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.     

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.     

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.     

Morphologic and morphometric analysis of Hepatozoon spp. (Apicomplexa,Hepatozoidae) of snakes

Hepatozoon species are the most abundant hemoparasites of snakes. Its identification has been based mainly on the morphologic characterization of the gamonts in the peripheral blood of the vertebrate host and also of the cysts found in the internal organs of the vertebrate and invertebrate hosts. Using a computerized image analysis system, we studied five species of Hepatozoon from recently captured snakes in Botucatu, State of S o Paulo, Brazil, to evaluate the importance of the morphology and morphometry of the gamonts for the characterization of Hepatozoon species and to analyze the morphologic changes induced in the erythrocytes by the parasite. The studied species were H. terzii of Boa constrictor amarali, Hepatozoon sp. of Crotalus durissusterrificus, H. philodryasi of Philodryas patagoniensis, and H. migonei and H. cyclagrasi of Hydrodynastes gigas. We observed three different groups, one of them including the species H. terzii, H. philodryasi and Hepatozoon sp. of C. durissus terrificus; and the other two consisting of H. migonei and H. cyclagrasi. Degree of alterations in the erythrocytes was variable and it may be useful for characterization of Hepatozoon species.     

A Hepatozoon species genetically distinct from H. canis infecting spotted hyenas in the Serengeti ecosystem, Tanzania

Health monitoring of spotted hyenas (Crocuta crocuta) in the Serengeti ecosystem, Tanzania, revealed Hepatozoon infection in all of 11 immature individuals examined following death from natural causes. Hepatozoon infection was probably an important factor contributing to mortality in two cases that exhibited clinical signs of ataxia, lethargy, ocular discharge, retching, and labored breathing before death. Whether Hepatozoon infection contributed to six deaths from fire, probable lion predation and unknown causes could not be determined. Four deaths from infanticide and starvation were unlikely to be associated with Hepatozoon infection. Histologic examination revealed lung tissue infected with cyst-like structures containing protozoan stages in all eight cases examined and interstitial pneumonia in most cases. Systemic spread of infection to several organs was found in three cases. Alignment of a 426 bp sequence from the parasite's 18s rRNA gene revealed a Hepatozoon species identical to that recently described from two domestic cats in Spain and only 7 bp substitutions when a 853 bp sequence was aligned to this cat Hepatozoon species. Previous reports of infection of wild carnivores in eastern and southern Africa with an unspecified Hepatozoon species similar in appearance to H. canis may have involved the species described in this study.     

Haemogregarine specificity in two communities of Florida snakes, with descriptions of six new species of Hepatozoon (Apicomplexa: Hepatozoidae) and a possible species of Haemogregarina (Apicomplexa: Haemogregarinidae)

Five species of snakes in Florida, from Palm Beach County in the south and Alachua County 450 km to the north, occur in similar habitat but have distinctive Hepatozoon species characteristic of each host species. In Palm Beach County, Diadophis punctatus is host to Hepatozoon punctatus n. sp., Thamnophis sauritus sackenii to Hepatozoon sauritus n. sp., and Nerodia fasciata pictiventris to Hepatozoon pictiventris n. sp. In Alachua County, N. fasciata pictiventris is parasitized by Hepatozoon fasciatae n. sp., Seminatrix p. pygaea by Hepatozoon seminatrici n. sp., and Thamnophis s. sirtalis by Hepatozoon sirtalis n. sp. Each Hepatozoon sp. has distinctive gamonts and sporogonic characters and, in the 4 species where known, meronts. Nerodia floridana is host to Haemogregarina floridana n. sp. in both localities, with generic identification tentative, based upon presence of erythrocytic meronts. The presence of sporocysts in the proboscis of 31% of Aedes aegypti infected by H. pictiventris is the first report of infective stages of a reptilian Hepatozoon species within the mouthparts of a dipteran vector. This study suggests that in Florida, at least, the diversity of the Hepatozoon community not only equals but probably exceeds the diversity of the snake communities present, and that host specificity in nature may be much greater than that postulated from previous studies.     

Molecular characterization of the 18S rDNA gene of an avian Hepatozoon reveals that it is closely related to Lankesterella

As a part of intensive study of blood parasite infections in a population of the passerine bird blue tit (Cyanistes caeruleus, Paridae), we detected a parasite species that, based on its morphological similarity, was tentatively identified as Hepatozoon parus, the only species of this parasite genus described from birds of this family. However, morphological measurements show that H. parus is slightly larger than the parasite detected in our population. A molecular characterization of the parasite species was conducted by amplification of the 18S rDNA gene, using primers that were reported previously to amplify in Hepatozoon sp. of water pythons. Additional primers were developed based on the new sequence obtained. The 1,484-bp fragment amplified reveals that the parasite from our bird population is more closely related to Lankesterella minima than to Hepatozoon species from other vertebrates according to analysis using the BLAST comparison method (93% identity). In addition, phylogenetic analyses using parsimony and Kimura procedures unequivocally related the parasite species detected by PCR with L. minima. The bootstrap values obtained were 97% and 100%, respectively. These results imply that this parasite is a species of a lankesterellid instead of Hepatozoon.     

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.     

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.     

Population estimates and distribution changes of landbirds on Silhouette Island,Seychelles

Cresswell, W., Irwin, M., Mee, A., Mellanby, R., McKean, M. & Milne, L. 1997. Population estimates and distribution changes of landbirds on Silhouette Island, Seychelles. Ostrich 68 (2–4): 50–57.

The density of landbird species within different habitats, and presence within 4 ha squares, on Silhouette Island were recorded in August 1996, repeating a previous survey (January 1979). Densities of the four most common species were calculated from point counts: Seychelles Sunbirds occurred at 2–5 birds/ha, Seychelles Bulbuls 0.5–4 birds/ha, Indian Mynahs 0.2–2 birds/ha and Seychelles Blue Pigeons 0.3 birds/ha depending on habitat. Population estimates were: Seychelles Sunbirds 5600, Seychelles Bulbuls 4000, Indian Mynahs 1100, Seychelles Blue Pigeons 600, and Seychelles Kestrel, approximately 15 pairs. The distributions of non-endemic Indian Mynahs, Barred Ground Doves and Madagascar Fodies were smaller in 1996 compared to 1979, while those of endemic Seychelles Bulbuls and Sunbirds were larger. Mynahs were more likely to occur in squares containing human activity, but habitat variables rather than human effects predicted the presence or absence of endemics. Mynahs were more likely to occur in a square, controlling for habitat variables, if Bulbuls were absent, and vice versa. There may be major seasonal changes in the relative distribution of introduced and endemic species. Alternatively, introduced species may have actually declined, probably because of a decrease in human population between the two surveys; endemics may then have increased because of reduced competition.     

Monozoic cysts of Hepatozoon canis

Small monozoic cysts found in the spleen of dogs infected with Hepatozoon canis are described from naturally and experimentally infected dogs. These forms of H. canis resemble cysts formed by other Hepatozoon species that infect frogs, lizards, and grey squirrels as intermediate hosts. The H. canis cyst stage differs in size and morphology from the large cysts of H. americanum, the second Hepatozoon species known to infect dogs.     

Hepatozoon ursi n. sp. (Apicomplexa: Hepatozoidae) in Japanese black bear (Ursus thibetanus japonicus)

Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. sp., in Japanese black bear (Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7+/-4.6 x 42.7+/-4.5 microm in size. Each mature schizont contained approximately 80-130 merozoites and 0-5 residual bodies. The merozoites were 7.0+/-0.7 x 1.8+/-0.3 microm in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9+/-0.3 x 3.3+/-0.2 microm. The analyses of the18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks (Haemaphysalis japonica and Haemaphysalis flava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 x 234.0 microm and 331.8 x 231.7 microm, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2+/-2.5 x 27.0+/-2.9 microm in size. Each sporocyst contained at least 8-16 sporozoites, with the sporozoites being 12.2+/-1.4 x 3.5+/-0.5 microm in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae.     

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.     

Intraerythrocytic development of species of Hepatozoon infecting ranid frogs: evidence for convergence of life cycle characteristics among apicomplexans

Intraerythrocytic development of the adeleorin apicomplexans Hepatozoon clamatae and Hepatozoon catesbianae were investigated in the bullfrog, Rana catesbeiana, the green frog, Rana clamitans melanota, and the Northern leopard frog, Rana pipiens. Merozoites emerging from hepatic meronts penetrated erythrocytes and underwent 1-3 rounds of binary fission to produce 2-8 merozoites. Following their release from infected erythrocytes, individual merozoites entered new cells and transformed into gamonts. Although this is the first report of intraerythrocytic development for a fully described species of Hepatozoon, a phylogenetic reanalysis of 11 species of Hepatozoon, 6 species representative of the 5 other hemogregarine taxa, 2 species of dactylosomatids, and 2 species of piroplasms, indicates that asexual reproduction of parasites within blood cells of vertebrates has arisen at least 3 times in the apicomplexan lineage that includes adeleorins and piroplasms. This method of asexual development, which is also observed in species of hemospororin genera such as Plasmodium, is discussed in the context of the evolution of apicomplexan life cycles. In addition to supporting the paraphyly of the genus Hepatozoon determined in an earlier study, this phylogenetic analysis featured a monophyletic group, consisting of the sister taxa Hemolivia and Karyolysus, that was the sister group to a clade consisting of the more derived hemogregarines, the dactylosomatids, and the piroplasms.     

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 analysis of Hepatozoon species (Apicomplexa: Adeleorina) infecting frogs of Nova Scotia, Canada, determined by ITS-1 sequences

Species of Hepatozoon are apicomplexan parasites infecting tetrapod vertebrates and hematophagous arthropods. Two species, Hepatozoon catesbianae and Hepatozoon clamatae, have been described inhabiting the erythrocytes of bullfrogs and green frogs. A number of characteristics typically used to distinguish between members of this genus are shared between these 2 species, prompting speculation as to whether or not these organisms are in fact distinct species. To test the species distinction, bullfrogs and green frogs were captured at various sites across Nova Scotia, blood samples were collected, and DNA was extracted from samples containing parasites. The internal transcribed spacer 1 (ITS-1) from geographically diverse samples of both species was amplified by PCR, sequenced, and analyzed. ITS-1 sequences from the 2 species revealed single-nucleotide polymorphisms at 6 sites. Phylogenetic analysis of these molecular data and cytopathological features place isolates of each species in separate monophyletic groups. Comparison of the ITS-1 sequences between isolates from Nova Scotia and Ontario revealed that ITS-1 sequences of H. catesbianae from a previous study were mischaracterized as being those of H. clamatae. Phylogenetic data based on molecular variation and cytopathological features from this study provide the strongest evidence to date supporting the distinction between these 2 species.     

Ecological observations on marine fungi from the Seychelles

HYDE, K. D. & JONES, E. 6. G., 1989. Ecological observations on marine fungi from the Seychelles . Submerged wood, wood trapped amongst rocks, sand-buried wood and exposed roots and branches of shoreline trees were collected from three intertidal beach sites in the Seychelles. These were examined for the presence of marine fungi, the species present identified and their frequency of occurrence noted. Leaves, seaweeds and seagrasses washed up on the seashore, and seafoam, were also examined for filamentous marine fungi. Species present are listed with their frequency of occurrence. Differences in species composition from one substratum to the next were noted, and the results discussed. Sixty-three species were collected: 35 Ascomycotina, 2 Basidiomycotina and 26 Deuteromycotina, all are new records for the Seychelles. The mycogeography and ecological niche of the fungi encountered are discussed.     

A new species of Hepatozoon (Apicomplexa: Adeleorina) from Python regius (Serpentes: Pythonidae) and its experimental transmission by a mosquito vector

Hepatozoon ayorgbor n. sp. is described from specimens of Python regius imported from Ghana. Gametocytes were found in the peripheral blood of 43 of 55 snakes examined. Localization of gametocytes was mainly inside the erythrocytes; free gametocytes were found in 15 (34.9%) positive specimens. Infections of laboratory-reared Culex quinquefasciatus feeding on infected snakes, as well as experimental infection of juvenile Python regius by ingestion of infected mosquitoes, were performed to complete the life cycle. Similarly, transmission to different snake species (Boa constrictor and Lamprophis fuliginosus) and lizards (Lepidodactylus lugubris) was performed to assess the host specificity. Isolates were compared with Hepatozoon species from sub-Saharan reptiles and described as a new species based on the morphology, phylogenetic analysis, and a complete life cycle.     

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.