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.

Leucocytozoon parasites remain poorly investigated in comparison to other haemosporidians. The host cell inhabited by their blood stages (gametocytes) remains insufficiently known. This study aimed to determine the blood cells inhabited by Leucocytozoon gametocytes in different species of Passeriformes and to test if this feature has a phylogenetic importance. We microscopically analyzed blood films stained with Giemsa from six different bird species and individuals and used PCR-based methods for parasite lineage identification. The DNA sequences obtained were applied for phylogenetic analysis. Leucocytozoon parasite from the song thrush Turdus philomelos (cytochrome b lineage STUR1), the blackbird Turdus merula (undetermined lineage), the garden warbler Sylvia borin (unknown lineage) inhabited erythrocytes, a parasite from the blue tit Cyanistes caeruleus (PARUS4) infects lymphocytes, while in the wood warbler Phylloscopus sibilatrix (WW6) and the common chiffchaff Phylloscopus collybita (AFR205) they were found inhabiting thrombocytes. Parasites infecting thrombocytes were closely related, while the parasites infecting erythrocytes were placed in three different clades, and the one found in lymphocytes was placed in a separate clade. This shows that the determination of host cells inhabited by Leucocytozoon parasites can be phylogenetically important and should be considered in future species descriptions. Noteworthy, phylogenetic analysis might be used for the prediction of which host cells parasite lineages might inhabit.

Amphibians and reptiles represent a considerable proportion of the vertebrate fauna in Brazil. Different blood parasitic infections have been reported in these groups, such as Haemogregarina, Hepatozoon, Trypanosoma and microfilariae. However, insufficient research on interactions between these parasites and their hosts has been carried out in some regions of the country. Samples were collected from populations of wild herpetofauna in different microhabitats throughout Brazil, totaling 111 samples of reptiles from the states of Mato Grosso and Pernambuco. We used an integrative approach, with classical microscopy, morphometry and molecular analysis, in order to identify hemoparasites present in the analyzed fauna. Genomic DNA was extracted for the PCR protocol based on the 18S ribosomal RNA gene for Hepatozoon spp. A total of 53 positives were obtained with molecular screening (47.7%), all confirmed as Hepatozoon spp. using DNA sequencing. Among positive samples, 23 slides were examined, confirming the presence of Hepatozoon spp. in 91.3% of the smears. The phylogenetic analysis performed with sequences from 43 samples resulted in a tree containing several distinct clades. Sequences were generally grouped according to the taxonomic order of the host. Co-infections with microfilariae and Trypanosoma spp. were also found in microscopy analyses. This study describes the presence of Hepatozoon caimani in a new host species (Paleosuchus palpebrosus) that can be a paratenic host in the natural environment. The existence of parasitic co-infections in alligator species underscores the significance of recognizing the impact of infections by various parasitic taxa on the host populations.

Leucocytozoon species are cosmopolitan and prevalent avian parasites, with some infections being lethal, mainly due to the exo-erythrocytic development of the parasite in bird tissues. The patterns of exo-erythrocytic development in Leucocytozoon spp. infections in wild birds remain poorly studied. This study investigated the development of Leucocytozoon spp. tissue stages in tits (Paridae). Great tits (Parus major), Blue tits (Cyanistes caeruleus), and Coal tits (Periparus ater) were screened for infections using an integrative approach that consisted of microscopic analysis of thin blood smears, histological techniques, chromogenic in situ hybridization (CISH), PCR-based methods, and phylogenetic analysis. In total, 41 individuals were analyzed (eight naturally infected that were selected and euthanized, and 33 found dead in the wild and opportunistically sampled). Among the naturally infected birds, all individuals that were microscopically positive for Leucocytozoon species were also PCR-positive for these parasites. Co-infections with Plasmodium spp. and Haemoproteus spp. were commonly found, mainly among the opportunistically sampled birds. Two morphotypes were identified, Leucocytozoon majoris (Laveran, 1902) and Leucocytozoon fringillinarum Woodcock, 1910. Tissue stages were present in three birds sampled exclusively during the non-breeding season, two of them with meronts developing in the kidneys and liver, and one individual with a megalomeront in the heart. All the exo-erythrocytic stages were confirmed to be Leucocytozoon spp. by CISH using a Leucocytozoon genus-specific probe. Phylogenetic analysis placed parasite lineages with different morphotypes in separate clades. The developmental patterns of exo-erythrocytic stages of Leucocytozoon spp. in naturally infected passerines are poorly understood, requiring further research.

Here we describe, in an unprecedented way for the Brazilian Amazon, one Apicomplexa species infecting an anuran in Pará, Brazil, using an integrative approach that includes taxonomy, providing morphology, morphometrics, and molecular data. Samples were collected between February 2021 and February 2022 in the community of Curupira, municipality of Santarém, and in the industrial area of Marabá, both in Pará state. Specimens of Rhinella marina and Rhinella diptycha were captured by hand during the active search, and blood samples were collected by cardiac puncture. One blood aliquot was used to prepare blood smears for microscopical analysis and another aliquot for further molecular analyses. Of the five R. marina caught, one (20%) presented parasites morphologically compatible with the parasites belonging to the genus Hepatozoon, while of the three R. diptycha caught, one (33.3%) presented parasites morphologically compatible with the parasites belong to the genera Lankesterella and Hemolivia. Based on molecular data of 18S rDNA sequences, the Hepatozoon sequence obtained clustered with other parasite species recovered from Brazilian amphibians. The Lankesterella sp. sequence was placed in a sister clade of Lankesterella species described in birds and close to L. minima, described in amphibians. All attempts to amplify Hemolivia parasite DNA were unsuccessful. We described Lankesterella oliviacatarinae n. sp. and reported infection by Hepatozoon sp. and Hemolivia sp. in bufonids anurans from the Brazilian Amazon. This study increases knowledge of Brazilian anuran hemoparasites and confirms the importance of using an integrative approach for the taxonomy of these parasite groups.