Finding a needle in a haystack: DNA Haemoproteus columbae enrichment using percoll density gradient and flow cytometry.

Isolation of genomic DNA of blood parasites in birds, reptiles, amphibians, and fishes is a challenging task, given that their red blood cells are nucleated; for that reason, parasite genomic DNA is only a fraction of the total extracted DNA, and it is challenging to obtain concentrated high-quality genetic material. Percoll Density Gradient (PDG) and flow cytometry are tools for separating and analyzing cell populations or even a single cell, and both represent potent approaches for isolating avian haemosporidians parasites. Our experimental design included several steps seeking to concentrate the parasite´s DNA. We used blood samples from a Rock pigeon infected with Haemoproteus columbae. After inducing parasite exflagellation and gametogenesis in vitro, we subjected the samples to a Percoll Density Gradient to separate the parasites from the rest of the blood cells. Following centrifugation, the layer containing extracellular parasites underwent a flow cytometry and cell sorting process, during which we selected two different subpopulations of cells for analysis. Based on qPCR analyses, we demonstrate parasite DNA enrichment in Percoll Density Gradient and flow cytometry samples; simultaneously, these samples showed the lowest concentration of Columba livia DNA. However, the concentration of parasite DNA was higher in the PDG than in the cell sorting sample. This study reports the concentration of the Haemoproteus parasite by flow cytometry without DNA-intercalating dyes, and this methodology can serve as a technique for DNA enrichment of blood parasites infecting nucleated red blood cells to improve techniques that allow obtaining complete genomes.

Hemoparasite occurrence and hematological/serum chemistry variations in Podocnemis vogli turtles: A comparative analysis between wild-residing infected specimens and captive non-infected counterparts.

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.

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.

Indigenous ecological calendars and seasonal vector-borne diseases in the Colombian Amazon: an intercultural and interdisciplinary approach / Calendarios ecológicos indígenas y enfermedades estacionales transmitidas por vectores en la Amazonia colombiana: un enfoque intercultural e interdisciplinario

Traditional ecological knowledge of indigenous groups in the southeastern Colombian Amazon coincides in identifying the two main hydrological transition periods (wet-dry: August-November; dry-wet: March-April) as those with greater susceptibility to disease in humans. Here we analyze the association between indigenous knowledge about these two periods and the incidence of two vector-borne diseases: malaria and dengue. We researched seven "ecological calendars" from three regions in the Colombian Amazon, malaria and dengue cases reported from 2007 to 2019 by the Colombian National Institute of Health, and daily temperature and precipitation data from eight meteorological stations in the region from 1990-2019 (a climatological normal). Malaria and dengue follow a seasonal pattern: malaria has a peak from August to November, corresponding with the wet-dry transition (the "season of the worms" in the indigenous calendars), and dengue has a peak in March and April, coinciding with the dry-wet transition. Previous studies have shown a positive correlation between rainfall and dengue and a negative correlation between rainfall and malaria. However, as the indigenous ecological knowledge codified in the calendars suggests, disease prediction cannot be reduced to a linear correlation with a single environmental variable. Our data show that two major aspects of the indigenous calendars (the time of friaje as a critical marker of the year and the hydrological transition periods as periods of greater susceptibility to diseases) are supported by meteorological data and by the available information about the incidence of malaria and dengue.(AU)

Los conocimientos ecológicos tradicionales de grupos indígenas del sureste de la Amazonia colombiana coinciden en identificar dos principales periodos de transición hidrológica (seco-húmedo: agosto-noviembre; húmedo-seco: marzo-abril) como los de mayor susceptibilidad a enfermedades en humanos. Aquí analizamos la asociación entre el conocimiento indígena sobre estos dos periodos y la incidencia de dos enfermedades transmitidas por vectores: malaria y dengue. Investigamos siete calendarios ecológicos de tres regiones en la Amazonia colombiana, casos de dengue y malaria reportados de 2007 hasta 2019 por el Instituto Nacional de Salud de Colombia y datos diarios de temperatura y precipitación de ocho estaciones meteorológicas en la región, de 1990 a 2019 (una normal climatológica). Malaria y dengue siguen un patrón estacional, la malaria tiene un pico de agosto a noviembre, correspondiendo con la transición húmedo-seco (el "tiempo de gusano" según los calendarios indígenas), mientras que dengue tiene un pico de marzo a abril, coincidiendo con la transición seco-húmedo. Estudios previos mostraron una correlación positiva entre precipitación y dengue, y una correlación negativa entre precipitación y malaria. Sin embargo, como lo sugiere el conocimiento ecológico codificado en los calendarios indígenas, la predicción de enfermedades no puede reducirse a una correlación lineal con una sola variable medioambiental. Nuestros datos muestran que dos aspectos principales de los calendarios indígenas (el tiempo de friaje como un marcador crítico anual y los periodos de transición hidrológica como épocas de mayor susceptibilidad a enfermedades) están soportados por datos meteorológicos e información disponible acerca de la incidencia de malaria y dengue.(AU)

Haemosporidian parasites in the ash-breasted Sierra finch (Geospizopsis plebejus): insights from an Andean dry forest population.

Haemosporidian genera Plasmodium, Haemoproteus and Leucocytozoon, responsible for avian malarial infections, are highly diverse and have a wide range of health effects and predictors, depending on the host and its environmental context. Here, we present, for the first time, detailed information on the identity, prevalence and parasitaemia of haemosporidians and other haemoparasites that infect the ash-breasted Sierra finch, Geospizopsis plebejus, in an Andean dry forest. We study the consequences of infection in the host body and health conditions and explore the environmental and intrinsic factors that influence infection status and parasitaemia. We conducted diagnoses by cytochrome b (cytb) sequencing and morphological identification, and estimated the levels of parasitaemia based on microscopy. We identified 6 cytb lineages infecting G. plebejus. Two of them were new lineages: Haemoproteus sp. GEPLE01 and GEPLE02. We also detected Haemoproteus sp. ZOCAP08, Haemoproteus sp. AMAVIR01, Plasmodium homopolare BAEBIC02 and Plasmodium cathemerium ZONCAP15. By microscopy, we detected Haemoproteus coatneyi, Haemoproteus erythrogravidus, P. homopolare and other unidentified species of Haemoproteus, Plasmodium, Babesia sp. and 1 microfilaria. We found no evidence of Leucocytozoon. Additionally, we detected several coinfections by sequencing and microscopy. The prevalence of haemosporidian infections was high (87.7%), and the mean parasitaemia was 61.65 infected cells per 10 000 erythrocytes examined. Prevalence and parasitaemia were higher for Haemoproteus than for Plasmodium. Haemoproteus sp. AMAVIR01 showed the highest prevalence (43.1%) and mean parasitaemia (94.39/10 000 erythrocytes) and might be associated with H. coatneyi. Immature individuals showed a lower prevalence than adults, supporting previous findings.

Hematological and blood chemistry parameters of a Podocnemis vogli and P. unifilis captive population in Colombia.

The Podocnemididae family is seriously affected by anthropogenic factors, which is why almost all of their family members are threatened, according to the IUCN red list. The biology and ecology of these species, as well as the hematological and serum chemistry reference intervals that allow clinical action and decision-making conservation programs, are poorly known. Based on this, the objective of this study was to establish the hematological and blood chemistry parameters of the Savannah side-necked turtle (Podocnemis vogli) and Yellow-spotted river turtle (Podocnemis unifilis) maintained in captivity at the Estación de Biología Tropical Roberto Franco (Villavicencio-Colombia). Forty-nine captive turtles of the species P. vogli (n = 28) and P. unifilis (n = 21) were sampled to determine hematological and serum chemistry parameters. Blood samples were taken from the jugular veins of both male and female turtles across both species. Student's t-test and Mann-Whitney-Wilcoxon tests were used to compare values between the parameters evaluated against genders and sizes. Reference intervals were calculated for the hematological and biochemical values of each species. Some assessed parameters demonstrated significant differences between the males and females of both species. Most of the analyzed parameters exhibited similar reference intervals in both species. In this study, we report values and propose the hematological and serum chemistry reference intervals for P. vogli and P. unifilis, which can be used in the clinical diagnosis of these reptiles and in future research.

Is Haemoproteus gabaldoni a valid species? An approach from morphology and molecular tools applied to parasites of Anseriformes.

Currently, there are three recognized species of haemoproteids infecting Anseriformes: Haemoproteus nettionis, H. macrovacuolatus, and H. greineri. Unfortunately, genetic information associated with a morphotype is available only for H. macrovacuolatus. We recently found a parasite morphologically compatible with Haemoproteus gabaldoni, a species Bennet (1993) described in a Cairina moschata (Muscovy duck) from Venezuela. This species was synonymized to H. nettionis by Valkiunas (2005), arguing not enough morphological differentiation between them; it was said that H. greineri could be as well a synonym of H. nettionis. In this study, we aimed to provide evidence to determine if Haemoproteus gabaldoni is a different species of H. nettionis and help to clarify other species status. We first performed morphological and morphometrical analyses and compared this information against the parahapantotypes of H. greineri, H. gabaldoni and material diagnosed as H. nettionis provided by the International Reference center for Avian Haematozoa (IRCAH), and H. macrovacuolatus from the Host-Parasite Relationship Study Group (GERPH, in Spanish Grupo de Estudio Relación Parásito Hospedero) biological collection. We used Principal Component Analysis (PCA) of dimensionless standard morphometrical variables from gametocytes. Furthermore, we amplified a small fragment of cytochrome b (cyt b) to compare the sequence with information in GenBank and Malavi through phylogenetic analyses and haplotype networks. PCA analyses revealed the presence of three distinct groups in the samples studied, supported in the morphological traits of each parasite species analyzed; phylogenetic analyses grouped parasite lineages separately according to the host and continent of provenance. Such results indicate that, H. gabaldoni, is a different species from H. nettionis. One more time, it is demonstrated the importance of linking barcode surveys to morphological studies. Finally, it is highlighted the importance of biological collections as repositories of worldwide biodiversity.

Hematology, Biochemistry Reference Intervals, and Morphological Description of Peripheral Blood Cells for a Captive Population of Crocodylus intermedius in Colombia.

The Orinoco crocodile (Crocodylus intermedius, Graves, 1918) is the most threatened crocodilian of South America. There is only scarce information available about the physiology of this neotropical crocodile. This study aimed to propose baseline hematological and biochemistry reference data and intervals and a morphological description of the peripheral blood cells of captive C. intermedius. Blood was collected from 318 clinically healthy individuals maintained in captivity at Villavicencio, Colombia. Eight of these individuals were sampled and resampled, and these data were compared. Reference intervals were proposed for hematological values [packed cell volume (PCV), red blood cell count, white blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin, and white blood cell count differential counts] and biochemistries [total solids, alanine aminotransferase (ALT), aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatine kinase, glucose, albumin, cholesterol, uric acid, creatinine, and lactate] including adults and juveniles, males and females' crocodiles. Blood cell morphology for the species is described. Significant differences between sex and age were observed. The intraindividual analysis concluded differences for total solids (P ≤ 0.01) and red blood cell counts (P ≤ 0.01). Some biochemical analytes showed a moderate correlation between them, such as ALT-alkaline phosphatase and ALT-uric acid. We present here novel and baseline data with special importance for the clinical diagnosis, improving the national reintroduction programs from either in situ and ex situ populations.

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.

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.

The apicoplast of Haemoproteus columbae: A comparative study of this organelle genome in Haemosporida.

Apicomplexa is a phylum of parasitic protozoa; among them are the order Haemosporida, vector-borne parasites that include those that cause malaria (genus Plasmodium). Most Apicomplexa species have a non-photosynthetic plastid or apicoplast. Given its unique metabolic pathways, this organelle is considered a target for malaria therapeutics. Regardless of its importance, there is a paucity of complete apicoplast genome data hindering comparative studies. Here, the Haemoproteus (Haemoproteus) columbae apicoplast genome (lineage HAECOL1) was obtained using next-generation sequencing. This genome was included in a comparative analysis with other plastids. This 29.8 kb circular genome shares the same structure found in Plasmodium parasites. It is A + T rich (87.7%), comparable but at the higher end of A + T content observed in Plasmodium species (85.5-87.2%). As expected, considering its high A + T content, the synonymous codon usage (RSCU) and the effective number of codons (ENc) showed a moderate codon bias. Several apicoplast genes have a phylogenetic signal. However, unlike mitochondrial genes, single-gene phylogenies have low support in haemosporidian clades that diverged recently. The H. columbae apicoplast genome suggests that the apicoplast function may be conserved across Haemosporida. This parasite could be a model to study this organelle in a non-mammalian system.

Molecular and morphological description of the first Hepatozoon (Apicomplexa: Hepatozoidae) species infecting a neotropical turtle, with an approach to its phylogenetic relationships.

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.

A phylogenetic study of Haemocystidium parasites and other Haemosporida using complete mitochondrial genome sequences.

Haemosporida are diverse vector-borne parasites associated with terrestrial vertebrates. Driven by the interest in species causing malaria (genus Plasmodium), the diversity of avian and mammalian haemosporidian species has been extensively studied, relying mostly on mitochondrial genes, particularly cytochrome b. However, parasites from reptiles have been neglected in biodiversity surveys. Reptilian haemosporidian parasites include Haemocystidium, a genus that shares morphological features with Plasmodium and Haemoproteus. Here, the first complete Haemocystidium mitochondrial DNA (mtDNA) genomes are studied. In particular, three mtDNA genomes from Haemocystidium spp. sampled in Africa, Oceania, and South America, are described. The Haemocystidium mtDNA genomes showed a high A + T content and a gene organization, including an extreme fragmentation of the rRNAs, found in other Haemosporida. These Haemocystidium mtDNA genomes were incorporated in phylogenetic and molecular clock analyses together with a representative sample of haemosporidian parasites from birds, mammals, and reptiles. The recovered phylogeny supported Haemocystidium as a monophyletic group apart from Plasmodium and other Haemosporida. Both the phylogenetic and molecular clock analyses yielded results consistent with a scenario in which haemosporidian parasites radiated with modern birds. Haemocystidium, like mammalian parasite clades, seems to originate from host switches by avian Haemosporida that allowed for the colonization of new vertebrate hosts. This hypothesis can be tested by investigating additional parasite species from all vertebrate hosts, particularly from reptiles. The mtDNA genomes reported here provide baseline data that can be used to scale up studies in haemosporidian parasites of reptiles using barcode approaches.

Accediendo al pasado: uso de especímenes de colección como fuentes de información genética para el género Bombus (Hymenoptera: Apidae)

Introducción: Recientemente ha tomado relevancia el uso de especímenes de museo como fuente de información genética para desarrollar estudios que resuelven preguntas taxonómicas, ecológicas, demográficas y evolutivas a diversas escalas temporales y geográficas. Sin embargo, material genético obtenido a partir de ejemplares depositados en colecciones biológicas es poco usado, debido al deterioro natural del ADN preservado en dichos ejemplares, de manera que la obtención de material genético de calidad es demandante en términos de tiempo y dinero. Objetivo: Usando material de museo, identificar una secuencia mini-barcode que pueda ser empleada en la determinación taxonómica, y que a su vez suministre información que permita la estimación de relaciones filogenéticas de especies del género Bombus. Métodos: Se estandarizó el protocolo de extracción de ADN a partir de la extremidad mesotoracica derecha y/o una muestra de músculo torácico de 96 especímenes depositados en la colección LABUN entre 7 y 38 años atrás. Las diferentes combinaciones de oligonucleótidos evaluadas permitieron amplificar fragmentos de 152 a 407 pares de bases (pb) del gen mitocondrial Cytochrome Oxidase I (COI). Usando como plantilla un grupo de 31 secuencias amplificadas a partir de especímenes recolectados recientemente, los fragmentos obtenidos de los especímenes del museo fueron ensamblados y analizados en un marco filogenético. Además, se realizó un análisis de red de haplotipos para evaluar en detalle las relaciones entre los haplotipos mitocondriales resultantes. Resultados: Se determinó un mayor éxito de extracción de ADN a partir de muestras de extremidad depositadas a partir del año 1982.Entretanto, la amplificación exitosa de fragmentos de más de 300 pares de bases (pb) se logró principalmente en muestras depositadas en fechas posteriores a 1999, lo que indica una mayor integridad del material genético recuperado de individuos de 19 años de recolección en adelante. Aunque todos los fragmentos evaluados pueden ser empleados como mini-barcode, solo con uno se obtiene una topología similar a la observada con el fragmento completo. Se detectó una gran variacion genética, particularmente al interior de las especies Bombus atratus y B. funebris, en las que se reveló una clara estructura filogeográfica. Conclusiones: Se obtuvieron nuevas secuencias de códigos de barras mediante extracción de ADN y protocolo de amplificación de muestras de museos. Además, se generó nueva información sobre la variabilidad genética intraespecífica, detectando la presencia de haplotipos mitocondriales únicos que podrían constituir Unidades Significativas Evolutivas sujetas a conservación. Dicha información es de vital importancia para formular estrategias de conservación para estos polinizadores en Colombia.

Introduction: The use of museum specimens as a source of genetic information to develop studies that resolve taxonomic, ecological, demographic, and evolutionary questions at various temporal and geographic scales, has recently become relevant. However, genetic material obtained from specimens deposited in biological collections is not used frequently due to the natural deterioration of the DNA preserved in these specimens. Getting quality genetic material is demanding in terms of time and money. Objective: By using museum material,to identify a mini-barcode sequence that can be used in the taxonomic determination and provides information that allows the estimation of phylogenetic relationships of species of the genus Bombus. Methods: The DNA extraction protocol for museum samples was standardized using the mesothoracic right leg and / or a sample of thoracic muscle of 96 specimens deposited in the LABUN collection between 7 and 38 years ago. Different combinations of oligonucleotides allowed to amplify fragments from 152 to 407 base pairs (bp) of the mitochondrial gene Cytochrome Oxidase I (COI). Using as a template a group of 31 sequences amplified from recently collected specimens, the fragments obtained from the museum specimens were assembled and analyzed in a phylogenetic framework. Additionally, a haplotype network analysis was performed in order to evaluate in detail the relationships between the resulting mitochondrial haplotypes. Results: The greatest success of DNA extraction was achieved from limb samples deposited since the year 1982 on. Meanwhile, successful amplification of fragments longer than 300 base pairs (bp) was achieved mostly in samples deposited on dates after 1999, which indicates greater integrity of the genetic material recovered from individuals of 19 years of collection and onwards. Although all the fragments evaluated can be used as mini-barcode, only with one primer pair, it was possible to obtain a topology similar to that observed with the complete fragment. A large genetic variation was detected, particularly within the Bombus atratus and B. funebris species, in which a clear phylogeographic structure was revealed. Conclusions: New barcode sequences were obtained through DNA extraction and amplification protocol from museum samples. Furthermore, new information on intraspecific genetic variability was generated, detecting the presence of unique mitochondrial haplotypes that could constitute management units subject of conservation. Such information is of vital importance to formulate conservation strategies for these pollinators in 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.

Experimental characterization of the complete life cycle of Haemoproteus columbae, with a description of a natural host-parasite system used to study this infection.

Characterization of complete life cycles of haemoparasites requires the maintenance of suitable susceptible vertebrate hosts and vectors for long periods in captivity, in order to follow the complete parasitic cycle in definitive and intermediate hosts. Currently, there are few host-parasite models established in avian haemosporidian research, and those have been developed mainly for species of Passeriformes and their parasites. This study aimed to develop an experimental methodology to access the complete life cycle of Haemoproteus columbae (cytb lineage HAECOL1), which parasitizes the Rock Pigeon (Columba livia) and louse fly (Pseudolynchia canariensis). A colony of louse flies, which are the natural vectors of this parasite, was established. Thirty newly emerged insects were exposed to H. columbae infection and used to infect naïve Rock Pigeons. The peak of parasitaemia (acute stage) was seen between 27 and 32 days p.i. when up to 70.8% of red blood cells were infected. The crisis occurred approximately 1 week after the peak, and the long-lasting chronic parasitaemia stage followed. Exo-erythrocytic meronts were seen mainly in the lungs where extensive tissue damage was reported, but also in the kidneys and spleen. In the vector, the sporogonic cycle of H. columbae was completed between 13 and 16 days p.i., at an average temperature ranging between 12 and 15 °C. This host-parasite model is tractable for maintenance in captivity. It is recommended for use in studies aiming for detailed characterization of host-parasite relationships in areas such as physiology, pathology, immunobiology, genetics, as well as for evaluative treatments and to follow the infection in any stage of parasite development both in the vertebrate or invertebrate host.

Disentangling Leucocytozoon parasite diversity in the neotropics: Descriptions of two new species and shortcomings of molecular diagnostics for leucocytozoids.

Avian communities from South America harbor an extraordinary diversity of Leucocytozoon species (Haemosporida, Leucocytozoidae). Here, of 890 birds sampled, 10 (1.2%) were infected with Leucocytozoon parasites. Among them, two new species were discovered and described. Leucocytozoon grallariae sp. nov. and Leucocytozoon neotropicalis sp. nov. were found in non-migratory highland passeriforms belonging to the Grallaridae and Cotingidae, respectively. They both possess gametocytes in fusiform host cells. However, due to combining microscopic examination and molecular detection, it was revealed that these parasites were present in co-infections with other Leucocytozoon species, which gametocytes develop in roundish host cells, therefore exhibiting two highly distant parasite lineages isolated from the same samples. Remarkably, the lineages obtained by cloning the mtDNA genomes were not captured by the classic nested PCR, which amplifies a short fragment of cytochrome b gene. Phylogenetic analyses revealed that the lineages obtained by the classic nested PCR clustered with parasites possessing gametocytes in roundish host cells, while the lineages obtained by the mtDNA genome PCR protocol were closely related to Leucocytozoon parasites possessing gametocytes in fusiform host cells. These findings suggest problems with the sensitivity of the molecular protocols commonly used to detect Leucocytozoon species. A detailed analysis of the primers used in the classic nested PCR revealed a match with DNA sequences from those parasites that possess gametocytes in roundish host cells (i.e., Leucocytozoon fringillinarum), while they differ with the orthologous regions in the mtDNA genomes isolated from the samples containing the two new species. Since these are mixed infections, none of the lineages detected in this study can be assigned accurately to the new Leucocytozoon morphospecies that develops in fusiform host cells. However, phylogenetic analyses allowed us to hypothesize their most probable associations. This study highlights the need for developing detection methods to assess the diversity of Leucocytozoon parasites accurately.

Blood parasites infecting the Hoatzin (Opisthocomus hoazin), a unique neotropical folivorous bird.

The Hoatzin (Opisthocomus hoazin) is the only extant member of the order Opisthocomiformes. This unique South American bird lives in the riparian lowland vegetation characteristic of the Amazon and Orinoco basins. Hoatzins nest in communal social units close to water bodies; they are strictly folivores being the only bird with pregastric fermentation in the crop. Because of the complex logistics involved in capturing this bird, there is a knowledge gap on its parasites. This study documents two distant lineages of haemosporidian parasites (Plasmodium spp.) in a juvenile and two adults sampled in the Cojedes state, Venezuela. Although negative by microscopy, the parasite identification was possible by using molecular methods. We estimated the phylogenetic relationships on the parasite cytochrome b (cytb, 480 bp) gene and the mitochondrial DNA. We found one of the parasites lineages in two individuals (nestling and adult), and the corresponding fragment of cytb was identical to a one found in Wood Stork (Mycteria americana) from Brazil. The other lineage, found in an adult, has an identity of 469 out of 478 bp (98%) with Plasmodium sp. GAL-2012 (isolate THAMB08) from Brazil. Although a morphological description of these parasites was not possible, this is the first molecular study focusing on Hoatzin haemosporidian parasites and the first documentation of Plasmodium infections in the Hoatzin from Venezuela. Furthermore, we reported microfilaria in two adults as well as hematological parameters for six individuals. Information on hematological parameters could contribute to establishing the necessary baseline to detect underlying conditions, such as infections, in this bird species.