52) which died during dredging procedure are collected from earthen fishery basins in Zarghan region. They have been died earlier during dredging procedure in different farms. Three species of helminths in total were found in gastrointestinal tract, including two nematodes (Serpinema microcephalus and Falcaustra araxiana) and one digenean trematod (Telorchis assula). Large intestines of all examined turtles were infected by F. araxiana (100 %, Mean intensity = 18) and this nematode were also found in gastric nodules. Nine turtles (17.3 %, 3 male, 6 female, Mean intensity = 3) were infected with Serpinema microcephalus. T. assula were found in 25 turtles (48.07 %, 5 male, 20 female, mean intensity = 5). Helminths were not found in any examined organs and no ectoparasite found eighter. F. araxiana is the most prevalent nematode in European pond turtles. Detection of Serpinema.microcephalus is in agreement with the fact which this parasite is common parasite of turtles in all over the world. T. assula might be transmitted between variety of reptiles so presence of the trematod should be considered as a risk factor for other reptiles.