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Soft ticks in the genus Ornithodoros occur throughout the Mojave Desert in southern Nevada, southeastern California, and parts of southwestern Utah and northwestern Arizona, USA, and are frequently observed parasitizing Mojave desert tortoises (Gopherus agassizii). However, limited research exists examining the relationship between ticks and desert tortoises. Mojave desert tortoises are listed as threatened by the US Fish and Wildlife Service, and as such, their populations are monitored and individual tortoise health is routinely assessed. These health assessments document the presence and abundance of ticks present on tortoises, but detailed examination of the relationship between ticks and tortoise health has been lacking. This study analyzed the relationship between tick presence and desert tortoise health assessments as a function of season, location, age (adult vs. juvenile), foraging behavior, evidence of clinical signs of disease, body condition score, and sex. Our results indicate that more ticks were found on tortoises in the summer than in any other season. Ticks were observed more frequently on captive tortoises versus wild tortoises, and more ticks were likely to be present on adult tortoises than on juveniles. Ticks were also more likely to be observed on tortoises that lacked evidence of foraging and on tortoises with observed clinical signs of disease. These findings provide valuable insights into the biology of ticks in relation to tortoises that may be useful for management of both captive and free-living threatened tortoise populations where ticks are detected. Our study also may improve understanding of potential tick-borne disease dynamics in the Mojave desert tortoise habitat, including Borrelia sp. carried by Ornithodoros ticks, which cause tick-borne relapsing fever in people., (© Wildlife Disease Association 2024.)

The ultrastructural features of the mature spermatozoon of Telorchis attenuatus (Digenea, Telorchiidae), an intestinal parasite of the red-eared turtle Trachemys scripta elegans (Testudines, Emydidae), are described using transmission electron microscopy (TEM). The mature spermatozoon of T. attenuatus is a filiform cell tapered at both ends and displays Bakhoum et al.'s type IV of digenean sperm cells. Spermatozoa of T. attenuatus have: (i) two axonemes of different lengths with the 9+'1' pattern of trepaxonematan Platyhelminthes, surrounded by a continuous submembranous layer of cortical microtubules at their anterior end, (ii) an external ornamentation of the plasma membrane following Quilichini et al.'s type 2 and associated with cortical microtubules, (iii) two bundles of parallel cortical microtubules with the maximum number situated in the anterior part of the sperm cell, (iv) spine-like bodies, (v) two mitochondria, and (vi) a large number of irregularly distributed glycogen granules. Furthermore, the morphology of the posterior spermatozoon extremity in T. attenuatus corresponds to the Quilichini et al.'s fasciolidean type. The results of the current study are especially compared to the existing information from other families within the superfamily Plagiorchioidea., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

The order Testudines comprises some of the most endangered groups of vertebrates. Under specific circumstances, infectious and parasitic diseases may affect the survival and fecundity of these animals, potentially threatening Testudines populations. In Sardinia, besides the three species of tortoises present in the wild (Testudo hermanni, Testudo graeca and Testudo marginata), many others are kept as pets. However, epidemiological studies on these animals have not been conducted so far. Thus, the aim of the work was to investigate the presence of gastrointestinal parasites in captive and wild tortoises of Sardinia, Italy, with particular regard to those of zoonotic importance. For the 215 animals examined (n = 36 wild caught and n = 179 private-owned), fecal samples were collected and processed by flotation and modified Ziehl-Neelsen technique. An overall prevalence of 81.4% for endoparasites was detected, with oxyurids being the most prevalent (74.4%), followed by Nyctotherus spp. (18.6%), Cryptosporidium spp. (12.6%), Angusticaecum spp. (2.8%), strongyles (0.9%), Balantidium spp. (0.9%), coccidia (0.9%), cestodes (0.5%), and Giardia spp. (0.5%). Data suggest that tortoises are affected by a great variety of endoparasites, and further molecular analysis are required to assess the impact of Cryptosporidium and Giardia species in these hosts. Therefore, regular health screenings are of importance for the management of these animals and for preventing emerging infectious diseases., Competing Interests: Declaration of competing interest The authors declare there are no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Hepatozoon fitzsimonsi (Dias, 1953) is a frequently found haemogregarine of southern African tortoises. At the time of this species' reassignment from the genus Haemogregarina to Hepatozoon, developmental stages such as sporocysts and sporozoites were observed in ticks associated with H. fitzsimonsi parasitised and non-parasitised tortoises. It was thus suggested that ticks may act as the potential vectors for this parasite. However, this earlier research was unable to confirm the identity of these sporogonic stages using molecular markers. In a separate study aimed at identifying tick species parasitising South African reptiles and molecularly screening these for the presence of Hepatozoon, that study identified H. fitzsimonsi in tortoise-associated ticks. Thus, the present study aimed to revisit the potential of ticks to act as vectors for H. fitzsimonsi in tortoises using a combined microscopy and molecular approach., Methods: Specimens of Kinixys natalensis, Kinixys spekii, Kinixys zombensis and Stigmochelys pardalis were collected from Bonamanzi and Ndumo Game Reserve, South Africa. Upon capture, animals were examined for ticks, and these were collected along with blood and other tissues. Adult ticks were dissected and visceral impression slides were prepared along with thin blood and tissue smears on clean microscope slides. Smears and impression slides were stained with Giemsa, screened and micrographs of parasites were captured. Two primer sets were employed to target fragments of the 18S rRNA gene of parasites found in both tortoises and ticks and the resulting sequences were then compared with other known H. fitzsimonsi and haemogregarine sequences from the GenBank database., Results: Peripheral blood gamont and liver merogonic stages were observed in S. pardalis, while the sporogonic stages were observed in the haemocoel of Amblyomma ticks. Gamont and sporocyst stages compared morphologically with previous descriptions of H. fitzsimonsi, identifying them as this species. Phylogenetic analysis revealed that the blood and tick sequences obtained in this study clustered in a monophyletic clade comprising known H. fitzsimonsi., Conclusions: The present study provides further support for ticks acting as the vectors of H. fitzsimonsi by molecularly identifying and linking observed developmental stages in tortoises (S. pardalis) with those in the invertebrate host (Amblyomma spp.)., (© 2024. The Author(s).)

Blastocystis inhabits the digestive tracts of a diverse range of hosts. Transmission patterns, including host specificity, and the clinical and public health significance of Blastocystis in humans remain poorly understood. This study aimed to investigate the distribution and genetic diversity of Blastocystis in herbivorous and carnivorous reptiles in Eastern Thailand. A total of 501 faecal samples were collected from 363 iguanas, 79 bearded dragons, 50 tortoises, and nine snakes in an animal breeding farm in Chonburi Province, Eastern Thailand. Detection and differentiation of Blastocystis was based on amplification, sequencing, and phylogenetic analysis of specific small subunit (SSU) ribosomal RNA genes from faecal DNA extracted from the samples. Altogether 101/501 samples (20 %) were polymerase chain reaction (PCR) and sequencing-positive for Blastocystis, 90 (89 %) of which were from iguanas; the remaining positive samples were from African spurred tortoise (n=6), Bearded dragon (n=3), Leopard tortoise (n=1), and Red-footed tortoise (n=1). Phylogenetic analysis revealed that most of the Blastocystis sequences from iguanas were largely similar, and they were distinct from those of the tortoises. Subtype 17 was found in the three bearded dragons and likely reflected Blastocystis from prey animals. This is the largest survey of Blastocystis in reptiles to date. Remarkable differences in Blastocystis colonization rates and genetic diversity were observed between iguanas and other reptile orders, and what was considered Blastocystis colonization was only observed in herbivorous reptiles., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Species of Haemogregarina are blood parasites known to parasitise vertebrate hosts, including fishes (Haemogregarina sensu lato) and freshwater turtles (Haemogregarina sensu stricto). Their vectors, include gnathiid isopods and leeches, respectively. In turtles, Haemogregarina balli has the best-characterized life cycle in the genus. However, no studies in Brazil have suggested a possible vector for any species of Haemogregarina from freshwater turtles. Therefore, in the present study, we provide insights into a leech vector based on specimens found feeding on two species of freshwater turtles, Podocnemis unifilis and Podocnemis expansa, using morphological and molecular data. In 2017 and 2019, freshwater turtles were collected in Goiás State, Brazil. Hosts were inspected for ectoparasites and leeches were collected from two specimens of P. expansa and nine specimens of P. unifilis. Leeches were subsequently identified as members of the genus Unoculubranchiobdella. Leech histological slides revealed haemogregarine-like structures, similar to post-sporogonic merogony, found near the gills and within the posterior sucker. Molecular analysis of the haemeogregarines resulted in the identification of three species of Haemogregarina: Haemogregarina embaubali, Haemogregarina goianensis, and Haemogregarina brasiliana. Therefore, our findings, based on morphology and DNA data suggest leeches of the genus Unoculubranchiondella as vectors for at least three species of Haemogregarina from Brazilian turtles., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Six species of freshwater turtles dominate the Chaco-Pampa Plain in southern South America and their parasites have been relatively understudied, with most records concentrated in Brazil. Particularly in Argentina, there are only scattered records of parasites for most of the turtles that inhabit the region, leaving a large knowledge gap. The purpose of the present contribution is to increase the knowledge of the internal parasites of six species of freshwater turtles from Argentina, after 15 years of fieldwork, by providing new hosts and additional geographic records for many host-parasite relationships. Some molecular sequences of the studied parasites were provided as a tool for better species identification. We processed 433 stomach and fecal samples from live individuals and visceral and soft tissue samples from 54 dissected turtles collected from a wide range and different ecoregions. We found 6230 helminths belonging to 18 taxa (one cestode, 11 digeneans and six nematodes). Fourteen new parasite-host associations are reported here, and for the first time parasites are recorded for Phrynops williamsi. This work contributes significantly to the knowledge of the parasitofauna in freshwater turtles in Argentina, providing a detailed list of parasites present in each turtle species and reporting molecular characters for future studies.

Methods: Sampling was performed at three sites in the Tapajós River mouth, Pará State, Brazil, and an aliquot of blood was collected from each turtle by post-occipital dorsal vein puncture. The aliquots were used for the determination of hematological parameters and for the study of hemoparasites in the blood samples., Results: A total of 37 turtles were sampled: 21 Podocnemis expansa, three P. sextuberculata, nine P. unifilis, and four P. erythrocephala. Parasitological analyses showed inclusions in erythrocytes compatible with Haemogregarina sp., representing an overall prevalence of parasitized animals of 64.8% with an average of 94,789 parasites/ml of blood. In this study, the evolutionary forms of Haemogregarina spp. from trophozoites, pre-meronts, meronts, and mature and immature gamonts were observed in three species of freshwater turtle species, and one morphotype of the parasite with high similarity to Haemogregarina podocnemis has been observed., Conclusion: Despite recent research on freshwater turtles, this study provides new information on the intensity of blood parasite infection and hematological parameters of free-living animals, highlighting the need for studies on infected and uninfected animals from the same population., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The big-headed Amazon River turtle, Peltocephalus dumerilianus, is endemic to the Orinoco and Amazon River basins. It is a food source for local communities, often unsustainably. Knowledge about P. dumerilianus' parasitological fauna and host-parasite relationships is limited. Thus, ecological aspects of gastrointestinal parasitism in this species were investigated. Helminths were found in the gastrointestinal tract of 21 turtles, morphologically identified, and infection descriptors calculated. All animals harbored helminths: nematodes Ancyracanthus pinnatifidus, Paratractis hystrix, Atractis trematophila, Klossinemella conciliatus indeterminate three Klossinemella species, and digeneans Nematophila grandis, Helicotrema spirale, and Telorchis hagmanni. The highest parasite load occurred in the large intestine, followed by the small intestine and stomach. Shell length directly correlated with parasite burden of heteroxenic helminths, with males having higher burden than females. This is the first record of A. trematophila, K. conciliatus, and T. hagmanni in P. dumerilianus, and new location record for A. trematophila, P. hystrix, N. grandis, H. spirale, and T. hagmanni. Three potentially new Klossinemella species are presented.

Peltocephalus dumerilianus (Schweigger) is a diurnal freshwater turtle widely distributed in the Orinoco and Amazon River basins in Brazil, Colombia, Peru, Ecuador, Venezuela, and French Guiana. During a helminthological survey of freshwater turtles from the Brazilian Amazon, numerous nematodes with characteristics of the genus Orientatractis Petter, 1966 were recovered from the stomach of Peltocephalus dumerilianus. These nematodes were different from all congeneric species, and we describe herein. Orientatractis bowseri n. sp. differs from all known species of the genus by having dorsal cuticular projections on the posterior region of the females, distribution of caudal papillae and by having papilla-like ornamentations on the cuticle distributed from the mid-esophagus to the mid portion of the body. This study represents the fourth specie of the genus Orientatractis reported in Brazil, the seventh species of Orientatractis in the Neotropical region, and the third found infecting freshwater turtles in Brazil., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Reptiles show a high occurrence of hemoparasites in the wild; however, little is known about the impact of such infections on their hosts' physiology and health status. Podocnemis vogli is an ancient turtle distributed in South America, frequently infected by blood parasites. Specifically, we analyzed the hematological and serum chemistry parameters of 78 wild turtles. We compared these values with those obtained from non-infected turtles of the same species in ex-situ conditions, considering factors such as sex and coinfections. Two orders of hemoparasites were detected under microscopic analyses: Adelorina, represented by Haemogregarina sp. (98.72 ± 0.28%), and Haemosporida, represented by Haemocystidium sp. (30.77 ± 1.16%), the latter genus being always in coinfection with Haemogregarina. Significant differences were observed in 20 parameters between infected (free living) and uninfected (living in captivity) turtles. The ALP, PCV, Hb, and MCV were significantly different by sex; albumin, cholesterol, creatinine, percentage of heterophils, lymphocytes, and basophils differed in coinfection. This is the first report of reference intervals of P. vogli in the wild and the first study comparing hematological and blood biochemistry values between hemoparasite-infected and uninfected P. vogli turtles. However, the limited knowledge of these parameters in wild reptiles and the wide range of interval values makes it difficult to determine any significant impact on turtle health. Nevertheless, this study highlights the need for more in-depth research to explore the potential effects of blood parasite infections on turtles, including immune response and coevolution studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The growing contact between men and wild animals, caused by the increase in the population in urban centers and the destruction of the habitat of these animals, has been leading to a greater circulation of pathogens between humans and wildlife. Chelonoidis carbonaria , a tortoise found throughout South America, is one of the animals most rescued from animal trafficking and illegal breeding. Considering this situation, this study aimed to verify the occurrence of hemoparasites in C. carbonaria . Materials and Methods: Blood samples from 73 C. carbonaria were collected from animals located in (1) a rural commercial breeding unit, (2) an urban zoo, and (3) a center of rescued animal screening. Genomic DNA was extracted from these animals and used in PCRs to detect specific genomic fragments of haemogregarines ( i.e. , Hepatozoon and Hemolivia ), and members of the Anaplasmataceae Family ( i.e. , Ehrlichia sp. and Anaplasma sp.). Blood samples were screened for hemopathogens by direct microscopy and were used for hematological assays, and serum samples were analyzed to determine the concentration of serum components. Results: It was found that 34.2% of the tortoises presented Sauroplasma sp. in their blood samples; these animals showed clinical biochemistry changes that indicate altered liver function. Two zoo animals were positive for Ehrlichia sp. in PCR, and also presented clinical biochemistry and hematological changes. Conclusion: The present project is pioneer in the detection of Ehrlichia sp. in C. carbonaria , and was able to identify changes in clinical biochemistry that can be a result of the infection by hemopathogens in this species.

Trachemys dorbigni is the most abundant freshwater turtle species in Rio Grande do Sul, southern Brazil. Chelonians are known to host a wide variety of pathogens, including viruses, bacteria, hemoparasites and helminths. Among these, nine genera of polystomatid flatworms (Monogenoidea; Polystomatidae) infect freshwater turtles: Apaloneotrema, Aussietrema, Fornixtrema, Manotrema, Pleurodirotrema, Polystomoidella, Polystomoides, Uropolystomoides and Uteropolystomoides. However, little is known about the biology of these parasites in the Neotropical Realm. Through investigative cystoscopy, specimens of Polystomatidae were located inside the urinary bladder of the host T. dorbigni. Retrieved specimens were fixed and stained whole mounts prepared for taxonomic identification. In the present paper, a new species of Polystomoides (Monogenoidea: Polystomatidae) parasitizing the urinary bladder of a freshwater turtle of the species T. dorbigni in Brazil is described. Polystomoides santamariensis n. sp. differs from the congeneric species on the length of the genital spines, which are longer. Given the enormous diversity of freshwater turtles around the world, it is likely that a large number of chelonian polystomatids are still unknown.

Toxoplasmosis is one of the most widespread zoonotic diseases, especially in warm and humid areas, and affects all mammals, including humans and many herbivores and carnivores. The present study investigated the Toxoplasma gondii ( T. gondii ) parasite in tortoises for the first time in Iraq using PCR technology. A total of 28 tortoises/ Testudo graeca ( T. graeca ) were collected between October 2018 and March 2019 from the study stations and then sent to the Animal House, which belongs to the Department of Biology, Faculty of Education, University of AL-Qadisiyah, Iraq, to perform the dissection. The body cavity was opened, and all organs were removed. The tortoises' liver, heart, and brain were removed and kept at -20ºC until use. Afterward, the samples were subjected to DNA extraction. The Nested-PCR technique was implemented using two pairs of primers, and then the PCR products were analyzed using 1.5% agarose gel electrophoresis. The amplification of the gene during the first cycle indicated that 10 samples gave positive results with a total percentage of (11.9%), including five liver samples, three heart samples, and two brain samples (17.85%, 10.71%, and 7.14%, respectively). On the other hand, during the second cycle of the reaction, the amplification of the gene was obtained in seven samples (8.33%). The highest percentage of the presence of the gene was recorded in the tortoises' liver (14.28%) and the lowest in their brain (3.57%). This study is among the first to investigate the molecular detection of T. gondii in wild tortoises ( T. graeca ) in Iraq. The findings imply that tortoises have a role in transmitting T. gondii and are believed to acquire infection by feeding on small invertebrate animals or plants contaminated with the oocysts of the parasite., Competing Interests: The authors declare that they have no conflict of interest.

While monogenean worms are mainly parasites of the gills and skin of fish, and to a lesser extent parasites of the oral cavity, urinary bladder, and/or conjunctival sacs of amphibians and freshwater turtles, Oculotrema hippopotamiStunkard, 1924 is the single monogenean polystome reported from a mammal, the common hippopotamus (Hippopotamus amphibius Linnaeus). Several hypotheses have been suggested in the last decade to explain the origin of this enigmatic parasite which infects the conjunctival sacs of H. amphibius. Based on a molecular phylogeny inferred from nuclear (28S and 18S) and mitochondrial (12S and COI) sequences of O. hippopotami and chelonian polystomes, we found a sister group relationship between O. hippopotami and Apaloneotrema moleri (Du Preez & Morrison, 2012). This result suggests lateral parasite transfer between freshwater turtles and hippopotamuses, thus likely reflecting one of the most exceptional known examples of host-switching in the course of vertebrate evolution. It also demonstrates that the proximity in the ecological habitat of parasites within host species is an important feature for their speciation and diversification. Because A. moleri and its host, the Florida softshell turtle (Apalone ferox (Schneider)), are restricted to the USA, we suggest that an ancestral stock of parasites may have been isolated on primitive African trionychids after they diverged from their American relatives, and then switched to hippopotamuses or anthracotheres in Africa., (Copyright © 2023 Australian Society for Parasitology. All rights reserved.)

The protistan genus Epistylis contains freshwater colonial species that attach to aquatic organisms in an epibiotic or parasitic relationship. They are known to attach to the epidermis and shells of aquatic turtles, but have not been reported to cause heavy infestations or morbidity in turtles. We documented heavy infestations of Epistylis spp. in several populations of Sonoran mud turtles (Kinosternon sonoriense) inhabiting livestock ponds in Arizona, USA, and rough-footed mud turtles (Kinosternon hirtipes) from livestock ponds in Texas, USA, over the course of several years. Severe Epistylis spp. infestations on mud turtles appeared to alter diving and swimming behavior when compared to uninfested conspecifics. Infestations were cleared in captivity using tap water or a 10% salt solution, and the turtles had no permanent damage to their shell or epidermis upon clearing. While several of the mud turtles we observed had poor body condition, it is possible that the severe infestations we observed were caused by a comorbidity associated with a pathogen, parasite, or poor habitat quality that made the turtles more susceptible to the Epistylis spp. infestation. Further research on causes for these severe infestations are warranted because they contribute to changes in behavior of the heavily infested turtles and may contribute to morbidity in Kinosternon spp. when mud turtles inhabit extremely warm, shallow, eutrophic aquatic habitats, such as livestock ponds., (© Wildlife Disease Association 2023.)

Nematodes collected from the stomach and large intestine of Rhinoclemmys punctularia (Daudin) from the eastern Amazon, Brazil, are assigned to a new genus, Vogtnema n. gen., allocated in the family Atractidae (Cosmocerdoidea). Vogtnema n. gen. differs from all other genera of Atractidae based on the combination of the following morphological characters: four lips V-shaped (two subdorsal and two subventral) without sclerotized structures associated, absence of sclerotized formations and/or pieces surrounding the oral opening, oral aperture surrounded by flange internal membranous structures; in males the larger spicule is alate. The type species Vogtnema asymmetrica n. gen., n. sp. has the five precloacal pairs of papillae situated on the right side of the body that are more prominent than those on the left side. We also provide an identification key to the genera of the family Atractidae., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The occurrence of protozoan parasites Giardia duodenalis and Cryptosporidium spp. such as the pathogenic bacteria Salmonella spp. and Escherichia coli was molecularly investigated in the following free ranging species of striped dolphins (Stenella coeruleoalba), Risso's dolphins (Grampus griseus) as well as loggerhead (Caretta caretta) and green (Chelonia mydas) sea turtles living in the Gulf of Taranto (Mediterranean Sea). Out of forty-one investigated individuals belonging to the 4 species, 13 (31.7%) were positive to one or more pathogens and zoonotic G. duodenalis assemblage A, Cryptosporidium parvum and S. enterica were identified in striped dolphins, loggerhead and green sea turtles. In this work, the presence of these opportunistic pathogens has been investigated in fecal samples of free ranging dolphin and sea turtle species for the first time. Moreover, this is the first record of C. parvum in loggerhead sea turtles. These results may provide baseline data for the potential role of cetaceans and sea turtles as potential sentinel species for zoonotic and terrestrial pathogens in the marine environment., (© 2022. The Author(s).)

Pentastomes are crustacean endoparasites in the lungs of herpetofauna and rarely mammals and birds. To date, the only pentastomes reported in chelonians, all aquatic turtles, are Diesingia megastomum from Brazil and Pelonia africana from South Africa. In March 2021, 185 juvenile tortoises (Chelonoidis niger) were confiscated after an attempted illegal exportation from the Galápagos. It is believed the tortoises were removed from nests on Santa Cruz Island. The young tortoises were individually wrapped in plastic and at seizure ten were dead and another 25 tortoises subsequently died. One of the tortoises, estimated to be 3 months old, had 11 pentastomes in the lungs. The pentastomes were identified as a Raillietiella sp. based on morphology. The specimens had a claviform body that tapers into a bifid tail, a 165.3 μm × 92.1 μm buccal cadre, and sharp tipped anterior and posterior hooks with the posterior hooks being larger than the anterior. The males have smooth copulatory spicules with a rounded, smooth base. All females were gravid. Molecular analysis confirmed that the parasites were a Raillietiella sp. Based on the COI gene, it was most similar (82.7% identical) to an undescribed Raillietiella species from a Caribbean anole (Anolis cristatellus) found in Florida followed by R. hebitihamata (81%), R. indica (80.7%), and R. orientalis (78.8-80.7%). Based on the 18S rRNA gene sequence (1799 bp), it was most similar (99.3% identical) to two undescribed Raillietiella species followed by R. aegypti from a berber skink (Eumeces schneideri) from Saudia Arabia. Phylogenetically, with both molecular targets, the Raillietiella sp. from the Galápagos tortoise grouped with other Raillietiella spp. and was basal within the group. Currently, the origin of this parasite (native to Galápagos or introduced) and the life cycle are unknown. Because some pentastome species, especially when in aberrant hosts, can be pathogenic, additional studies of parasites in native and introduced reptile and amphibian species in the Galápagos are needed to better understand the risk this parasite poses to Galápagos tortoises., Competing Interests: Declaration of Competing Interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from myabsley@uga.edu, (Copyright © 2022 Elsevier B.V. All rights reserved.)

Eretmochelys imbricata (hawksbill turtle) is classified as a critically endangered species at world level; in Brazil, it is listed among the threatened species. Spirorchiids are parasites of the circulatory system of chelonians which may cause serious lesions in the various tissues of the host due to deposition of eggs in the bloodstream. In this context, the aim of the present study was to describe the pathology caused by spirorchiid eggs in E. imbricata from the Brazilian over a five year period. A total of 29 animals were analyzed, of which nine (31%) presented lesions associated to spirorchiids eggs. The lesions were: granulomatous enteritis in six (66.66%), granulomatous splenitis in five (55.55%), granulomatous hepatitis in three (33.33%), granulomatous pneumonia in three (33.33%), granulomatous pancreatitis in two (22.22%), and granulomatous adrenalitis in one specimen (11.11%). Concluded the main lesion in Eretmochelys imbricata was giant-cell granulomatous inflammatory reaction to the parasite's eggs. Animals exhibiting poor physical health were more susceptible to presenting such lesions.

Ticks carry a diverse community of microorganisms including non-pathogenic symbionts, commensals, and pathogens, such as viruses, bacteria, protozoans, and fungi. The assessment of tick-borne microorganisms (TBM) in tortoises and their ticks is essential to understand their eco-epidemiology, and to map and monitor potential pathogens to humans and other animals. The aim of this study was to characterize the diversity of microorganisms found in ticks collected from the spur-thighed tortoise (Testudo graeca) in North Africa and Anatolia. Ticks feeding on wild T. graeca were collected, and pathogens were screened by polymerase chain reaction using group-specific primers. In total, 131 adult Hyalomma aegyptium ticks were collected from 92 T. graeca in Morocco (n = 48), Tunisia (n = 2), Algeria (n = 70), and Turkey (n = 11). Bacteria and protozoa detected included Hemolivia mauritanica (22.9%), Midichloria mitochondrii (11.4%), relapsing-fever borreliae (8.4%), Ehrlichia spp. (7.6%), Rickettsia spp. (3.4%), Borrelia burgdorferi s.l. (0.9%), Francisella spp. (0.9%), and Wolbachia spp. (0.8%). The characterization of Rickettsia included R. sibirica mongolitimonae (Algeria), R. aeschlimannii (Turkey), and R.africae (Morocco). Hemolivia mauritanica and Ehrlichia spp. prevalence varied significantly with the sampling region/country. We did not detect significant associations in microorganism presence within ticks, nor between microorganism presence and tick mitochondrial DNA haplogroups. This is the first report of Francisella persica-like, relapsing fever borreliae, M. mitochondrii, and Wolbachia spp. in H. aegyptium ticks collected from wild hosts from the South and Eastern Mediterranean region, and of R. sibirica mongolitimonae and R. africae in H. aegyptium from Algeria and Morocco, respectively. Given that T. graeca is a common species in commercial and non-commercial pet trade, the evaluation of the role of this species and its ticks as hosts for TBM is particularly relevant for public health., (© 2021 Wiley-VCH GmbH.)

Molecular tools have revolutionized assessments of blood parasites in freshwater turtles. In the Iberian Peninsula and North Africa, two native species of terrapins occur, Emys orbicularis (Linnaeus) and Mauremys leprosa (Schweigger). Both have been identified as hosts for the blood parasite Haemogregarina stepanowi Danilewsky, 1885, which has also been found in related species. However, recent assessments of M. leprosa have identified several distinct genetic lineages of these parasites in this host, while only three haemogregarine lineages were identified in E. orbicularis in Tunisia. Here, we screened 215 individuals of E. orbicularis from the Iberian Peninsula, Menorca Island and Morocco for haemogregarine parasites using partial 18S rRNA gene sequences to estimate relationships. Three unrelated lineages of parasites were detected, one presumed H. stepanowi and two lineages previously known from M. leprosa. A considerable undescribed diversity of parasites exists within these vertebrate host species, while mixed infection and host-sharing is also widespread. Considering that E. orbicularis is near threatened in this region, it is of great importance to identify the parasites infecting it, and to further assess the potential deleterious impact of these diverse parasites on their hosts., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Tortoise ticks, Hyalomma aegyptium, are considered so strongly associated with their hosts that they are even used as indirect indicators for them. In such a case, a robust pattern of congruence between host and parasite could be expected, with phylogeographic breaks within the host being reflected in their parasites. We sequenced two mitochondrial partial gene regions (12S rRNA and Cytochrome Oxidase 1) from ticks across northern Africa and Anatolia, and compared patterns of variation with those identified in its main host, Testudo graeca. Two distinct haplogroups were identified, both of which were found distributed across much of northern Africa. This pattern does not reflect the known variation within the host, which has multiple, geographically disjunct subspecies in this region, but rather the major climatic zones. This relationship can be explained by adaptive processes to environmental conditions influenced by the climate, as well as by the spatial structure of the communities of tick potential hosts in larval and nymphal stages. Extensive anthropogenic movement of tortoises may also obscure congruence patterns between H. aegyptium ticks and their hosts., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Polystomatids are platyhelminth parasites that infect mainly amphibians and freshwater turtles. For more than seven decades, chelonian polystomes were classified into three genera according to the number of hamuli, i.e. absent for Neopolystoma, one pair for Polystomoidella and two pairs for Polystomoides. Following re-examination of morphological characters, seven new genera were erected the past six years, namely Apaloneotrema, Aussietrema, Fornixtrema, Manotrema, Pleurodirotrema, Uropolystomoides and Uteropolystomoides. However, the polyphyly of Neopolystoma and Polystomoides on the one hand, and the nested position of Uteropolystomoides within a clade encompassing all Neopolystoma and Polystomoides spp. on the other, still raised questions about the validity of these genera. We therefore re-examined several types, paratypes and voucher specimens, and investigated the molecular phylogeny of polystomes sampled from the oral cavity of North American turtles to re-evaluate their systematic status. We show that all Polystomoides Ward, 1917, sensu Du Preez et al., 2022, Neopolystoma Price, 1939, sensu Du Preez et al., 2022 and Uteropolystomoides Tinsley, 2017 species, display vaginae that are peripheral and extend well beyond the intestine. We thus reassign all species of the clade to Polystomoides and propose nine new combinations; however, although Uteropolystomoides is nested within this clade, based on its unique morphological features, we propose to keep it as a valid taxon. Polystomoides as redefined herein groups all polystome species infecting either the oral cavity or the urinary bladder of cryptodires, with peripheral vaginae and with or without two pairs of small hamuli. Uteropolystomoides nelsoni (Du Preez & Van Rooyen 2015), originally described from Pseudemys nelsoni Carr is now regarded as Uteropolystomoides multifalx (Stunkard, 1924) n. comb. infecting three distinct Pseudemys species of North America., (© A. Chaabane et al.,published by EDP Sciences, 2022.)

Distribution, prevalence and parasitaemia of apicomplexan parasites of the genus Haemogregarina were studied in 858 freshwater turtles (735 Mauremys leprosa leprosa and 123 Mauremys leprosa saharica) throughout 30 localities from Morocco. Blood smears were collected from the turtles and partial 18S rRNA sequence data used to infer genetic diversity and phylogenetic relationships. Of the 858 individuals analyzed by microscopy 22.7% were infected, from 16 M. leprosa leprosa localities. Individuals of M. leprosa saharica sampled south of the High Atlas Mountains were not infected, probably due to the absence or rarity of the leech vectors in these localities. Within M. leprosa leprosa, we did not identify any patterns between geography and prevalence, which varied between 10% (Oued Nfiss) to 100% (Oued Tassaout). Five distinct genetic lineages were identified, indicating the likely presence of multiple species of haemogregarines, one of which probably corresponds to Haemogregarina stepanowi. Mixed infections were also detected. Additional studies are needed to better understand the ecology and epidemiology of this parasite in turtles, as well as the host-parasite relationship with their definitive hosts, leeches., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The objective of this study was to determine the structure of the helminth fauna and identify the macroscopic and histopathological alterations associated with parasitic infections in Phrynops geoffroanus. Freshwater turtles of both sexes were captured during the dry and rainy seasons in four municipalities along the Capibaribe River. The study included 63 animals, of which 79.37% (50/63) were parasitized by one or more helminths. In total, 933 helminths of seven taxa were recovered: Serpinema monospiculatus, Spiroxys figueiredoi, Nematophila grandis, Polystomoides brasiliensis, Cheloniodiplostomum testudinis, Telorchis birabeni, and Prionosomoides scalaris. Monogeneans and digenetic trematodes were more sensitive to environmental pressures, since the prevalences varied significantly between areas. Nematodes proved to be more resistant to environmental pressure and caused severe injuries to their hosts: nodules in the stomach and small intestine, adhesions in the liver capsule, and pulmonary emphysema. Parasitic granulomas were recorded at the infection sites and in the lungs and liver, the latter caused by migration of S. figueiredoi larvae. This is the first record of P. brasiliensis, N. grandis, C. testudinis, and T. birabeni parasitizing P. geoffroanus in the state of Pernambuco. Histopathology proved to be an important tool for studies on the impact of parasites at the individual, population, and ecosystem levels. Considering the use of the Capibaribe River for public water supply, fishing, and other activities, within the One Health perspective, this study demonstrates that the anthropogenic impact affects parasites and their hosts, in addition to the human population that uses this ecosystem., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

International trade of animals may represent a gateway for the spreading of zoonotic pathogens and their vectors. Amongst animals, reptiles are commonly illegally imported worldwide, being Italy in the fifth position of importation of these animals. Thus, the current study analysed the pathogens associated with Hyalomma aegyptium ticks, which were collected from illegally imported tortoises from North Africa to Italy. All tick DNA samples were tested by conventional PCR for the presence of Anaplasma spp., Babesia spp., Borrelia spp., Coxiella burnetti, Ehrlichia spp., Hepatozoon spp., Rickettsia spp. and microfilariae of filarioids. Out of 22% (n=161) of ticks screened, 78.9% (n=127) were males and 21.1% (n=34) females. Among them, three male specimens collected from two different turtles (1.9%; 95% CI; 0.5-5.5) scored positive for Anaplasma spp./Ehrlichia spp., whereas all females were negative. BLAST analysis of the sequences obtained from positive samples revealed 99-99.3% nucleotide identity with the sequence of Ehrlichia ewingii available in GenBank. The finding of E. ewingii in ticks from imported reptiles warrants the need for imposing strict rules in the international trade of reptiles to effectively reduce the introduction of exotic pathogens and their vectors in new geographic areas., (Copyright © 2021. Published by Elsevier B.V.)

The role of invertebrates in some acanthocephalan life cycles is unclear because juvenile acanthocephalans are difficult to identify to species using morphology. Most reports suggest acanthocephalans from turtle definitive hosts use ostracods as intermediate hosts and snails as paratenic hosts. However, laboratory studies of the life cycle suggest that ostracods and snails are both required hosts in the life cycle. To elucidate the role of ostracods and snails in acanthocephalan life cycles better, we collected 558 freshwater snails of 2 species, including Planorbella cf. Planorbella trivolvis and Physa acuta, from 23 wetlands in Oklahoma, U.S.A., and examined them for acanthocephalan infections. Additionally, we examined 37,208 ostracods of 4 species, Physocypria sp. (morphotype 1), Cypridopsis sp., Stenocypris sp., and Physocypria sp. (morphotype 2) for juvenile acanthocephalans from 2 wetlands in Oklahoma. Juvenile acanthocephalans were morphologically characterized, and the complete internal transcribed spacer (ITS) region of nuclear rDNA was sequenced from acanthocephalans infecting 11 ostracod and 13 snail hosts. We also sampled 10 red-eared slider turtles, Trachemys scripta elegans, and 1 common map turtle, Graptemys geographica, collected from Oklahoma, Arkansas, and Texas and recovered 1,854 adult acanthocephalans of 4 species. The ITS of 17 adult acanthocephalans of 4 species from turtle hosts were sequenced and compared to juvenile acanthocephalan sequences from ostracod and snail hosts from this study and GenBank to determine conspecificity. Of the 23 locations sampled for snails, 7 (30%) were positive for juvenile acanthocephalans in the genus Neoechinorhynchus. The overall prevalence and mean intensity of acanthocephalans in Planorbella cf. P. trivolvis and P. acuta were 20% and 2 (1-6) and 2% and 1 (1), respectively. In contrast, only 1 of 4 species of ostracods, Physocypria sp. (morphotype 1), was infected with larval/juvenile Neoechinorhynchus spp. with an overall prevalence of 0.1% and a mean intensity of 1 (1-2). Although 4 species of acanthocephalans infected turtle definitive hosts, including Neoechinorhynchus chrysemydis, Neoechinorhynchus emydis, Neoechinorhynchus emyditoides, and Neoechinorhynchus pseudemydis, all the ITS sequences from cystacanths infecting snail hosts were conspecific with N. emydis. In contrast, the ITS sequences from larval/juvenile acanthocephalans from ostracods were conspecific with 2 species of acanthocephalans from turtles (N. emydis and N. pseudemydis) and 1 species of acanthocephalan from fish (Neoechinorhynchus cylindratus). These results indicate that N. emydis infects freshwater snails, whereas other species of Neoechinorhynchus appear not to infect snail hosts. We document new ostracod and snail hosts for Neoechinorhynchus species, including the first report of an ostracod host for N. pseudemydis, and we provide novel molecular barcodes that can be used to determine larva, juvenile, and adult conspecificity of Neoechinorhynchus species., (© American Society of Parasitologists 2021.)

Although intranuclear coccidiosis was first identified in chelonians less than 30 years ago, it is now considered an important emerging disease. Symptoms include anorexia, weakness and weight loss, potentially leading to death of the infected animal. The use of molecular tools has led to improved diagnosis and has also led to an increase in known host species. Here we report a putative intranuclear coccidium in Mauremys leprosa (Schweigger), from Morocco, based on 18S rDNA sequence analysis. This is, to the best of our knowledge, the first report of this parasite from a freshwater terrapin species.

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., (Copyright © 2021. Published by Elsevier B.V.)

The genus Desmogonius (Digenea: Pronocephalidae) is known only from sea turtles and currently contains two species: D. desmogonius Stephens, 1911 and D. loossi Chattopadhyaya, 1972. The present study describes a third species, Desmogonius baldassinae n. sp. found in a juvenile green sea turtle (Chelonia mydas) from Brazil. The new species is larger than its congeners in all dimensions, with vitellinic fields ending posterior to the ovary. In addition, we present a key for species identification and an emended diagnosis of the genus Desmogonius.

Background: The emergence of tick-borne disease is increasing because of the effects of the temperature rise driven by global warming. In Turkey, 19 pathogens transmitted by ticks to humans and animals have been reported. Based on this, this study aimed to investigate tick-borne pathogens including Hepatozoon spp., Theileria spp., Babesia spp., Anaplasma spp., Borrelia spp., and Bartonella spp. in tick samples (n = 110) collected from different hosts (dogs, cats, cattle, goats, sheep, and turtles) by molecular methods., Methods: To meet this objective, ticks were identified morphologically at the genus level by microscopy; after DNA isolation, each tick sample was identified at the species level using the molecular method. Involved pathogens were then investigated by PCR method., Results: Seven different tick species were identified including Rhipicephalus sanguineus, R. turanicus, R. bursa, Hyalomma marginatum, H. anatolicum, H. aegyptium, and Haemaphysalis erinacei. Among the analyzed ticks, Hepatozoon spp., Theileria spp., Babesia spp., and Anaplasma spp. were detected at rates of 6.36%, 16.3%, 1.81%, and 6.36%, respectively while Borrelia spp. and Bartonella spp. were not detected. Hepatozoon spp. was detected in R. sanguineus ticks while Theileria spp., Babesia spp., and Anaplasma spp. were detected in R. turanicus and H. marginatum. According to the results of sequence analyses applied for pathogen positive samples, Hepatozoon canis, Theileria ovis, Babesia caballi, and Anaplasma ovis were identified., Conclusion: Theileria ovis and Anaplasma ovis were detected for the first time to our knowledge in H. marginatum and R. turanicus collected from Turkey, respectively. Also, B. caballi was detected for the first time to our knowledge in ticks in Turkey.

Haemogregarines (Adeleorina) have a high prevalence in turtles. Nevertheless, there is only one Hepatozoon species described that infects Testudines so far; it is Hepatozoon fitzsimonsi which infects the African tortoise Kinixys belliana. Colombia harbours a great diversity of chelonians; however, most of them are threatened. It is important to identify and characterize chelonian haemoparasite infections to improve the clinical assessments, treatments and the conservation and reintroduction programs of these animals. To evaluate such infections for the Colombian wood turtle Rhinoclemmys melanosterna, we analysed blood from 70 individuals. By using the morphological characteristics of blood stages as well as molecular information (18S rRNA sequences), here we report a new Hepatozoon species that represents the first report of a hepatozoid species infecting a semi-aquatic continental turtle in the world. Although the isolated lineage clusters within the phylogenetic clades that have morphological species of parasites already determined, their low nodal support makes their position within each group inconclusive. It is important to identify new molecular markers to improve parasite species identification. In-depth research on blood parasites infecting turtles is essential for increasing knowledge that could assess this potential unknown threat, to inform the conservation of turtles and for increasing the state of knowledge on parasites.

As presently recognized, Herpetodiplostomum is a small genus of proterodiplostomid digeneans parasitic in crocodilians. Most members of Cheloniodiplostomum, a genus of proterodiplostomids that parasitize chelonians, were originally placed within Herpetodiplostomum. The 2 genera were distinguished based on the presence/absence of papillae on the holdfast organ and anterior extent of vitellarium. Our study of Herpetodiplostomum and Cheloniodiplostomum revealed a lack of consistent morphological differences between the genera. Therefore, we consider Cheloniodiplostomum to be a junior synonym of Herpetodiplostomum. Recent molecular phylogenetic studies included a number of proterodiplostomid taxa; however, DNA sequence data are not available for any Herpetodiplostomum or Cheloniodiplostomum species. Herein, we describe a new Herpetodiplostomum species from Geoffroy's side-necked turtle Phrynops geoffroanus from Mato Grosso State, Brazil. The new species can be distinguished from other Herpetodiplostomum species based on the distribution of vitellarium, level of development of holdfast papillae, and ratio of prosoma:opisthosoma width, among other characters. We provide the first molecular phylogeny of the Proterodiplostomidae that includes a Herpetodiplostomum species. The limited geographic distribution of Herpetodiplostomum spp. within the geographic ranges of caimans suggests a secondary host switching event from crocodilians to chelonian definitive hosts in the evolution of Herpetodiplostomum., (© American Society of Parasitologists 2021.)

A new glossiphoniid leech species, Placobdelloides tridens sp. n., is discovered on the Malayan Giant Turtle (Orlitia borneensis) at the Nakhon Ratchasima Zoo in Thailand. The morphological study of this new species revealed that it is distinguished from P. siamensis, a turtle leech species that can be found commonly in Thailand. Placobdelloides tridens presented the following diagnostic morphological characteristics: a pear-shaped and triannulate body, well-developed rod-like papillae on the dorsal surface, smooth posterior and anterior suckers with nominal pits inside, a single pair of dark contiguous eyes, light yellow-brown to greenish dorsal color, absence of median line, male and female gonopore separated by a single annulus and a unique trident shape at the tip of the crop ceca. The phylogenetic relationships of P. tridens sp. n., was clarified, and shown to be a sister clade to the P. siamensis and P. sirikanchanae clade. Furthermore, this is a new host record for P. siamensis, which was found on O. borneensis, Batagur affinis and B. borneoensis in the Khao Kheow Open Zoo, Chonburi, Thailand.

Background: In free-ranging reptile populations, bacterial, fungal, viral and parasitic pathogens may affect hosts through impairment in movements, thermoregulation, reproduction, survival, and population dynamics. The speckled dwarf tortoise (Chersobius [Homopus] signatus) is a threatened species that is mostly restricted to the Succulent Karoo biome in South Africa, and little information on pathogens of this species is available yet. We derived baseline parameters for five males and five females that were captured to genetically enhance a conservation breeding program in Europe. Upon collection of the tortoises, ticks were removed and identified. Immediately upon arrival in Europe, ocular, nasal, oral and cloacal swabs were taken for viral, bacteriological and mycological examinations. Fecal samples were collected before and 1 month after fenbendazole treatment, and analyzed for parasites. A panel of PCR, aiming to detect herpesviruses, adenoviruses and iridoviruses, was carried out., Results: Samples were negative for viruses, while bacteriological examination yielded detectable growth in 82.5% of the swabs with a mean load of 16 × 10 7  ± 61 × 10 8 colony forming units (CFU) per swab, representing 34 bacterial species. Cloacal and oral swabs yielded higher detectable growth loads than nasal and ocular swabs, but no differences between sexes were observed. Fungi and yeasts (mean load 5 × 10 3  ± 13 × 10 3 CFU/swab) were detected in 25% of the swabs. All pre-treatment fecal samples were positive for oxyurid eggs, ranging from 200 to 2400 eggs per gram of feces, whereas after the treatment a significantly reduced egg count (90-100% reduction) was found in seven out of 10 individuals. One remaining individual showed 29% reduction, and two others had increased egg counts. In five tortoises, Nycthocterus spp. and coccidian oocysts were also identified. Soft ticks were identified as Ornithodoros savignyi., Conclusions: Our baseline data from clinically healthy individuals will help future studies to interpret prevalences of microorganisms in speckled dwarf tortoise populations. The study population did not appear immediately threatened by current parasite presence.

The hawksbill turtle Eretmochelys imbricata is a critically endangered species with a worldwide distribution. Limited information is available about the naturally occurring intestinal parasites of this species and what impact these parasites may have on the health of the hawksbill turtle. Diaschistorchis pandus was identified postmortem in 5 hawksbill turtles from Grenada, West Indies, using morphologic characterization. Sanger sequencing was performed for conserved ribosomal regions (5.8S, ITS2, 28S) and the mitochondrial cytochrome c oxidase subunit 1 gene (COI). Phylogenetic analysis of the 28S rRNA gene sequence data shows D. pandus clustering with other trematodes in the family Pronocephalidae, corroborating morphological classification. No genetic sequences have been previously reported for this trematode species, which has limited the collection of objective epidemiological data about this parasite of marine turtles., (© American Society of Parasitologists 2021.)

Mycoplasma agassizii is a common cause of upper respiratory tract disease in Mojave desert tortoises (Gopherus agassizii). So far, only two strains of this bacterium have been sequenced, and very little is known about its patterns of genetic diversity. Understanding genetic variability of this pathogen is essential to implement conservation programs for their threatened, long-lived hosts. We used next generation sequencing to explore the genomic diversity of 86 cultured samples of M. agassizii collected from mostly healthy Mojave and Sonoran desert tortoises in 2011 and 2012. All samples with enough sequencing coverage exhibited a higher similarity to M. agassizii strain PS6T (collected in Las Vegas Valley, Nevada) than to strain 723 (collected in Sanibel Island, Florida). All eight genomes with a sequencing coverage over 2x were subjected to multiple analyses to detect single-nucleotide polymorphisms (SNPs). Strikingly, even though we detected 1373 SNPs between strains PS6T and 723, we did not detect any SNP between PS6T and our eight samples. Our whole genome analyses reveal that M. agassizii strain PS6T may be present across a wide geographic extent in healthy Mojave and Sonoran desert tortoises., Competing Interests: The authors have declared that no competing interests exist.

Cucullanus carettae Baylis, 1923 (Nematoda: Cucullanidae) is found worldwide in loggerhead turtles (Caretta caretta). Regarding the Mediterranean, C. carettae has been identified in the Tyrrhenian and the Ionian Sea and a unique description of a Cucullanus sp. specimen in loggerheads from the Adriatic Sea has been reported in the literature so far. In the framework of a bio-monitoring project of the Abruzzo and Molise coasts, a parasitological survey was performed on stranded and by-caught sea turtles, at the Istituto Zooprofilattico of Abruzzo and Molise "G. Caporale." During necropsy, the gastrointestinal system of 72 stranded loggerhead turtles was analyzed for the presence of endoparasites and fecal samples were collected for coprological examination. Adult C. carettae (n = 123) was found in the upper intestine of one loggerhead turtle, associated with chronic lymphoplasmocytic enteritis. Additionally, five stool samples (6.9%) were positive for Cucullanus sp. eggs. Molecular characterization of adult nematodes was carried out to study phylogenetic relationships among the Cucullanus species. To our knowledge, this is the first morphological and molecular identification of C. carettae in loggerhead turtles from the Adriatic Sea. Additional studies on the distribution of this parasite in the Mediterranean are encouraged.

Herein, we describe several newly-collected specimens of Neopolystoma cf. orbiculare from the urinary bladder of 2 alligator snapping turtles, Macrochelys temminckii (Troost in Harland, 1835) (Cryptodira: Chelydridae Gray, 1831) from Comet Lake (30°35'46.94″N, 88°36'3.12″W), Pascagoula River, Mississippi. Our specimens differed from all previous descriptions of N. orbiculare and its junior subjective synonyms by the combination of having intestinal ceca adorned with triangular pockets and that terminate dorsal to the haptor, distinctive hooklets each having a handle and guard of approximately equal length and having a much longer and curved blade, 16 genital coronet spines that each possess 1-2 flanges per spine, pre-testicular vaginal pores, and vaginal ducts that are anterior to the junction of the oviduct and genito-intestinal canal. Some of our specimens were enantiomorphic (4 and 3 had a dextral and sinistral ovary, respectively). Nucleotide sequences (large subunit ribosomal DNA [28S], small subunit ribosomal DNA [18S], and cytochrome oxidase subunit 1 mitochondrial gene [COI]) for our specimens were most similar to GenBank sequences ascribed to N. orbiculare. Single-gene and concatenated phylogenetic analyses confirmed that NeopolystomaPrice, 1939 is polyphyletic and that our isolates share a recent common ancestor with those ascribed to N. orbiculare. This is the first record of a polystomatid from Mississippi, from the Pascagoula River, and from the alligator snapping turtle (and only the second species of Neopolystoma reported from any snapping turtle)., (© American Society of Parasitologists 2021.)

The gopher tortoise tick, Amblyomma tuberculatum, is known to parasitize keystone ectotherm reptile species. The biological success of ticks requires precise mechanisms to evade host hemostatic and immune responses. Acquisition of a full blood meal requires attachment, establishment of the blood pool, and engorgement of the tick. Tick saliva contains molecules which counter the host responses to allow uninterrupted feeding on the host. RNASeq of the salivary glands of Amblyomma tuberculatum ticks were sequenced resulting in 138,030 pyrosequencing reads which were assembled into 29,991 contigs. A total of 1875 coding sequences were deduced from the transcriptome assembly, including 602 putative secretory and 982 putative housekeeping proteins. The annotated data sets are available as a hyperlinked spreadsheet. The sialotranscriptome assembled for this tick species made available a valuable resource for mining novel pharmacological activities and comparative analysis., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

In this study, two potentially new species of turtle blood flukes (TBFs) (Digenea: Spirorchiidae) have been recorded from South Brazil. The spirorchiid parasites infect the vascular system of turtles, thereby compromising their health. The life cycle of these parasites is not well studied. The larval stage of cercaria is found in intermediate gastropod hosts, with some species presenting similar morphological characteristics, which can result in misinterpretations when using only morphological taxonomy for species identification. In this study, we recorded a single morphotype belonging to the family Spirorchiidae in Biomphalaria occidentalis in an urban aquatic ecosystem in Brazil. However, molecular data (28S rDNA and cytochrome oxidase subunit I) confirmed the presence of two species of Spirorchiidae in the sampled environment; both phylogenetically close to genera previously studied in freshwater turtles from the Peruvian Amazon. In this study, species characterization was possible because of molecular tools. We recommend using more than one molecular marker in future studies focusing on TBFs, which need attention about their evolutionary history and ecology to understand their distribution in South America.

Placobdelloides siamensis is a glossiphoniid leech with a short life cycle. In a laboratory setting, ten mature P. siamensis left their host (a turtle) after feeding for approximately three days and initiated copulation. The adults spent 3-4 days gestating before depositing eggs (272.8±62.9 eggs/clutch; range: 186-359 eggs/clutch). The eggs then changed from a creamy white to a creamy brownish color before hatching. Hatching occurred after incubation on the parent's ventral surface for 5-7 days. The transparent brood, with a single pair of red eyes, spent a couple of weeks under the venters of their parents. After this period, they left their parents and grew to maturity in 10-15 days; leeches were considered mature when their color was similar to that of their parents and they performed their first copulation. In addition, the mature leeches survived for 163 days on one feeding., Competing Interests: The authors have declared that no competing interests exist.

During spring 2018, we captured 101 spur-thighed tortoises, Testudo graeca, from seven localities in central Morocco. All tortoises were examined for the presence of blood parasites Hemolivia mauritanica and Hyalomma aegyptium ticks, the known vectors. We looked for H. mauritanica infection by examination of blood smears and by genetic analysis with PCR using extractions from both tortoises and ticks. On all tortoises collected, 71.29% were infested with at least one tick, with a mean infestation intensity of 7.12 ticks/tortoise and maximum of 15.55 ticks/tortoises in Had Draa locality (Essaouira region). Although the definitive host is present and abundant in all tortoise populations, all blood samples were Hemolivia-negative. Our results support and confirm the finding of studies previously conducted in other populations of Morocco and indicate that H. mauritanica has a narrower distribution range than its tick vector.

Polystomes (Monogenea: Polystomatidae) of freshwater turtles are currently represented by five genera, namely Neopolystoma, Polystomoides, Polystomoidella, Uropolystomoides and Uteropolystomoides. These parasites can infect the urinary, oral and/or the conjunctival sac systems of their hosts, showing strict site specificity. A recent phylogenetic study showed that the two most diverse genera within chelonian polystomes, i.e. Neopolystoma and Polystomoides, are not monophyletic. Furthermore, polystomes infecting the conjunctival sacs of their host, except for one species, formed a robust lineage. A fusiform egg shape has been reported for conjunctival sac polystomes and it was assumed that this characteristic could be a good character for the systematics of polystomes. Our objective in the present work was, therefore, to study more in depth the morphology of polystomes collected from the conjunctival sacs of chelonians to find characters defining a putative new genus. To achieve this objective, more specimens were collected in 2018 and 2019 from turtles sampled in North Carolina and Florida (USA) to extend taxon sampling for the phylogenetic analysis. Morphological characters of relevant polystome specimens were re-examined from several collections from Asia, Australia, Europe, South Africa, South America and North America. Based on a Bayesian tree inferred from the analysis of four concatenated genes, namely 12S, 18S, 28S and COI, polystomes found in the conjunctival sacs were grouped in three distinct lineages, the first one including a single species infecting an Australian pleurodire turtle; the second one including eleven species infecting cryptodire turtles of South America, North America and Asia; and the last one including a single species infecting a softshell cryptodire turtle of North America. Based on observations of live specimens by Dr. Sylvie Pichelin and our morphological analysis, the conjunctival sac polystomes from Australian turtles are small, cannot extend their body significantly, have a spherical ovary and egg, have a large genital bulb and possess latero-ventral vaginae at the level of the testis. Based on observations of live specimens and morphological analysis of whole mounted specimens, polystomes of the second lineage share the following morphological characteristics: the ability to stretch out and double their length, a long oval ovary, a separate egg-cell-maturation-chamber, fusiform to diamond-shaped eggs with acute tips, small genital bulb and vaginae peripheral on the side of the body at the level of the testis. The polystome species of the third lineage occupies a basal position, has the ability to stretch out and possess an elongated ovary, a large fusiform egg with rounded tips, a small genital bulb and small latero-ventral vaginae at the level of the ovary. These three distinct conjunctival sac polystome lineages are herein described as separate new genera, Aussietrema, Fornixtrema and Apaloneotrema, respectively.

Sea turtles are difficult to sample because of their protected status; however, museum collections and sea turtle stranding networks provide unique opportunities for parasitological research. Four gastrointestinal tracts from stranded, endangered green turtles, Chelonia mydas, were collected between 1993 and 1995 from the upper Texas coast and opportunistically sampled for parasite fauna. Two new species of Telorchis, a common freshwater amphibian and reptilian intestinal parasite genus, were found and described. Telorchis marinus n. sp. differs from Telorchis mydas n. sp. by its short body length, lack of pharyngeal glands, long esophagus relative to total body length, short and straight cirrus sac, short ventral sucker to ovary length relative to total body length, and an ovary located in the anterior one-third of body; it differs from its congeners in the number of ovary lengths between the ventral sucker and ovary, the number of ventral sucker lengths the cirrus sac extends beyond the posterior margin of the ventral sucker, and the vitelline field extent. Telorchis mydas differs from its congeners in the number of ovary lengths between the ventral sucker and ovary, the number of ventral sucker lengths the cirrus sac extends beyond the posterior margin of the ventral sucker, and the combination of having its ovary position near the midbody and a long, sinuous cirrus sac that is 35-44% of the total body length. Given the taxonomic complexities within Telorchis, a revised key to North American species is provided using morphological characteristics to assist future researchers in delineating true species and appropriate synonymies with molecular explorations. We reject the majority of synonymies in the genus until molecular data are available; we accept the synonymies of Telorchis necturi as Telorchis stunkardi and Telorchis gutturosi as Telorchis chelopi. Both Telorchis linstowi and Telorchis stossichi should be considered as species inquirenda. This is the first confirmed report of Telorchis from a marine host and the first report on parasites of cheloniid sea turtles in Texas, and this study adds to the ever-growing evidence that collections are essential to understanding biodiversity., (© American Society of Parasitologists 2020.)

Long-term monitoring of host-parasite interactions is important for understanding the consequences of infection on host fitness and population dynamics. In an eight-year survey of the loggerhead sea turtle (Caretta caretta) population nesting in Cabo Verde, we determined the spatiotemporal variation of Ozobranchus margoi, a sanguivorous leech best known as a vector for sea turtle fibropapilloma virus. We quantified O. margoi association with turtles' δ 15 N and δ 13 C stable isotopes to identify where infection occurs. We then measured the influence of infection on reproduction and offspring fitness. We found that parasite prevalence has increased from 10% of the population in 2010, to 33% in 2017. Stable isotope analysis of host skin samples suggests transmission occurs within the host's feeding grounds. Interestingly, we found a significant interaction between individual size and infection on the reproductive success of turtles. Specifically, small, infected females produced fewer offspring of poorer condition, while in contrast, large, infected turtles produced greater clutch sizes and larger offspring. We interpret this interaction as evidence, upon infection, for a size-dependent shift in reproductive strategy from bet hedging to terminal investment, altering population dynamics. This link between infection and reproduction underscores the importance of using long-term monitoring to quantify the impact of disease dynamics over time.

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.

Emys orbicularis (Linnaeus, 1758) was considered as a specific host of Placobdella costata (Fr. Mûller, 1846). However, since the parasite was recorded from outside the distribution area of its host, some authors suggested a possible relationship with other hosts. Although two accidental associations were found with another turtle, Mauremys leprosa (Schweigger, 1812), the obtained data remain insufficient to better understand this discovered host-parasite ecological system. In this context, the present study was carried out to evaluate the relationship between the Mediterranean pond turtle, M. leprosa, and the freshwater rhynchobdellid leech, P. costata (Hirudinida: Glossiphoniidae), in aquatic ecosystems of Morocco. During the period from April to June 2018, we found leeches attached to turtles in five out of 30 populations sampled with a prevalence of infection significantly higher in adult than that in juvenile turtles. Moreover, the males are the most infested with 51% of the total, followed by females (33.3%) and juveniles (15.7%). The obtained results indicated that 51 turtles were infested by 139 leeches with a mean intensity of infestation of 4.17 ± 0.47 leeches/turtle (up to 10 leeches/turtle). It was higher in males than that in females in almost all sites. The posterior limbs are the most preferred attachment site, and the body condition of turtles was not affected by the intensity of infestation but it is rather a function of altitude. Our findings proved that M. leprosa-P. costata association is more than accidental and that M. leprosa is rather the main host of P. costata in aquatic ecosystems of Morocco.