Morphometric and molecular characterization of an unpigmented haemosporidian parasite in the Neotropical turnip-tailed gecko (Thecadactylus rapicauda).

Morphological traits from blood stages have been the gold standard for determining haemosporidian parasite species. However, the status of some taxa and the value of such traits in parasites from reptiles remain contentious. The scarce sampling of these species worsens the situation, and several taxa lack molecular data. A survey was performed in the Magdalena Department in Colombia, where 16 species of reptiles were captured. A peculiar haemosporidian parasite was found in the Turnip-tailed gecko Thecadactylus rapicauda. This haemosporidian does not show malarial pigment in blood stages under light microscopy; thus, it fits the Garnia genus's characters belonging to the Garniidae. However, the phylogenetic analyses using a partial sequence of cytochrome b and the mitochondrial DNA placed it within the Plasmodium clade. Our findings suggest that many putative Garnia species belong to the genus Plasmodium, like the one reported here. This study either shows that visible malarial pigment in blood stages is not a diagnostic trait of the genus Plasmodium or malarial pigment might be present in an undetectable form under a light microscope. In any case, the current taxonomy of haemosporidian parasites in reptiles requires revision. This study highlights the importance of using molecular and morphological traits to address taxonomic questions at the species and genus levels in haemosporidian parasites from reptiles.

Plasmodium parasites in reptiles from the Colombia Orinoco-Amazon basin: a re-description of Plasmodium kentropyxi Lainson R, Landau I, Paperna I, 2001 and Plasmodium carmelinoi Lainson R, Franco CM, da Matta R, 2010.

Colombia is a megadiverse country with about 600 species of reptiles; however, there are few studies on species of hemoparasites found in this taxonomic group. Here, we document the presence of Plasmodium spp. in four species of reptiles from the northern part of the Orinoco-Amazon region in Colombia. Individuals analyzed in this study were captured in localities between 200 and 500 m altitude, in the department of Guaviare. Each sample was screened for haemosporidian parasites by using morphology and a nested polymerase chain reaction (PCR) protocol that targets the mitochondrial cytochrome b (cytb) gene. Four morphotypes of the genus Plasmodium were found; two of these species are re-described using morphological and molecular data (cytb). For the other two morphotypes, it was not possible to assign a described species. Among those, Plasmodium screened one species was only detected by microscopy. Considering the potential species diversity, it is possible that commonly used primers may not detect all species, reinforcing the importance of using microscopy in haematozoa surveys. There was no correspondence between the morphological traits associated with the subgenera and the phylogenetic relationships that we found in our analyses. Additionally, we found an expansion in the geographical distribution of these two species, and a new host for P. kentropyxi, demonstrating that studies of tropical herpetofauna and their parasites deserve more attention.

The genetic and morphological diversity of Haemogregarina infecting turtles in Colombia: Are mitochondrial markers useful as barcodes for these parasites?

Adeleorinid parasites commonly infect turtles and tortoises in nature. Currently, our knowledge about such parasites is extremely poor. Their characterization is based on morphological and molecular approaches using the 18S rDNA molecular marker. However, there is a limitation with the 18S rDNA due to its slow rate of evolution. For that reason, the goals of this study were to 1) design primers for new molecular mitochondrial markers to improve the phylogenetic reconstructions of adeleorinid parasites and 2) to determine the morphological and genetic diversity of Haemogregarina infecting turtles and tortoises in Colombia. Turtles from 16 species representing six families were examined for the presence of haemoparasites. We analyzed 457 samples using PCR, and 203 of them were also analyzed by microscopy. Using a mitochondrial genome of Haemogregarina sequenced in this study, we designed primers to amplify fragments of the cytochrome oxidase I (coxI), cytochrome oxidase III (coxIII), and cytochrome b (cytb) mitochondrial markers in adeleorinid parasites. Lineages obtained from nuclear and mitochondrial molecular markers clustered according to the turtle lineages from which they were isolated. It is noteworthy that we found different evolutionary lineages within the same morphotype, which may indicate heteroplasmy and/or cryptic diversity in Haemogregarina. Due to this situation, we could not make a species delimitation, even when integrating the different lines of evidence we had in this study. However, the primers presented here are useful for diagnosis and, moreover, according to the available information, all three genes retain phylogenetic signals; thereby fragments amplified can be used in reconstructing evolutionary relationships. This effort contributes to the knowledge of the diversity of these parasites infecting continental turtles from Colombia.

Haemocystidium spp., a species complex infecting ancient aquatic turtles of the family Podocnemididae: First report of these parasites in Podocnemis vogli from the Orinoquia.

The genus Haemocystidium was described in 1904 by Castellani and Willey. However, several studies considered it a synonym of the genera Plasmodium or Haemoproteus. Recently, molecular evidence has shown the existence of a monophyletic group that corresponds to the genus Haemocystidium. Here, we further explore the clade Haemocystidium spp. by studying parasites from Testudines. A total of 193 individuals belonging to six families of Testudines were analyzed. The samples were collected in five localities in Colombia: Casanare, Vichada, Arauca, Antioquia, and Córdoba. From each individual, a blood sample was taken for molecular analysis, and peripheral blood smears were made, which were fixed and subsequently stained with Giemsa. The prevalence of Haemocystidium spp. was 1.55% (n = 3/193); all infected individuals belonged to Podocnemis vogli (Savanna Side-necked turtle) from the department of Vichada. This is the first report of Haemocystidium spp. in Colombia and in this turtle species. The phylogenetic analysis of a mitochondrial cytb fragment revealed Haemocystidium spp. as a monophyletic group and as a sister taxon of Haemoproteus catharti and the genus Plasmodium. Haemocystidium spp. are difficult to identify by morphology only. As a result, it is possible that some of the taxa, such as Haemocystidium (Simondia) pacayae, represent a species complex. The parasite found in our study is morphologically indistinguishable from Haemocystidium (Simondia) pacayae reported in Peru. However, the new lineage found in P. vogli shows a genetic distance of 0.02 with Hae. pacayae and 0.04 with Hae. peltocephali. It is proposed that this divergent lineage might be a new species. Nevertheless, additional molecular markers and ecological features could support this hypothesis in the future.

Do blood parasites increase immature erythrocytes and mitosis in amphibians? / ¿Causan los parásitos sanguíneos un aumento en los eritrocitos inmaduros y la mitosis en los anfibios?

Abstract Introduction: In amphibians, blood may act as a hematopoietic tissue. However, the knowledge concerning hematological features is scarce, there is not much information that allows an analysis about the possible explanations of this physiological feature. Objective: This study aimed to evaluate the relationship between immature red blood cells (RBCs) mitosis and the presence of blood parasites in amphibians. Methods: We sampled 116 amphibians (31 species) in six Colombian localities. Blood was taken by cardiac puncture or maxillary vein puncture. Smears were prepared, fixed, and Giemsa stained for microscopical analysis. The variables analyzed were the percentage of immature RBCs, mitotic cells in peripheral blood, and blood parasite infection. Data were analyzed using Wilcoxon's rank test and exact Fisher statistical tests. Results: Sixty-two individuals showed mitosis in peripheral blood, and these mitotic RBCs shared morphological features with immature RBCs. Overall, parasite prevalence was 30.1 %, distributed as follows: Trypanosoma (24.1 %), Hepatozoon-like (6 %), Dactylosoma (4.3 %), Karyolysus-like (0.9 %), and Filarioidea (2.6 %). A positive association between the percentage of immature RBCs and the presence of mitotic RBCs was found, and also between the blood parasite infection and the percentage of immature RBCs. Conclusions: In this study, we found that the presence of blood parasites, immature RBCs, and RBCs mitosis are frequent events in amphibians' peripheral blood, and our analysis suggests an association between those features. Thus, the release of immature RBCs and the mitosis of those cells in peripheral blood may be a physiological response to blood parasite infection. Further studies characterizing hematology in amphibians and wildlife, in general, are desirable.

Resumen Introducción: En anfibios, la sangre puede actuar como un tejido hematopoyético. Sin embargo, el conocimiento acerca de las características hematológicas es escaso y no hay información que permita un análisis acerca de las posibles explicaciones a este rasgo fisiológico. Objetivo: La intención de este estudio fue evaluar la relación entre la presencia de eritroblastos, mitosis de glóbulos rojos (GRs) y la infección por hemoparásito en sangre periférica de anfibios. Métodos: Se muestrearon 116 anfibios (31 especies) en seis localidades de Colombia. Se tomaron muestras de sangre mediante punción cardiaca o punción a la vena maxilar. Se prepararon extendidos sanguíneos, se fijaron y tiñeron con Giemsa para su posterior análisis por microscopía. Se analizaron variables como porcentaje de GRs inmaduros, células mitóticas en sangre periférica e infección por hemoparásitos. Los datos fueron analizados mediante el test de rango de Willcoxon y el test exacto de Fisher. Resultados: sesenta y dos individuos evidenciaron mitosis en sangre periférica y dichas mitosis compartían características morfológicas con GRs inmaduros. La prevalencia general de parásitos fue del 30.1 %, distribuido de la siguiente forma: Trypanosoma (24.1 %), Hepatozoon-like (6 %), Dactylosoma (4.3 %), Karyolysus-like (0.9 %), y Filarioidea (2. 6 %). Hay una asociación positiva entre el porcentaje de GRs inmaduros y la presencia de células mitóticas, también se encontró una relación entre la infección por hemoparásitos y el porcentaje de GRs inmaduros. Conclusiones: En este estudio encontramos que la presencia de parásitos sanguíneos, GRs inmaduros y mitosis de GRs son eventos frecuentes en sangre periférica de anfibios, y nuestros resultados sugieren una asociación entre dichas características. Por tanto, la liberación de GRs inmaduros y la mitosis de estas células en sangre periférica podría ser una respuesta fisiológica a infecciones parasitarias. Posteriores estudios que caractericen la hematología en anfibios y en vida silvestre en general, son deseables.