Molecular and morphological description of a Hepatozoon species in reptiles and their ticks in the Northern Territory, Australia

Ticks, representing 3 species of Amblyomma, were collected from the water python (Liasis fuscus) and 3 additional reptile species in the Northern Territory, Australia, and tested for the presence of Hepatozoon sp., the most common blood parasites of snakes. In addition, blood smears were collected from 5 reptiles, including the water python, and examined for the presence of the parasite. Hepatozoon sp. DNA was detected in all tick and reptile species, with 57.7% of tick samples (n = 187) and 35.6% of blood smears (n=35) showing evidence of infection. Phylogenetic analysis of the 18S rRNA gene demonstrated that half of the sequences obtained from positive tick samples matched closest with a Hepatozoon species previously identified in the water python population. The remaining sequences were found to be more closely related to mammalian and amphibian Hepatozoon species. This study confirms that species of Amblyomma harbor DNA of the same Hepatozoon species detected in the water pythons. The detection of an additional genotype suggests the ticks may be exposed to 2 Hepatozoon species, providing further opportunity to study multiple host-vector-parasite relationships.     

High prevalence of Hepatozoon spp. (Apicomplexa, Hepatozoidae) infection in water pythons (Liasis fuscus) from tropical Australia

Molecular methods were used to identify blood parasites frequently observed in blood smears of water pythons (Liasis fuscus) captured in our study area in the Northern Territory of Australia. A nested polymerase chain reaction (PCR) using primers amplifying the 18s ribosomal RNA (rRNA) nuclear gene resulted in a short PCR product (180 bp) matching this region in the genus Hepatozoon. However, because of the short sequence obtained. 2 new primers were designed based on 18s rRNA sequences of 3 Hepatozoon taxa available in GenBank. Using these primers, approximately 600 bp of the parasite's 18s rRNA gene was amplified successfully and sequenced from 2 water python samples. The new primers were used to investigate the prevalence of blood parasites in 100 pythons. In 25 of these samples we did not observe any blood parasites when examining stained slides. All the samples revealed a 600-bp PCR product, demonstrating that pythons in which we did not visually observe any parasites were infected by Hepatozoon spp. We also analyzed the nucleotide sequences of blood parasites in 4 other reptile taxa commonly encountered in our study area. The sequences obtained from water pythons and from 1 of these taxa were identical, suggesting that the parasite is capable of infecting hosts at different taxonomic levels.     

Phylogenetic analysis of the genus Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina)

A phylogenetic analysis of species of Hepatozoon Miller, 1908 was performed using 16 morphological, morphometric and developmental characters. An adeleorin parasite of gastropod molluscs, Klossia helicina Schneider, 1875 and four haemogregarines of other genera, Karyolysus lacertae (Danilewsky, 1886), Cyrilia lignieresi (Laveran, 1906), Desseria myoxocephali (Fantham, Porter & Richardson, 1942) and Haemogregarina balli Paterson & Desser, 1976 were used as the outgroup to 12 species of Hepatozoon. A single most parsimonious interpretation of the data was found, with the resulting cladogram having 28 transformations and a consistency index of 0.75. The proposed phylogeny revealed Hepatozoon as a paraphyletic group. One monophyletic lineage contained 10 of the 12 species of Hepatozoon, including all of those species that undergo gametogenesis and sporogony in the haemocoel of arthropods. Within this lineage, gamonts of those species found in vertebrate leucocytes instead of erythrocytes formed a clade basal to the remainder of the species. Species of Hepatozoon which undergo gametogenesis and sporogonic development in the gut epithelium of acarines, and which produce aflagellate microgametes, as well as the four outgroup taxa of haemogregarines, formed a monophyletic group and were the sister group to the remainder of the species of Hepatozoon. The biological and morphological diversity of these parasites suggests that species of the genus Hepatozoon, which are members of the paraphyletic haemogregarine complex, could be partitioned into at least two genera of adeleorin parasites.     

Molecular characterization of an ancient Hepatozoon species parasitizing the 'living fossil' marsupial 'Monito del Monte' Dromiciops gliroides from Chile

The Microbiotheriid Dromiciops gliroides , also known as 'Monito del Monte', is considered to be a threatened species and the only living representative of this group of South American marsupials. During the last few years, several blood samples from specimens of 'Monito del Monte' captured at Chiloé island in Chile have been investigated for blood parasites. Inspection of blood smears detected a Hepatozoon species infecting red blood cells. The sequences of DNA fragments corresponding to small subunit ribosomal RNA gene revealed two parasitic lineages belonging to Hepatozoon genus. These parasite lineages showed a basal position with respect to Hepatozoon species infecting rodents, reptiles, and amphibians but are phylogenetically distinct from Hepatozoon species infecting the order Carnivora. In addition, the Hepatozoon lineages infecting D. gliroides are also different from those infecting other micro-mammals living in sympatry, as well as from some that have been described to infect an Australian species of bandicoot. The potential vector of this parasite appears to be the host-specific tick Ixodes neuquenensis because the sequencing of a long amplicon determined the presence of one of the two lineages found in the marsupial.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 568–576.     

Phylogenetic relationships of Hepatozoon (Apicomplexa: Adeleorina) based on molecular, morphologic, and life-cycle characters

To evaluate higher-level affinities of Hepatozoon species within Apicomplexa, we sequenced the 18S rRNA gene from 2 parasites (Hepatozoon americanum and Hepatozoon canis) of dogs and 1 (Hepatozoon catesbianae) of bullfrogs. Sequences from other apicomplexans among the Sarcocystiidae, Eimeriidae, Theileriidae, Plasmodiidae, Cryptosporiidae, and Babesiidae, a Perkinsus species and 2 dinoflagellates were obtained from GenBank. Phylogenetic analysis indicated that Plasmodium, Cryptosporidium, and Hepatozoon form a monophyletic group distinct from representatives of other apicomplexan families. Although equivocal, our analysis indicated that Plasmodium and Cryptosporidium are sister taxa and that Hepatozoon is basal to them. To evaluate phylogenetic affinities among H. americanum, H. canis, and other species of Hepatozoon, we examined 18 morphologic and life-cycle features of 13 species currently assigned to Hepatozoon. This analysis indicates paraphyly of Hepatozoon (as currently arranged) because Hepatozoon lygosomarum was found most closely related to Hemolivia mauritanicum. These results, combined with results of previous studies, support elevating Hepatozoon to familial level (Hepatozoidae) as originally suggested by Wenyon in 1926. Both DNA sequence data and morphologic and life-cycle characters support a sister-group relationship between H. americanum and H. canis.     

Diversity of haemogregarine parasites infecting Brazilian snakes from the Midwest and Southeast regions with a description of two new species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae)

Although Brazil is a hotspot for snake species, there is a lack of information on the biodiversity of haemoparasites infecting these hosts. Thus, the present study aimed to bring new insights on the diversity of species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) infecting Brazilian snakes from the Midwest and Southeast regions. The snakes were captured from 2018 to 2020 from the states of Mato Grosso, Mato Grosso do Sul, Goiás, and São Paulo. Three to five blood smears were made and the remaining blood sample was stored for further molecular analysis. Moreover, histopathological slides of the organs were stained with haematoxylin-eosin. Regarding molecular diagnosis, PCR was performed targeting different regions of the 18S rRNA gene of apicomplexan parasites. From the 13 free-living snakes screened, ten (76.92%) were found infected with Hepatozoon spp. Based on morphological and morphometric tools, five different morphotypes of species of Hepatozoon gamonts were detected. Molecular data and phylogenetic analysis support the morphological data, identifying five species of Hepatozoon from snakes, of which three species belong to previously described species, Hepatozoon cevapii, Hepatozoon cuestensis, and Hepatozoon quagliattus, with a genetic similarity of 100% (based on the 18S rRNA genetic marker). The present study identifies and describes two new species of Hepatozoon, Hepatozoon annulatum sp. nov. infecting the snake Leptodeira annulata and Hepatozoon trigeminum sp. nov. infecting the snake Oxyrhopus trigeminus. Thus, based on morphological and molecular data the present study provides new insights on haemogregarine diversity infecting Brazilian snakes from the Midwest and Southeast regions.     

Epizootiology of blood parasites in an Australian lizard: a mark-recapture study of a natural population

The dynamics of a naturally endemic blood parasite (Hepatozoon hinuliae) were studied in a lizard (Eulamprus quoyii) host population, using 2 years of longitudinal data. We investigated how parasite abundance in the population varied over time, examined whether certain host sub-populations were more prone to infection, and compared parasite loads in relation to host reproductive behaviour. We recorded blood parasite infections of 331 individuals, obtained in 593 captures. Prevalence (the proportion of the host population infected) of blood parasites was high; approximately 66% of the lizard population was infected. Probability of infection increased with host age and size, but did not differ between the sexes. Within individuals, parasite load (the intensity of infection within individuals) did not vary over time, and was independent of host reproductive behaviour. Parasite load was significantly higher in males compared to females.     

Molecular survey of Apicomplexa in Podarcis wall lizards detects Hepatozoon, Sarcocystis, and Eimeria species

The occurrence of apicomplexan parasites in Podarcis sp. wall lizards from the Iberian Peninsula and Balearic islands was studied by amplification and sequencing of the 18S rRNA gene. Species from 3 genera, Hepatozoon , Sarcocystis , and Eimeria , were found. The phylogenetic analysis of the 18S rRNA gene provides unexpected insights into the evolutionary history of these parasites. All Hepatozoon spp. specimens were recovered as part of a clade already identified in lizards from North Africa. The Sarcocystis species, detected in Podarcis lilfordi from Cabrera Island in the Balearic Islands, appears related to Sarcocystis gallotiae , known only from endemic Gallotia sp. lizards from the Canary Islands. Based on the lack of snake predators on this island, this parasite presumably presents an atypical transmission cycle that uses the same host species as both intermediate and final host through cannibalism, like S. gallotiae . Eimeria sp. is reported for the first time from Podarcis spp. lizards. This study shows the power of detecting multiple different apicomplexan parasites through screening of tail tissue samples and blood drops that are often collected in reptiles for other purposes.     

Molecular characterization of Hepatozoon species in reptiles from the Seychelles

Hepatozoon parasites were examined for the first time in reptiles from the Seychelles Islands. Although both prevalence and intensity were low, Hepatozoon species were detected in individuals from 2 endemic species, the lizard Mabuya wrightii and the snake Lycognathophis seychellensis. This was confirmed using visual identification and through sequencing part of the 18s rRNA gene. Phylogenetic analysis indicates that the Hepatozoon on the Seychelles form a monophyletic lineage, although more data are clearly needed to stabilize estimates of relationships based on this marker.     

A new species of <Emphasis Type="Italic">Hepatozoon</Emphasis> Miller, 1908 (Apicomplexa: Adelerina) from the snake <Emphasis Type="Italic">Philodryas nattereri</Emphasis> Steindachner (Squamata: Dipsadidae) in northeastern Brazil

Based on both unique morphological characteristics of the gamont, distinct changes caused to the host erythrocyte and analysis of partial 18S rRNA gene sequences, a new parasite of the genus Hepatozoon Miller, 1908 is described from the snake Philodryas nattereri Steindachner (Squamata: Dipsadidae) in northeastern Brazil. The new species, Hepatozoon musa n. sp., is characterized by large and curved mature gamonts (18.9 ± 0.9 μm in length and 3.8 ± 0.3 μm in width) that considerably engorge infected host erythrocytes and displace the nucleus laterally, which become longer and thinner. Phylogenetic estimates indicate the new species is more closely related to the recently described Hepatozoon cuestensis O’Dwyer, Moço, Paduan, Spenassatto, Silva & Ribolla, 2013, from Brazilian rattlesnakes. These recent findings highlight the need for further studies of Hepatozoon to better determine the biodiversity of this common but poorly-studied parasite group.     

Diversity and host assemblage of avian haemosporidians in different terrestrial ecoregions of Peru

Characterizing the diversity and structure of host–parasite communities is crucial to understanding their eco-evolutionary dynamics. Malaria and related haemosporidian parasites are responsible for fitness loss and mortality in bird species worldwide. However, despite exhibiting the greatest ornithological biodiversity, avian haemosporidians from Neotropical regions are quite unexplored. Here, we analyze the genetic diversity of bird haemosporidian parasites (Plasmodium and Haemoproteus) in 1,336 individuals belonging to 206 bird species to explore for differences in diversity of parasite lineages and bird species across 5 well-differentiated Peruvian ecoregions. We detected 70 different haemosporidian lineages infecting 74 bird species. We showed that 25 out of the 70 haplotypes had not been previously recorded. Moreover, we also identified 81 new host–parasite interactions representing new host records for these haemosporidian parasites. Our outcomes revealed that the effective diversity (as well as the richness, abundance, and Shannon–Weaver index) for both birds and parasite lineages was higher in Amazon basin ecoregions. Furthermore, we also showed that ecoregions with greater diversity of bird species also had high parasite richness, hence suggesting that host community is crucial in explaining parasite richness. Generalist parasites were found in ecoregions with lower bird diversity, implying that the abundance and richness of hosts may shape the exploitation strategy followed by haemosporidian parasites. These outcomes reveal that Neotropical region is a major reservoir of unidentified haemosporidian lineages. Further studies analyzing host distribution and specificity of these parasites in the tropics will provide important knowledge about phylogenetic relationships, phylogeography, and patterns of evolution and distribution of haemosporidian parasites.     

Phylogenetic relationships among Hepatozoon species from snakes, frogs and mosquitoes of Ontario, Canada, determined by ITS-1 nucleotide sequences and life-cycle, morphological and developmental characteristics

The molecular biological characteristics of Hepatozoon species infecting various species of snakes, frogs and mosquitoes were investigated by determining the nucleotide sequences of the first internal transcribed spacer region. A phylogenetic analysis was performed on seven isolates of Hepatozoon infecting snakes, including Hepatozoon sipedon and four morphologically similar but not identical forms, and two isolates of Hepatozoon catesbianae infecting Green frogs (Rana clamitans melanota). This analysis, which utilised data from first internal transcribed spacer nucleotide sequences, morphological and morphometric features of gamonts, oocysts and sporocysts, and previously determined life-cycle and host-specificity characteristics, revealed that H. sipedon is a polymorphic species with a wide host and geographic range. Four synapomorphies. including two nucleotide substitutions and two morphological character state changes, supported a monophyletic group of six isolates of H. sipedon from the central region of Ontario which was the sister group for an isolate (HW1) from the southern part of the province. Based on the results of this study, an evaluation of which criteria are useful for describing species of Hepatozoon is presented, with the intent of curtailing the practice of naming species based on morphological features of gamonts or on incomplete life-cycle data.     

New molecular data on mammalian Hepatozoon species (Apicomplexa: Adeleorina) from Brazil and Spain

Molecular techniques were used to examine the phylogenetic relationships among Hepatozoon species isolated from 13 foxes and 15 opossums from Brazil, and from 15 dogs, 20 foxes, 45 rodents, and 330 domestic cats from Spain. Hemogregarine infection was confirmed by amplification of the 18S rRNA gene and later sequencing. No hemogregarine infections were found in opossums. The prevalence of Hepatozoon in canids ranged from 26.6% (symptomatic domestic dogs) to 90% (Spanish foxes). Four different H. canis genotypes were detected, as well as an H. americanum-related protozoan (97% identical to the USA strain). Two Spanish cats were parasitized by a Hepatozoon species (0.6% prevalence) that showed 96% sequence identity to H. canis. DNA amplification assays performed on Spanish rodents showed 2 bank voles (Clethrionomys glareolus) to be infected by a Hepatozoon species (4.44% prevalence) with 95% sequence identity to Hepatozoon sp. from cats. Phylogenetic analysis showed Hepatozoon to be a monophyletic genus, in which species from carnivorous mammals (Hepatozoon sp. from cats, H. americanum and H. canis) appear as a sister lineage of that of lower vertebrates and rodents. This association suggests that H. americanum evolved in ticks and carnivores (either canids, or felids, or both) rather than in other ectoparasites and other types of mammal.     

The first detection of Babesia species DNA from Japanese black bears (Ursus thibetanus japonicus) in Japan

In this study, we tried to detect protozoan blood parasites from the liver or blood of 156 Japanese black bears (Ursus thibetanus japonicus) in Iwate Prefecture of Japan by polymerase chain reaction. Two amplicons (approximately 540 bp and 480 bp) were detected by amplification for V4 hyper-variable regions of the 18S rRNA gene. Approximately 540-bp products were obtained in 119 samples (76.3%) and were considered to be DNA of Hepatozoon ursi. Approximately 480-bp products were obtained in 22 samples (14.1%) and were considered to be DNA of Babesia species. The nucleotide sequences (1635 bp) of the 18S rRNA gene of Babesia sp. were very similar (99.3%) to those (AY190123, AY190124) of Babesia sp. detected previously from Ixodes ovatus. Phylogenetic analysis showed that Babesia sp. detected in this study closely related to Babesia sp. derived from raccoons in Japan and the U.S.A. This is the first report of Babesia species detected from Japanese black bears.     

Low haemosporidian diversity and one key-host species in a bird malaria community on a mid-Atlantic island (São Miguel, Azores)

When host species colonize new areas, the parasite assemblage infecting the hosts might change, with some parasite species being lost and others newly acquired. These changes would likely lead to novel selective forces on both host and its parasites. We investigated the avian blood parasites in the passerine bird community on the mid-Atlantic island of S?o Miguel, Azores, a bird community originating from continental Europe. The presence of haemosporidian blood parasites belonging to the genera Haemoproteus, Plasmodium, and Leucocytozoon was assessed using polymerase chain reaction. We found two Plasmodium lineages and two Leucocytozoon lineages in 11 bird species (84% of all breeding passerine species) on the island. These lineages were unevenly distributed across bird species. The Eurasian Blackbird (Turdus merula) was the key-host species (total parasite prevalence of 57%), harboring the main proportion of parasite infections. Except for Eurasian Blackbirds, all bird species had significantly lower prevalence and parasite diversity compared to their continental populations. We propose that in evolutionary novel bird communities, single species may act as key hosts by harboring the main part of the parasite fauna from which parasites "leak" into the other species. This would create very different host-parasite associations in areas recently colonized by hosts as compared to in their source populations.     

Haemosporidian blood parasites in European birds of prey and owls

Avian blood parasites have been intensively studied using morphological methods with limited information on their host specificity and species taxonomic status. Now the analysis of gene sequences, especially the mitochondrial cytochrome b gene of the avian haemosporidian species of Haemoproteus, Plasmodium, and Leucocytozoon, offers a new tool to review the parasite specificity and status. By comparing morphological and genetic techniques, we observed nearly the same overall prevalence of haemosporidian parasites by microscopy (19.8%) and polymerase chain reaction (PCR) (21.8%) analyses. However, in contrast to the single valid Leucocytozoon species (L. toddi) in the Falconiformes we detected 4 clearly distinctive strains by PCR screening. In the Strigiformes, where the only valid Leucocytozoon species is L. danilewskyi, we detected 3 genetically different strains of Leucocytozoon spp. Two strains of Haemoproteus spp. were detected in the birds of prey and owls examined, whereas the strain found in the tawny owl belonged to the morphospecies Haemoproteus noctuae. Three Plasmodium spp. strains that had already been found in Passeriformes were also detected in the birds of prey and owls examined here, supporting previous findings indicating a broad and nonspecific host spectrum bridging different bird orders.     

Transmission of hepatozoon canis to dogs by naturally-fed or percutaneously-injected Rhipicephalus sanguineus ticks.

Hepatozoon canis is an apicomplexan protozoan parasite of dogs, prevalent in Asia, Africa, and southern Europe. Experimental transmission of H. canis to dogs was performed with laboratory-reared Rhipicephalus sanguineus nymphs that fed on a naturally infected dog or were percutaneously injected with canine blood containing H. canis gamonts. Dogs were inoculated by oral ingestion of adult ticks containing H. canis oocysts. Transstadial transmission of H. canis was recorded, whereas transovarial transmission could not be demonstrated. Oocysts were detected in 85% of the adult ticks that had engorged as nymphs on an infected dog and in 61% of the adult ticks resulting from nymphs injected percutaneously with blood from the same dog. Nine of 12 dogs (75%) inoculated with naturally fed or percutaneously injected ticks became parasitologically positive, and all showed seroconversion. Meronts were initially detected in the bone marrow 13 days postinoculation and gamonts 28 days after infection. The variation in the time of initial detection of parasitemia among infected dogs and the rapid appearance of gamonts in dogs immunosuppressed with corticosteroids suggest that immune mechanisms play an important role in controlling H. canis parasitism. The artificial acquisition of Hepatozoon parasites by percutaneous injection of ticks, demonstrated here for the first time, may serve as a useful tool for studies on transmission, vector-host relationships, and the immunology of infection with Hepatozoon species.     

Novel Hepatozoon in vertebrates from the southern United States

Novel Hepatozoon spp. sequences collected from previously unrecognized vertebrate hosts in North America were compared with documented Hepatozoon 18S rRNA sequences in an effort to examine phylogenetic relationships between the different Hepatozoon organisms found cycling in nature. An approximately 500-base pair fragment of 18S rDNA common to Hepatozoon spp. and some other apicomplexans was amplified and sequenced from the tissues or blood of 16 vertebrate host species from the southern United States, including 1 opossum (Didelphis virginiana), 2 bobcats (Lynx rufus), 1 domestic cat (Felis catus), 3 coyotes (Canis latrans), 1 gray fox (Urocyon cinereoargenteus), 4 raccoons (Procyon lotor), 1 pet boa constrictor (Boa constrictor imperator), 1 swamp rabbit (Sylvilagus aquaticus), 1 cottontail rabbit (Sylvilagus floridanus), 4 woodrats (Neotoma fuscipes and Neotoma micropus), 3 white-footed mice (Peromyscus leucopus), 8 cotton rats (Sigmodon hispidus), 1 cotton mouse (Peromyscus gossypinus), 1 eastern grey squirrel (Sciurus carolinensis), and 1 woodchuck (Marmota monax). Phylogenetic analyses and comparison with sequences in the existing database revealed distinct groups of Hepatozoon spp., with clusters formed by sequences obtained from scavengers and carnivores (opossum, raccoons, canids, and felids) and those obtained from rodents. Surprisingly, Hepatozoon spp. sequences from wild rabbits were most closely related to sequences obtained from carnivores (97.2% identical), and the sequence from the boa constrictor was most closely related to the rodent cluster (97.4% identical). These data are consistent with recent work identifying prey-predator transmission cycles in Hepatozoon spp. and suggest this pattern may be more common than previously recognized.     

Low genetic and morphological differentiation between an introduced population of dunnocks in New Zealand and an ancestral population in England

Species invasions and exotic species introductions can be considered as ??unplanned experiments??, which help us to understand the evolution of organisms. In this study, we investigated whether an exotic bird species, the dunnock (Prunella modularis), has diverged genetically and morphologically from its native source population (Cambridge, England) after introduction into a new environment (Dunedin, South Island of New Zealand; exotic population). We used a set of microsatellite markers and three morphological traits to quantify the divergence between these two populations. We quantified neutral genotypic differentiation between the populations, and also used an individual-based Bayesian clustering method to assess genetic structure. We compared morphological divergence using univariate and principal components analyses. We found that individuals from the Dunedin population are genetically distinct from the Cambridge population, but levels of differentiation are very low. Overall within-population levels of genetic diversity are low compared to other bird species, and effective population sizes are small; indicating that the native population probably has a historically low level of genetic diversity, and that the introduced population retained most of that diversity after its introduction into New Zealand. We found little evidence of morphological divergence, and the evolutionary rate of change in these traits is below the average for other taxa. Our study adds support to the growing literature showing that invasive species maintain most of their initial genetic diversity after multiple founder events, even when population size is severely reduced. Moreover, our morphological data indicate slow evolutionary rates in species introduced to similar habitats.