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The knowledge of the diversity and geographic distribution of parasite species is the first step towards understanding processes of global epidemiology and species conservation. Despite recent increases in research on haemosporidian and haemogregarine parasites of reptiles and amphibians, we still know little about their diversity and parasite-host interactions, especially in the Iberian Peninsula, where a few studies have been conducted. In this study, the haemosporidian and haemogregarine diversity and phylogenetic relationships of the parasites in southwestern Iberian amphibians and reptiles were assessed using PCR approaches on blood samples of 145 individuals from five amphibian and 13 reptile species. The amphibians did not present any of both groups of parasites studied. Regarding reptiles, five Hepatozoon , one Haemogregarina , and one Haemocystidum haplotypes were found infecting four different species, revealing new host records for these parasites. Among them, we found one new Haemocystidium haplotype and three new and a previously reported Hepatozoon haplotype from a north African snake. The latter finding suggests that some Hepatozoon parasites may not be host-specific and have large geographic ranges even crossing geographical barriers. These results increased the knowledge about the geographic distribution and the number of known host species of some reptile apicomplexan parasites, highlighting the great unexplored diversity of them in this region.

The eight-year study (2015–2023) was performed on a large sample of poached European pond turtles infected with Haemogregarina stepanowi and held in a pond that belongs to a quarantine section of Belgrade Zoo. The protected species of European pond turtles have been found in poor health, with general weakness, anorexia, and low motility. Comprehensive cytological, hematological, molecular, and postmortem evaluations have been performed. Initially, Diff Quick staining of the blood smears revealed rounded or elongated erythrocytes, often bearing premeront or U-shaped gamont of the hemogregarines inside. The reduced erythrocyte numbers, hemoglobin, and hematocrit values found in the examined population of infected turtles indicated anemia. Macroscopically, shell necrosis and massive skin hemorrhages were the most prominent findings observed in diseased turtles. Microscopically, the lungs, liver, kidneys, and spleen revealed hyperemia, hemorrhages, and the presence of parasitic stages in tissue samples in 31 of 40 necropsied turtles. Cytological and microscopic examination of the samples proved to be sufficient for establishing the infection, but molecular analyses of the 18S sequence were used for phylogenetic studies. Over the years, the number of diseased and dead turtles has decreased, which could be hypothetically attributed to the elimination of leeches as the definitive host.

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.

In parasite–host interactions host species may differ in their ability to fight parasitic infections, while other ecological interactions, including competition, may differentially alter their physiological state, making them even more susceptible to parasites. In this study, we analyse the haemogregarine blood parasites infecting two competing lizard species, Iberolacerta horvathi and Podarcis muralis, and explore host–parasite relationships under different host competition scenarios. Both species were infected with haemogregarine parasites belonging to the genus Karyolysus. Using the 18S rRNA gene, six new Karyolysus haplotypes were identified clustering with other Central and Eastern European samples, and widely shared between both lizard hosts. Haemogregarine infections were detected at all sampled sites with over 50% of individuals parasitized. Overall, I. horvathi was more frequently and also more intensely parasitized than P. muralis, with higher infection rates observed in syntopy. Males of both species tended to be more frequently infected and showed a higher infection intensity than conspecific females. The results suggest that parasitisation by haemogregarines may be relevant in the dynamics of the competitive relationship between these lizard species. More studies, including immunological response analysis, and the identification of the vectors are needed to better understand host–parasite relationships and competition.

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.

Haemogregarines (Apicomplexa: Adeleiorina) are a diverse group of haemoparasites reported from almost all vertebrate classes. The most commonly recorded haemogregarines to parasitize anurans are species of Hepatozoon Miller, 1908. To date 16 Hepatozoon species have been described from anurans in Africa, with only a single species, Hepatozoon hyperolli (Hoare, 1932), infecting a member of the Hyperoliidae. Furthermore, only two Hepatozoon species are known from South African anurans, namely Hepatozoon theileri (Laveran, 1905) and Hepatozoon ixoxo Netherlands, Cook and Smit, 2014, from Amietia delalandii (syn. Amietia quecketti) and three Sclerophrys species, respectively. Blood samples were collected from a total of 225 individuals representing nine hyperoliid species from several localities throughout northern KwaZulu-Natal, South Africa. Twenty frogs from three species were found positive for haemogregarines, namely Afrixalus fornasinii (6/14), Hyperolius argus (2/39), and Hyperolius marmoratus (12/74). Based on morphological characteristics, morphometrics and molecular findings three new haemogregarine species, Hepatozoon involucrum Netherlands, Cook and Smit n. sp., Hepatozoon tenuis Netherlands, Cook and Smit n. sp. and Hepatozoon thori Netherlands, Cook and Smit n. sp., are described from hyperoliid hosts. Furthermore, molecular analyses show anuran Hepatozoon species to be a separate monophyletic group, with species isolated from African hosts forming a monophyletic clade within this cluster.

Apicomplexa is a phylum that includes all parasitic protozoa sharing unique ultrastructural features. Haemogregarines are sophisticated apicomplexan blood parasites with an obligatory heteroxenous life cycle and haplohomophasic alternation of generations. Haemogregarines are common blood parasites of fish, amphibians, lizards, snakes, turtles, tortoises, crocodilians, birds, and mammals. Haemogregarine ultrastructure has been so far examined only for stages from the vertebrate host. PCR-based assays and the sequencing of the 18S rRNA gene are helpful methods to further characterize this parasite group. The proper classification for the haemogregarine complex is available with the criteria of generic and unique diagnosis of these parasites.

Apicomplexan haemoparasites of the genera Schellackia Reichenow, 1919, and Karyolysus Labbé, 1894, seem to be common in lizards and widespread across the world. For decades, their identification has been based on morphological descriptions and life cycle patterns, with molecular characterizations, applied only recently. We used molecular characterization to confirm the identification of haemoparasites detected by microscopy in blood smears of Lacerta schreiberi Bedriaga, 1878, a lizard of the Iberian Peninsula. Since blood samples other than blood smears were not available from the studied lizards, 264 engorged ticks Ixodes ricinus (Linneaus, 1758) collected from them were used as an alternative non-invasive source of haemoparasite DNA for molecular genetic analyses. Of the 48 blood smears microscopically examined, 31 were positive for blood parasites (64.6% prevalence). We identified trophozoites and gamonts similar to Karyolysus lacazei (Labbé, 1894) (24/48; 50%) and Schellackia-like sporozoites (20/48; 41.7%). Mixed infections with both species occurred in 13 blood smears (27.1%). Sequence data were obtained for both parasites from engorged ticks. Phylogenetic analyses placed our unique haemogregarine sequence within the Karyolysus clade, nevertheless, within substantial polytomy. Thus, according to its morphology and effect on the host cell, we refer to this haemogregarine as Karyolysus cf. lacazei. Besides the Schellackia sequences being identical to a previously identified haplotype, we also obtained sequences of three new closely related haplotypes.

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.). Graphical Abstract

Oceanic islands are hotspots of biodiversity due to their high levels of endemism, with the Canary Islands being a notable example. A previous molecular study on the biogeography and host associations of haemogregarines (Apicomplexa: Adeleorina) infecting lizards from this archipelago detected seven parasite haplogroups. These haplogroups exhibited high host-specificity and geographical structure, suggesting that they might correspond to distinct biological identities. In this study, along with sequencing a longer fragment of the 18S rRNA, we further explore the distinctiveness of these parasites by analysing their morphology, effects on host erythrocytes and parasitaemia levels. These lines of evidence together with their genetics, host associations, frequency of occurrence and geographical distribution support them as different biological entities. As such, we describe seven new species: Karyolysus canariensis sp. nov., Karyolysus galloti sp. nov., Karyolysus stehlini sp. nov., Karyolysus gomerensis sp. nov., Karyolysus atlanticus sp. nov., Karyolysus tinerfensis sp. nov. and Karyolysus makariogeckonis sp. nov. These new taxa are further examples of endemic diversity in the Canarian archipelago. They also contribute to clarify the taxonomy within the Apicomplexa, a phylum estimated to have one of the lowest percentages of described species.