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.

DEPREDACION POR INSECTOS DE NIDOS Y NEONATOS PROTEGIDOS DE Podocnemis vogli (SABANAS INUNDABLES DEL CASANARE) / Prédation by insects of protected nests and hatchlings of Podocnemis vog/i (flooded savannas of Casanare)

RESUMEN La galápaga sabanera (Podocnemis vogli) es una tortuga de ambientes acuáticos, principalmente lénticos, que centra su época reproductiva en la temporada seca, en la que sus huevos son una importante fuente de alimento en la red trófica y para el hombre, de manera que frecuentemente son afectados por depredadores naturales. Con el propósito de reducir el efecto negativo de la depredación natural sobre las nidadas de P vogli en la Reserva Natural de la Sociedad Civil La Esperanza en Paz de Ariporo, se implementó una estrategia de protección directa con malla metálica y se realizó el seguimiento a las posturas durante tres periodos de anidación entre 2016 y 2018. Como resultado del uso de esta estrategia para proteger las nidadas, se evidenció el efecto de otros depredadores y parásitos menos conspicuos como las hormigas legionarias (Labidus) y las larvas de moscas parasitarias (Phoridae). En ambos casos la afectación se registró en los neonatos recién eclosionados de galápaga sabanera al interior de los nidos, algunos de ellos hallados en un estado de salud precaria o muertos. Este es el primer reporte de depredación de los neonatos de P. vogli por estas especies de insectos sobre las cuales las estrategias físicas convencionales de protección fueron ineficientes.

ABSTRACT The savanna side-necked turtle (Podocnemis vogli) is a river turtle of mainly lentic aquatic environments that centers its reproductive period in the dry season; its eggs are a significant source of nourishment in the food web that includes humans as well. Therefore, by monitoring and installing metal mesh we protected the nests during three spawning seasons (2016-2018). After using the mentioned protection strategy other possible predators or parasites, such as the insects, became evident. We observed that when monitoring clutches, organisms such as ants and parasitic flies took advantage of the newborn hatchlings inside the nests. In both cases, the affectations by both species caused considerable injuries in the neonates, which eventually showed a precarious state of health and even died rapidly. This is the first time that predation on P. vogli neonates by these insect species has been reported, in which physical protection strategies are not functional.

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.

Maturation Phenotype of Peripheral Blood Monocyte/Macrophage After Stimulation with Lipopolysaccharides in Irritable Bowel Syndrome.

BACKGROUND/AIMS: Abnormal immune regulation and increased intestinal permeability augmenting the passage of bacterial molecules that can activate immune cells, such as monocytes/macrophages, have been reported in irritable bowel syndrome (IBS). The aim was to compare the maturation phenotype of monocytes/macrophages (CD14+) from IBS patients and controls in the presence or absence of Escherichia coli lipopolysaccharides (LPS), in vitro. METHODS: Mononuclear cells were isolated from peripheral blood of 20 Rome II-IBS patients and 19 controls and cultured with or without LPS for 72 hours. The maturation phenotype was examined by flow cytometry as follows: M1-Early (CD11c+CD206-), M2-Advanced (CD11c-CD206+CX3CR1+); expression of membrane markers was reported as mean fluorescence intensity (MFI). The Mann-Whitney test was used and significance was set at P < 0.05. RESULTS: In CD14+ cells, CD11c expression decreased with vs without LPS both in IBS (MFI: 8766.0 ± 730.2 vs 12 920.0 ± 949.2, P < 0.001) and controls (8233.0 ± 613.9 vs 13 750.0 ± 743.3, P < 0.001). M1-Early cells without LPS, showed lower CD11c expression in IBS than controls (MFI: 11 540.0 ± 537.5 vs 13 860.0 ± 893.7, P = 0.040), while both groups showed less CD11c in response to LPS (P < 0.01). Furthermore, the percentage of "Intermediate" (CD11c+CD206+CX3CR1+) cells without LPS, was higher in IBS than controls (IBS = 9.5 ± 1.5% vs C = 4.9 ± 1.4%, P < 0.001). Finally, fractalkine receptor (CX3CR1) expression on M2-Advanced cells was increased when treated with LPS in controls but not in IBS (P < 0.001). CONCLUSIONS: The initial phase of monocyte/macrophage maturation appears to be more advanced in IBS compared to controls. However, the decreased CX3CR1 in patients with IBS, compared to controls, when stimulated with LPS suggests a state of immune activation in IBS.

Description of Leucocytozoon quynzae sp. nov. (Haemosporida, Leucocytozoidae) from hummingbirds, with remarks on distribution and possible vectors of leucocytozoids in South America.

We describe Leucocytozoon quynzae sp. nov. (Haemosporida, Leucocytozoidae), which is the first Leucocytozoon parasite identified to species level in hummingbirds. It was found in the Amethyst-throated Sunangel (Heliangelus amethysticollis, Trochilidae, Apodiformes) captured in the Palacio Forest, which belongs to the damping zone of Chingaza National Natural Park, Cundinamarca, Colombia, at 2,900 m above sea level where the transmission occurs; the new species were found both in the high Andean forest and Paramo ecosystem. This parasite is described based on the morphology of its blood stages, a fragment of the mitochondrial cytochrome b gene, and the complete mitochondrial genome. Illustrations of blood stages of the new species are given, and the phylogenetic analysis places this lineage in a well-supported clade with other lineages of unidentified to species level leucocytozoids reported in the Trochilidae birds elsewhere. The new species possess gametocytes in roundish host cells; it can be readily distinguished from other similar leucocytozoids, primarily due to (1) a comma-like shape of the host cell nucleus, which extended one half or less of the circumference of the gametocyte and (2) a large number of prominent volutin granules in the cytoplasm. Identical mitochondrial cytochrome b sequence of Leucocytozoon quynzae was found in different hummingbird species at the type locality and also was reported in one passerine bird at the highlands of Peru. Leucocytozoon quynzae is the first leucocytozoid parasite described from South American birds; its transmission occurs both at low temperatures and high elevations. We discuss some patterns of distribution of avian leucocytozoids in South America and the role of Gigantodax spp. (Diptera, Simuliidae) as potential vectors of Leucocytozoon parasites in the Andean Region.

From cytokines to toll-like receptors and beyond - current knowledge and future research needs in irritable bowel syndrome.

The irritable bowel syndrome (IBS) is a complex disorder in which psychosocial, cultural and biological factors, interact. Recent knowledge in the pathophysiology of IBS, seem to combine issues such as a low grade inflammation or immune activation and dysbiosis that can trigger or exacerbate IBS. On the other hand, stress mediated through the hypothalamic-pituitary-adrenal axis can produce motility abnormalities that can modify the microbiota as well, with the subsequent immune activation in the mucosa and stimulation of nerve terminals, generating symptoms of IBS. Also, we speculate that, stress, dysbiosis or an underlying genetic predisposition, may increase the epithelial permeability leading to a contact between pathogens-associated molecular patterns and toll-like receptors in the deeper layers of the gut, developing a host immunity response and IBS generation. We believe that the role of toll-like receptors in IBS and elucidating the communication processes between the immune and the nervous system, warrant future research.