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Hepatozoon Miller, 1908 parasites in the Colubridae snakes Clelia clelia (Daudin, 1803) and Drymarchon corais (Boie, 1827) from the Eastern Amazonia

Abstract

Based on the genetic, morphological, and morphometric data of blood gamonts, we identified Hepatozoon parasites in colubrid snakes sampled in the Eastern Amazon region. Hepatozoon trigeminum was detected in the mussurana snake Clelia clelia and exhibited wide and elongated gamonts (mean dimensions: 14.25±0.65 × 4.31±0.43 μm) with an evident parasitophorous vacuole. Hepatozoon odwyerae sp. nov. was described in the indigo snake Drymarchon corais, whose gamonts have elongated and thin bodies (mean dimensions: 13.41±0.79 × 3.72±0.35 μm) with one end more tapered than the other. Phylogenetic analyses, based on the amplification of a 441 bp fragment of the 18S rRNA gene, revealed that the novel sequences of Hepatozoon spp. from our study were closely related to hemogregarine lineages found in lizards and snakes from Brazil, forming a well-supported monophyletic clade with them. The present study provides the first species description of Hepatoozon in D. corais and a new record of a host species for C. clelia using the integrated taxonomic data. We also highlight the importance of further investigations into the diversity of Hepatozoon in snakes, a rich but underestimated group of parasites, especially in the Amazonian biome.

Key words
Apicomplexa; colubrid snakes; hemoparasites; integrative taxonomy; 18S rRNA

INTRODUCTION

The phylum Apicomplexa Levine, 1970 comprises the greatest diversity of protozoan parasites, with approximately 6,000 named species (Votýpka et al. 2017VOTÝPKA J, MODRÝ D, OBORNÍK M, ŠLAPETA J & LUKEŠ J. 2017. Apicomplexa. In: ARCHIBALD J, SIMPSON A & SLAMOVITS C (Eds). Handbook of the Protists, Cham: Springer, p. 567-624.). However, they remain poorly known in terms of biodiversity, and probably only 0.1% of the species in this phylum have been described (Morrison 2009MORRISON DA. 2009. Evolution of the Apicomplexa: where are we now? Trends Parasitol 25: 375-382., Duszynski 2021DUSZYNSKI DW. 2021. Biodiversity of the Coccidia (Apicomplexa: Conoidasida) in vertebrates: what we know, what we do not know, and what needs to be done. Folia Parasitol 68: 1-18.). All the organisms in this taxon are parasites (Levine 1988LEVINE ND. 1988. Progress in taxonomy of the Apicomplexan protozoa. J Protozool 35: 518-520.) and frequently infect humans and domestic and wild animals. They possess a set of structures known as the apical complex, which facilitates the entry and survival of these parasites in the cells of their host (Tardieux & Baum 2016TARDIEUX I & BAUM J. 2016. Reassessing the mechanics of parasite motility and host-cell invasion. J Cell Biol 214: 507-515.).

Hepatozoon Miller, 1908 (Adeleorina, Hepatozoidae) is one of the most well-known genera of this phylum and can be found infecting blood and tissues from a wide range of vertebrate hosts around the world, such as amphibians, reptiles, birds, and mammals (Smith 1996SMITH TG. 1996. The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol 82: 565-585., O’Donoghue 2017O’DONOGHUE P. 2017. Haemoprotozoa: making biological sense of molecular phylogenies. Int J Parasitol Parasites Wildl 6: 241-256.). As these intracellular parasites have a heteroxenous life cycle, invertebrates, including ticks, lice, fleas, dipterans and even leeches, act as definitive hosts and vectors (Smith 1996SMITH TG. 1996. The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol 82: 565-585., Viana et al. 2010VIANA LA, SOARES P, PAIVA F & LOURENÇO-DE-OLIVEIRA R. 2010. Caiman-biting mosquitoes and the natural vectors of Hepatozoon caimani in Brazil. J Med Entomol 47: 670-676.). In snakes, infection occurs after the ingestion of the invertebrate host containing mature oocysts, or after feeding on an infected intermediate vertebrate host (i.e., frogs, lizards and rodents) (Sloboda et al. 2007SLOBODA M, KAMLER M, BULANTOVÁ J, VOTÝPKA J & MODRÝ D. 2007. A new species of Hepatozoon (Apicomplexa: Adeleorina) from Python regius (Serpentes: Pythonidae) and its experimental transmission by a mosquito vector. J Parasitol 93: 1189-1198., Tomé et al. 2012TOMÉ B, MAIA JP & HARRIS DJ. 2012. Hepatozoon infection prevalence in four snake genera: influence of diet, prey parasitemia levels, or parasite type? J Parasitol 98: 913-918.), as well as by vertical transmission (Kauffman et al. 2017KAUFFMAN KL, SPARKMAN A, BRONIKOWSKI AM & PALACIOS MG. 2017. Vertical transmission of Hepatozoon in the garter snake Thamnophis elegans. J Wildl Dis 53: 121-125.).

The taxonomy and systematics of Hepatozoon are the subject of considerable controversy (Smith 1996SMITH TG. 1996. The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol 82: 565-585., Hrazdilová et al. 2021HRAZDILOVÁ K, ČERVENÁ B, BLANVILLAIN C, FORONDA P & MODRÝ D. 2021. Quest for the type species of the genus Hepatozoon–phylogenetic position of hemogregarines of rats and consequences for taxonomy. Syst Biodivers 19: 1-10.). As highlighted by Zechmeisterová et al. (2021)ZECHMEISTEROVÁ K, JAVANBAKHT H, KVIČEROVÁ J & ŠIROKÝ P. 2021. Against growing synonymy: Identification pitfalls of Hepatozoon and Schellackia demonstrated on North Iranian reptiles. Eur J Protistol 79: 125780., the current main problem for the identification of such hemogregarines is the insufficient data used to perform species description, which often leads to synonymy. Moreover, the molecular phylogenies, mainly established using the 18S rDNA gene marker, have shown that this genus is not a monophyletic taxon (Kvičerová et al. 2014KVIČEROVÁ J, HYPŠA V, DVOŘÁKOVÁ N, MIKULÍČEK P, JANDZIK D, GARDNER MG, JAVANBAKHT H, TIAR G & ŠIROKÝ P. 2014. Hemolivia and Hepatozoon: haemogregarines with tangled evolutionary relationships. Protist 165: 688-700., Karadjian et al. 2015KARADJIAN G, CHAVATTE JM & LANDAU I. 2015. Systematic revision of the adeleid haemogregarines, with creation of Bartazoon ng, reassignment of Hepatozoon argantis Garnham, 1954 to Hemolivia, and molecular data on Hemolivia stellata. Parasite 22: 31., Hrazdilová et al. 2021HRAZDILOVÁ K, ČERVENÁ B, BLANVILLAIN C, FORONDA P & MODRÝ D. 2021. Quest for the type species of the genus Hepatozoon–phylogenetic position of hemogregarines of rats and consequences for taxonomy. Syst Biodivers 19: 1-10.). Some attempts have been made to deal with this issue, such as the raising of a new genus, ‘Bartazoon’ (Karadjian et al. 2015KARADJIAN G, CHAVATTE JM & LANDAU I. 2015. Systematic revision of the adeleid haemogregarines, with creation of Bartazoon ng, reassignment of Hepatozoon argantis Garnham, 1954 to Hemolivia, and molecular data on Hemolivia stellata. Parasite 22: 31.), which was not accepted by the scientific community, and a search for the sequence of the type species, Hepatozoon muris (Miller, 1908), which has yet to be found (Hrazdilová et al. 2021HRAZDILOVÁ K, ČERVENÁ B, BLANVILLAIN C, FORONDA P & MODRÝ D. 2021. Quest for the type species of the genus Hepatozoon–phylogenetic position of hemogregarines of rats and consequences for taxonomy. Syst Biodivers 19: 1-10.). Nonetheless, there is a strong consensus on the necessity of studies involving both morphological and molecular methods to detect and characterize Hepatozoon species, especially from wild hosts, to improve understanding of the diversity and phylogenetic relationships of these haemoparasites (Maia et al. 2016MAIA JP, CARRANZA S & HARRIS DJ. 2016. Comments on the systematic revision of adeleid haemogregarines: are more data needed? J Parasitol 102: 549-553., Zechmeisterová et al. 2021ZECHMEISTEROVÁ K, JAVANBAKHT H, KVIČEROVÁ J & ŠIROKÝ P. 2021. Against growing synonymy: Identification pitfalls of Hepatozoon and Schellackia demonstrated on North Iranian reptiles. Eur J Protistol 79: 125780.).

Snakes are extraordinarily rich in species of Hepatozoon, with over 130 species described so far, at least 40 of which have been recorded in Brazil (Smith 1996SMITH TG. 1996. The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol 82: 565-585., Úngari et al. 2018ÚNGARI LP, SANTOS ALQ, O’DWYER LH, SILVA MRL, SANTOS TCR, CUNHA MJR, PINTO RMC & CURY MC. 2018. Molecular characterization and identification of Hepatozoon species Miller, 1908 (Apicomplexa: Adeleina: Hepatozoidae) in captive snakes from Brazil. Parasitol Res 117: 3857-3865.). Few, however, have genetic data available. Most of these records come from colubrid hosts (Pessôa et al. 1974PESSÔA SB, DE BIASI P & PUORTO G. 1974. Nota sobre a frequência de hemoparasitas em serpentes no Brasil. Mem Inst Butantan 38: 69-117., Smith 1996SMITH TG. 1996. The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol 82: 565-585.). Colubridae Oppel, 1811 is a large and diversified snake family that encompasses seven subfamilies, of which only two, Dipsadinae Bonaparte, 1838 and Colubrinae Oppel, 1811, occur in Brazil and throughout the Amazon region (Pyron et al. 2013PYRON RA, BURBRINK FT & WIENS JJ. 2013. A phylogeny and revised classification of Squamata, including 4161 species of lizards and snakes. BMC Evol Biol 13: 93., Fraga et al. 2013FRAGA RD, LIMA AP, PRUDENTE ADC & MAGNUSSON WE. 2013. Guide to the snakes of the Manaus region—Central Amazonia. Editora INPA, Manaus.). The aims of the present study were, therefore, to investigate the occurrence of Hepatozoon spp. in the colubrid snakes, Clelia clelia (Daudin, 1803) and Drymarchon corais (Boie, 1827), and to characterize the specimens detected, using both morphological and molecular analyses.

MATERIALS AND METHODS

Study area and sample collection

In January 2016, one C. clelia specimen was captured in the Virola-Jatobá Sustainable Development Project (SDP) (-03° 9’ 28.15” S; -51° 27’ 51.67” W) in the municipal region of Anapu, Pará, Brazil. The SDP has an area of 38423.97 ha., with its resident human population surviving through sustainable forest management (Maestri et al. 2021MAESTRI MP, RUSCHEL AR, PORRO R, AQUINO MGC & MILÉO RC. 2021. Manejo florestal comunitário do Projeto de Desenvolvimento Sustentável Virola Jatobá: cenários para a exploração de Vouacapoua americana Aublet. Biodivers Bras 11: 1-17.). This area is formed by dense ombrophilous forest with medium and large trees (Correa et al. 2018CORREA JKC, HERNÁNDEZ-RUZ EJ, DE SANTANA MIGLIONICO MT & VIANA LA. 2018. A new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the twist-necked turtle Platemys platycephala (Schneider) (Testudines: Chelidae) in Brazil. Syst Parasitol 22: 93-103.). A specimen of D. corais was captured from the peri-urban region of the municipal district of Altamira (-03° 12’ 01.8” S; -52°11’ 29.0” W), Pará, Brazil, in May 2016. After mechanical containment of the animals, blood samples were collected by caudal vein puncture. For microscopic examination, blood smears were prepared, fixed in methanol, and stained with May-Grunwald-Giemsa solution (10%). The remaining blood samples were stored in EDTA tubes at −20 °C until DNA extraction. Snakes were euthanized with injectable anesthetic for veterinary use and fixed in 10% formalin, stored in 70% ethanol, and deposited in the collection of the Adriano Giorgi Laboratory of Zoology of the School of Biological Science, Altamira Campus, Pará, Brazil, as C. clelia (LZA 1416) and D. corais (LZA 1331). All procedures for snakes handling, sampling, and accessing genetic data were approved by the ethics committee on animal use from UNIFAP (protocol number 02/2020) and authorized by the Ministry of Environment of Brazil (SISBIO number 32401; SISGEN number AB23235).

Microscopic analyses

The search for parasites was performed under a Leica DM4B microscope (Leica Microsystems, Heerbrugg, Switzerland) at magnifications of × 400 and × 1000. Positive slides were carefully examined, and images were captured with an attached Leica DMC4500 digital camera and processed with the LAS V4.8 software platform (Leica Microsystems Suiza Limited 2015). The length, width and area of the gamonts and nuclei were measured using this system, and parasitemia was estimated by counting the number of parasites observed in 2,000 erythrocytes (Godfrey et al. 1987GODFREY Jr RD, FEDYNICH AM & PENCE DB. 1987. Quantification of haematozoa in blood smears. J Wildlife Dis 23: 558-565.).

DNA extraction, amplification, and sequencing

Genomic DNA extraction was performed following the phenol-chloroform protocol described by Sambrook et al. (1989)SAMBROOK J, FRITSCH EF & MANIATIS T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory.. The DNA isolated after the procedure was suspended in 30 µl of ultra-pure sterile water, and DNA quality was verified by electrophoresis on 1% agarose gel. Hepatozoon spp. detection was performed by conventional Polymerase Chain Reaction (PCR) using the HepF300 (5’-GTT TCT GAC CTATCA GCT TTC GAC G-3’) and Hep900 (5’-CAA ATC TAA GAA TTT CAC CTC TGA C-3’) primers, which amplifies approximately 600 bp of the 18S rRNA gene (Ujvari et al. 2004UJVARI B, MADSEN T & OLSSON M. 2004. High prevalence Hepatozoon spp. (Apicomplexa, Hepatozoidae) infection in water pythons (Liasis fuscus) from tropical Australia. J Parasitol 90: 670-672.). PCRs were performed in a total volume of 15 µl containing 1.5 µl of MgCl2 (25 mM), 1.25 µl of 10X PCR buffer (75 mM Tris-HCl, 50 mM KCl, 20 mM (NH4)2SO4), 1.25 µl of dNTPs (10 mM), 0.3U of Taq polymerase, 1.5 µl of each primer (10 uM), 1 µl of DNA (≈ 30-50 ng/µl) and 6.7 µl of nuclease free water. The PCR conditions were as follows: 92 °C for 1 min, 35 cycles at 92 °C for 1 min, 50 °C for 50 s, 72 °C for 1.5 min, and a final extension step at 72 °C for 7 min. Amplified products were purified and sequenced in a forward direction using the BigDye™ Terminator v.3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) and the ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Phylogenetic analysis

The obtained sequences were edited using the BioEdit software package v7.2.5 (Hall 1999HALL TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 95-98.) and compared in the GenBank database using the BLASTn software (http://www.ncbi.nlm.nih.gov/BLAST). The two sequences obtained in this study were aligned with 48 sequences published in GenBank® using the MUSCLE algorithm and the Geneious v.7.1.3 software package (Biomatters; http://www.geneious.com). Phylogenetic reconstructions were based on Bayesian Inference and Maximum Likelihood methods. The jModelTest v.2.1.10 (Darriba et al. 2012DARRIBA D, TABOADA GL, DOALLO R & POSADA D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9: 772.) was used to select the best evolution model for phylogenetic analysis. Based on the Akaike Information Criterion (AIC), HKY + G was the best model chosen for Bayesian Inference and TPM2uf + G was chosen for maximum likelihood. Bayesian Inference was implemented using MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003RONQUIST F & HUELSENBECK JP. 2003. MrBayes: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574.). Markov chain Monte Carlo (MCMC) simulations were run for 10,000,000 generations in two parallel runs, sampling one tree every 1,000 generations, with a burn-in of 25%. Maximum likelihood analysis was inferred using PhyML v.3.0 (Guindon et al. 2010GUINDON S, DUFAYARD J, LEFORT V, ANISIMOVA M, HORDIJK W & GASCUEL O. 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59: 307-321.), with 1000 bootstrap replicates. An estimate of the nucleotide divergence was obtained using a 441 bp alignment from the Hepatozoon sp. sequences obtained in this study and Hepatozoon spp. sequences isolated from Squamata available in the GenBank database. This analysis was performed using p-distance in the MEGA 6.0 software package (Tamura et al. 2013TAMURA K, STECHER G, PETERSON D, FILIPSKI A & KUMAR S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30: 2725-2729.).

RESULTS

Hepatozoon gamonts were detected in the blood samples from C. clelia and D. corais, with parasitemia values of 146 and 36 / 2,000 erythrocytes in the blood, respectively. Two different gamonts morphologies were found in each host (Figure 1, Table I).

Figure 1
Hepatozoon gamonts in the peripheral blood of snakes from the Eastern Amazon, Brazil. (a-c) Hepatozoon trigeminum from Clelia clelia. (d-f) Hepatozoon odwyerae sp. nov., from Drymarchon corais. Arrows indicate parasites; (n) indicates gamont nucleus; and (pv) indicates parasitophorous vacuole. Micrographs are from Giemsa-stained thin blood films. Scale bar is 10 μm.
Table I
Morphometric analysis of Hepatozoon spp. gamonts and their host cells detected in colubrid snakes from the Eastern Amazonia, Brazil. Measurements are presented as mean ± standard deviation (SD) followed by the range (maximum and minimum values).

In C. clelia (Figure 1a-c), gamonts were elongated and wide (14.25±0.65 × 4.31±0.43 μm; Table I), with both ends rounded and one pole more curved than the other, with an evident parasitophorous vacuole (PV), appearing as a ‘halo’, and a basophilic and granulous cytoplasm. The nucleus of this parasite was large and square-shaped (4.50±0.62 × 2.63±0.41 μm; Table I), eccentric and slightly displaced towards the curved end, with condensed dark stained chromatin filaments. This gamonts induce displacement of the host cell nucleus. No other cytopathological effect on host cells (dimensions: 19.05±1.15 × 10.94±0.79 μm) was noticed when compared with non-parasitized erythrocytes (dimensions: 18.00±1.25 × 9.58±0.94 μm) (Table I). For gamonts observed in D. corais, the morphological data are shown in the species description section below.

The blood samples from C. clelia and D. corais were also PCR-positive for Hepatozoon spp. In the BLAST search, the sequence obtained from C. clelia (OM032593) exhibited 99,60% of similarity with Hepatozoon trigeminum Úngari, Netherlands, Silva and O’Dwyer, 2022ÚNGARI LP, NETHERLANDS EC, SANTOS ALQ, ALCANTARA EP, EMMERICH E, SILVA RS & O’DWYER LH. 2021. Description of a New Species Hepatozoon quagliattus sp. nov. (Apicomplexa: Adeleorina: Hepatozoidae), infecting the Sleep Snake, Dipsas mikanii (Squamata: Colubridae: Dipsadinae) from Goiás State, Brazil. Acta Parasitol 66: 871-880. (ON262424) from Oxyrhopus trigeminus Duméril, Bibron and Duméril, 1854, 99.02% with Hepatozoon cuestensis O’Dwyer, Moço, Paduan, Spenassatto, Silva and Ribolla, 2013 (KC342524) from Crotalus durissus Linneus, 1758, and 98.64% with Hepatozoon sp. (AY252108) from Varanus scalaris Mertens, 1941. Hepatozoon sp. obtained from D. corais (OM032594), meanwhile, exhibited 99.79% of identity with Hepatozoon sp. (MF497768) from the Boa constrictor Linnaeus, 1758, 99.61% with Hepatozoon ameivae (Carini and Rudolphi, 1912) (MN833642), and 98.45% with Hepatozoon musa Borges-Nojosa, Borges-Leite, Maia, Zanchi-Silva, Braga and Harris, 2017 (KX880079) from Philondryas natterei (Steindachner, 1870). The genetic distance between the two Hepatozoon species obtained in this study was 1.21%. The pairwise distance between Hepatozoon sp. from C. clelia and the Hepatozoon spp. sequences from other squamates ranged from 0 to 7.51% (Table II). The p distance between Hepatozoon sp. from D. corais and the other Hepatozoon species ranged from 0.24 to 7.51% (Table II). Nucleotide divergence among the named Hepatozoon species from snakes and lizards ranged 0 to 8.23% (Table II).

Table II
Pairwise distances among partial 18S rDNA sequences of Hepatozoon spp. detected from snakes and lizards.

The phylogenetic tree (Figure 2) based on 441 bp partial sequences of the 18S rRNA gene revealed that the Hepatozoon sequences (OM032593; OM032594) generated in this study grouped in a clade composed of Hepatozoon spp. detected in snakes and lizards from Brazil. The sequence from C. clelia (OM032593) was positioned in a branch with the lineage of H. trigeminum (ON262424) (Fig. 2). In addition, Hepatozoon sp. (OM032594) obtained from D. corais clustered with Hepatozoon sp. (MF497768) from B. constrictor and formed a sister clade to H. ameivae from the Ameiva ameiva lizard (Linnaeus, 1758) (MN833642).

Figure 2
Consensus phylogenetic tree from Bayesian inference and maximum likelihood methods based on the 18S rRNA gene partial sequences (441bp) of Hepatozoon spp. isolated from Clelia clelia (OM032593) and Drymarchon corais (OM032594), and sequences deposited in in the GenBank database. Numbers at the nodes indicate posterior probabilities/bootstrap, represented on the Bayesian Inference tree. Posterior probabilities/bootstrap lower than 50 are not shown. Sequences obtained in this study are indicated in bold.

Considering the genetic divergence, phylogenetic positions and morphological features we identified the sequence isolated from C. clelia as H. trigeminum, and we proposed that the parasite found in D. corais is a new species of Hepatozoon.

Taxonomic summary

Genus Hepatozoon Miller, 1908

-Hepatozoon odwyerae sp. nov.

Type host: Drymarchon corais (Boie, 1827) (Colubridae: Colubrinae), Caninana, Indigo Snake.

Other hosts: Unknown.

Type locality: Peri-urban region in the municipal district of Altamira (-03° 12’ 01.8” S; -52° 11’ 29.0” W), Pará, Brazil.

Other locality: Unknown.

Type material: Hapantotype (two blood slides) from Drymarchon corais were deposited in the collection of the Adriano Giorgi Laboratory of Zoology of the School of Biological Sciences, Altamira Campus, Pará, Brazil (LZA – 1417 and 1418).

Site of infection: Blood erythrocytes.

Prevalence: one of one individual of Drymarchon corais was infected.

Parasitemia: The parasitemia was 36 parasites for every 2,000 erythrocytes (1.8%).

Vector: Unknown.

Gene sequence: The 18S ribosomal gene sequences were deposited in the GenBank database under accession number OM032594.

Zoobank registration: In accordance with section 8.5 of the International Code of Zoological Nomenclature (ICZN), details of the new species were submitted to Zoobank. The Life Science Identifier (LSID) for H. odwyerae sp. nov. is urn:lsid:zoobank.org:pub:AB980FE4-644B-427F-8B91-DFE2C1BDA5A1.

Etymology: The specific name, “odwyerae”, is a tribute to the parasitologist Prof. Lucia Helena O´Dwyer, from Universidade Estadual Paulista (Unesp), São Paulo, Brazil, in order to honor her scientific contribution to the knowledge of hemogregarines from Brazilian wildlife, especially with regard to snakes.

Description

Gamonts (Figure 1d-f, Table I): Elongated, thin and slightly curved; with one end tapered and the other rounded and flared; within a large PV; and with a purple-stained uniform cytoplasm. Nuclear chromatin usually diffuses in the cytoplasm; when condensed is elongated and with dark staining at one end or in the middle of the parasite. Gamonts measured (n = 30): 13.41±0.79 × 3.72±0.35μm; and area 43.04±4.04 μm2. Nucleus size (n = 30): 4.16±0.40 × 1.92±0.36 μm; and area 5.85±0.98 μm2.

Effects on the host cell: Gamonts cause displacement of the host cell nucleus to the lateral or superior region of the cell. No other morphological effects on host cells (dimensions: 18.48±1.01 × 10.40±0.53 μm) were noted when compared to non-parasitized erythrocytes (dimensions: 17.95±0.91 × 9.42±039 μm) (Table I).

Remarks: Sambom and Seligmann (1907) described a hemogregarine in a congeneric host Drymarchon couperi (Holbrook, 1842) –syn., Coluber corais couperi Holbr. –, kept at the London Zoo. This parasite was later named as Hepatozoon rarefaciens (Sambon and Seligmann, 1907SAMBON LW & SELIGMANN CG. 1907. Descriptions of five New Species of Haemogregarines from Snakes. Proc Zool Soc Lond 77: 283-284.) and found by Ball et al. (1967)BALL GH, CHAO J & TELFORD JR SR. 1967. The life history of Hepatozoon rarefaciens (Sambon and Seligmann, 1907) from Drymarchon corais (Colubridae), and its experimental transfer to Constrictor constrictor (Boidae). J Parasitol 53: 897-909. in high prevalences (n = 19/20) in specimens from North America. Hepatozoon rarefaciens gamonts have large and wide (15.4 x 5.5 µm) bodies, with both ends rounded, and cause remarkable hypertrophy in infected erythrocytes (Ball et al. 1967BALL GH, CHAO J & TELFORD JR SR. 1967. The life history of Hepatozoon rarefaciens (Sambon and Seligmann, 1907) from Drymarchon corais (Colubridae), and its experimental transfer to Constrictor constrictor (Boidae). J Parasitol 53: 897-909.). Thus, they differ remarkably from the small gamonts of H. odwyerae sp. nov. (13.41 × 3.72 μm). Compared to phylogenetically closely related hemogregarine species, H. ameivae, H. cuestensis, H. musa, H. trigeminum and Hepatozoon sp. (MF497768), previously detected in squamates from Brazil, H. odwyerae sp. nov. gamonts were morphologically and morphometrically different. Hepatozoon ameivae gamonts are longer and wider (14.28 × 4.50 µm) than those of H. odwyerae sp. nov., and, as previously mentioned, this parasite visibly interacts with the host cell nucleus, which is also not seen in the H. odwyerae sp. nov. parasites (Picelli et al. 2020PICELLI AM, SILVA MRL, RAMIRES AC, SILVA TRR, PESSOA FAC, VIANA LA & KAEFER IL. 2020. Redescription of Hepatozoon ameivae (Carini and Rudolph, 1912) from the lizard Ameiva ameiva (Linnaeus, 1758). Parasitol Res 119: 2659-2666.). Hepatozoon cuestensis and H. musa (O’Dwyer et al. 2013O’DWYER LH, MOÇO TC, PADUAN KS, SPENASSATTO C, SILVA RJ & RIBOLLA PEM. 2013. Descriptions of three new species of Hepatozoon (Apicomplexa, Hepatozoidae) from rattlesnakes (Crotalus durissus terrificus) based on molecular, morphometric and morphologic characters. Exp Parasitol 135: 200-207., Borges-Nojosa et al. 2017BORGES-NOJOSA DM, BORGES-LEITE MJ, MAIA JP, ZANCHI-SILVA D, BRAGA RR & HARRIS DJ. 2017. A new species of Hepatozoon Miller, 1908 (Apicomplexa: Adeleina) from the snake Philodryas nattereri Steindacher (Squamata: Dipsadidae) in northeastern Brazil. Syst Parasitol 94: 65-72.) have longer and thinner gamonts (17.05 × 3.6 µm and 18.94 × 3.76 µm, respectively) than H. odwyerae sp. nov. Additionally, both ends of H. musa gamonts are rounded and considerable curved, with the evident nuclei located in the middle of the parasite (Borges-Nojosa et al. 2017BORGES-NOJOSA DM, BORGES-LEITE MJ, MAIA JP, ZANCHI-SILVA D, BRAGA RR & HARRIS DJ. 2017. A new species of Hepatozoon Miller, 1908 (Apicomplexa: Adeleina) from the snake Philodryas nattereri Steindacher (Squamata: Dipsadidae) in northeastern Brazil. Syst Parasitol 94: 65-72.), differing from those in D. corais in this study. Regarding the other species found here in C. clelia, H. trigeminum has larger gamonts (14.25 × 4.31 µm) with very visible nuclei, which is not observed in H. odwyerae sp. nov. Finally, gamonts of the unnamed Hepatozoon sp. (MF497768) from the snake B. constrictor possess similar dimensions (13.3 × 4.6 µm) with H. odwyerae sp. nov., but in contrast to the latter both ends are rounded, there is a large and condensed nucleus, and their presence does not lead to displacement of the erythrocyte nucleus (Úngari et al. 2018ÚNGARI LP, SANTOS ALQ, O’DWYER LH, SILVA MRL, SANTOS TCR, CUNHA MJR, PINTO RMC & CURY MC. 2018. Molecular characterization and identification of Hepatozoon species Miller, 1908 (Apicomplexa: Adeleina: Hepatozoidae) in captive snakes from Brazil. Parasitol Res 117: 3857-3865.).

DISCUSSION

To the best of our knowledge, this is the first study to combine molecular and morphological data to identified two Hepatozoon species, H. trigeminum an H. odwyerae sp. nov., in C. clelia and D. corais snakes. These findings reinforce the fact that snakes are unique hosts for Hepatozoon parasites, and how much we have yet to learn about this diversity (Smith 1996SMITH TG. 1996. The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol 82: 565-585., Poulin 2014POULIN R. 2014. Parasite biodiversity revisited: frontiers and constraints. Int J Parasitol 44: 581-589.). So far, in Brazil, there were previously only seven named species of Hepatozoon spp. from snakes with available sequences (H. cevapii, H. cuestensis, Hepatozoon massardii O’Dwyer, Moço, Paduan, Spenassatto, Silva and Ribolla, 2013, H. musa, Hepatozoon quagliattus Úngari, Netherlands, Silva and O´Dwyer, 2021, Hepatozoon annulatum and Hepatozoon trigeminum Úngari, Netherlands, Silva and O’Dwyer, 2022), with H. cevapii the only one registered in the Amazonian biome (Paula et al. 2021PAULA FR, PICELLI AM, SILVA MRL, CORREA JKC & VIANA LA. 2021. Hepatozoon cevapii (Apicomplexa: Hepatozoidae) in the Thamnodynastes lanei snake (Colubridae, Tachymenini) from the Eastern Amazon, Brazil. Parasitol Res 120: 2981-2987.). Indeed, including our new sequences, this represents 20% (n = 8/40) of species morphologically described in Brazil (Smith 1996SMITH TG. 1996. The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol 82: 565-585., Úngari et al. 2022ÚNGARI LP, NETHERLANDS EC, SANTOS ALQ, ALCANTARA EP, EMMERICH E, SILVA RS & O’DWYER LH. 2022. 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). Parasitol Int 89: 102587.), a contradictory situation considering the modern molecular technologies and the improvements in parasitological research (Morand 2018MORAND S. 2018. Advances and challenges in barcoding of microbes, parasites, and their vectors and reservoirs. Parasitology 145: 537-542., Selbach et al. 2019SELBACH C ET AL. 2019. Parasitological research in the molecular age. Parasitology 146: 1361-1370.).

Other studies had already detected the presence of Hepatozoon parasites in both host species, but only using light microscopy techniques. Hemogregarines were reported in C. clelia in Costa Rica (Moreno & Bolanos 1977MORENO E & BOLANOS R. 1977. Haemogregarinas en serpientes de Costa Rica. Rev Biol Trop 25: 47-57.) and in French Guiana (Thoisy et al. 2000THOISY B, MICHEL JC, VOGEL & VIÉ JC. 2000. A survey of hemoparasite infections in free-ranging mammals and reptiles in French Guiana. J. Parasitol 86: 1035-1040.), with prevalences of 29% (n = 2/7) and 100% (n = 3/3), respectively. However, in the Costa Rica study, gamont description was carried out based on morphological group without specifying which morphology was observed in C. clelia (Moreno & Bolanos 1977MORENO E & BOLANOS R. 1977. Haemogregarinas en serpientes de Costa Rica. Rev Biol Trop 25: 47-57.). In the survey carried out in French Guiana, however, no information related to the morphology of the parasite was provided (Thoisy et al. 2000THOISY B, MICHEL JC, VOGEL & VIÉ JC. 2000. A survey of hemoparasite infections in free-ranging mammals and reptiles in French Guiana. J. Parasitol 86: 1035-1040.). Lutz (1901)LUTZ A. 1901. Sobre os drepanídios das serpentes. Uma contribuição para o conhecimento dos hemosporídios. In: BENCHIMOL JL & SÁ MR (Eds) Adolpho Lutz: Yellow fever, malaria and protozoology. 2005. Rio de Janeiro: Editora FIOCRUZ, p. 831-840., meanwhile, was the first to mention a hemogregarine parasitizing D. corais and other ophidians in Brazil, at the time he named Drepanidium serpentium Lutz, 1901, and which was later revised by Smith (1996)SMITH TG. 1996. The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol 82: 565-585. to Hepatozoon serpentium ([Lutz, 1901] Sambon, 1907). Nevertheless, when carefully analyzing Lutz’s study (1901), it is difficult to be certain about the validity of this species, or compare it with H. odwyerae sp. nov., as despite the morphological data presented, the author considered different gamont morphologies, obtained from several host species, as a single species, and did not mention which of the gamonts was seen in the blood from D. corais. Thus, in these studies, there are unfortunately not enough data to allow comparisons with H. trigeminum and H. odwyerae sp. nov.

Here we provide the first record of H. trigeminum in a new host species, C. clelia, at relatively high parasitemia level (7.3%). This hemogregarine species was recently described by Úngari et al. (2022)ÚNGARI LP, NETHERLANDS EC, SANTOS ALQ, ALCANTARA EP, EMMERICH E, SILVA RS & O’DWYER LH. 2022. 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). Parasitol Int 89: 102587. infecting, with a parasitemia of 1.4%, a single specimen of the O. trigeminus snake from municipality of Cocalinho, State of Mato Grosso, in Brazilian Midwest. Hepatozoon parasites are widely recognized for their low specificity to vertebrate hosts (Paula et al. 2021PAULA FR, PICELLI AM, SILVA MRL, CORREA JKC & VIANA LA. 2021. Hepatozoon cevapii (Apicomplexa: Hepatozoidae) in the Thamnodynastes lanei snake (Colubridae, Tachymenini) from the Eastern Amazon, Brazil. Parasitol Res 120: 2981-2987., Úngari et al 2022), so it is not unexpected to find H. trigeminium in two different host species. Clelia clelia and O. trigeminus are Dipsadinae snakes belonging to the Pseudoboini tribe and are phylogenetically closely related (Pyron et al. 2013PYRON RA, BURBRINK FT & WIENS JJ. 2013. A phylogeny and revised classification of Squamata, including 4161 species of lizards and snakes. BMC Evol Biol 13: 93.), in addition their geographic distribution in Brazil overlaps in some localities, as in the region where the two studies were carried out (Nogueira et al. 2019NOGUEIRA CC ET AL. 2019. Atlas of Brazilian Snakes: Verified point-locality maps to mitigate the Wallacean shortfall in a megadiverse snake fauna. South Am J Herpetol 14: 1-274., Costa et al. 2022COSTA HC, GUEDES TB & BÉRNILS RS. 2022. Lista de répteis do Brasil: padrões e tendências. Herpetologia Brasileira 10: 110-279.). This may make it easier for different species to share potential vectors and potential prey (paratenic hosts), which could aid H. trigeminum in switching hosts successfully.

According to the morphological description made by Úngari et al. (2022)ÚNGARI LP, NETHERLANDS EC, SANTOS ALQ, ALCANTARA EP, EMMERICH E, SILVA RS & O’DWYER LH. 2022. 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). Parasitol Int 89: 102587., the H. trigeminum forms observed in the blood of C. clelia resemble immature gamonts, although in our study the parasites are slightly smaller in their average dimensions (this study: body 14.25 × 4.31 μm, and nucleus 4.50 × 2.63 μm; Úngari et al. (2022)ÚNGARI LP, NETHERLANDS EC, SANTOS ALQ, ALCANTARA EP, EMMERICH E, SILVA RS & O’DWYER LH. 2022. 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). Parasitol Int 89: 102587.: body 14.53 × 4.71 μm, and nucleus 4.63 × 4.57 μm). Furthermore, the authors describe the nucleus of this parasitic form as oval and here, according to our perception, it appears to be more square-shaped. However, these variations are very subtle and may be related to the number of parasites measured (this study: 30; Úngari et al. (2022)ÚNGARI LP, NETHERLANDS EC, SANTOS ALQ, ALCANTARA EP, EMMERICH E, SILVA RS & O’DWYER LH. 2022. 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). Parasitol Int 89: 102587.: 25), the tools used in both studies, the subjective effect of the observer performing the description, or due gamont plasticity in different host species (Perkins et al. 2011PERKINS SL, MARTINSEN ES & FALK BG. 2011. Do molecules matter more than morphology? Promises and pitfalls in parasites. Parasitology 138: 1664-1674., Paula et al. 2021PAULA FR, PICELLI AM, SILVA MRL, CORREA JKC & VIANA LA. 2021. Hepatozoon cevapii (Apicomplexa: Hepatozoidae) in the Thamnodynastes lanei snake (Colubridae, Tachymenini) from the Eastern Amazon, Brazil. Parasitol Res 120: 2981-2987., Úngari et al. 2022ÚNGARI LP, NETHERLANDS EC, SANTOS ALQ, ALCANTARA EP, EMMERICH E, SILVA RS & O’DWYER LH. 2022. 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). Parasitol Int 89: 102587.).

Regarding the natural history of C. clelia and D. corais, both species are widely distributed through South America. In Brazil, however, C. clelia occurs mainly in the Amazonian region, while D. corais was recorded in all ecoregions, except the Araucaria Forest and Pampas Grasslands (Nogueira et al. 2019NOGUEIRA CC ET AL. 2019. Atlas of Brazilian Snakes: Verified point-locality maps to mitigate the Wallacean shortfall in a megadiverse snake fauna. South Am J Herpetol 14: 1-274.). The mussurana C. clelia is a large Dipsadinae snake, reaching up to 2.3 m in length, and is primarily terrestrial and nocturnal, inhabiting forested areas (Gaiarsa et al. 2013GAIARSA MP, ALENCAR LR & MARTINS M. 2013. Natural history of Pseudoboine snakes. Pap Avulsos Zool 53: 261-283.). Although it is considered a generalist species (Alencar et al. 2013ALENCAR LR, GAIARSA MP & MARTINS M. 2013. The evolution of diet and microhabitat use in pseudoboine snakes. South Am J Herpetol 8: 60-66.), it is well known for its preference for feeding on other snakes (ophiophagy), including highly venomous species (i.e., Bothrops spp.) (Delia 2009DELIA J. 2009. Another crotaline prey item of the Neotropical snake Clelia clelia (Daudin 1803). Herpetol Notes 2: 21-22., Fraga et al. 2013FRAGA RD, LIMA AP, PRUDENTE ADC & MAGNUSSON WE. 2013. Guide to the snakes of the Manaus region—Central Amazonia. Editora INPA, Manaus.). The indigo snake D. corais, meanwhile, is a diurnal Colubrinae species with semi-arboreal habits, found in forests and open areas, with few studies suggesting that it has a generalist diet, preying mainly on anurans and eventually other snakes (Bernarde & Abe 2010BERNARDE PS & ABE AS. 2010. Hábitos alimentares de serpentes em Espigão do oeste, Rondônia, Brasil. Biota Neotrop 10: 167-173., Prudente et al. 2014PRUDENTE ALC, MENKS AC, SILVA FM & MASCHIO GF. 2014. Diet and reproduction of the western indigo snake Drymarchon corais (serpentes: Colubridae) from the Brazilian Amazon. Herpetol Notes 7: 99-108., Pelegrini et al. 2019PELEGRINI SJS, VENÂNCIO NM & KUNIY AA. 2019. Note on an event of double predation: Leptodeira annulata (Linnaeus, 1758) (Serpentes: Dipsadidae) being predated by Drymarchon corais Boie, 1827 (Serpentes: Colubridae), being predated in turn by Bothrops atrox (Linnaeus, 1758) (Serpentes: Viperidae). Herpetol Notes 12: 1193-1195.). Despite other items in the diet of these snakes (frogs, lizards, and small mammals) (Gaiarsa et al. 2013GAIARSA MP, ALENCAR LR & MARTINS M. 2013. Natural history of Pseudoboine snakes. Pap Avulsos Zool 53: 261-283., Prudente et al. 2014PRUDENTE ALC, MENKS AC, SILVA FM & MASCHIO GF. 2014. Diet and reproduction of the western indigo snake Drymarchon corais (serpentes: Colubridae) from the Brazilian Amazon. Herpetol Notes 7: 99-108.), which are the most indicated paratenic hosts of Hepatozoon (Lainson et al. 2003LAINSON R, PAPERNA I & NAIFF RD. 2003 Development of Hepatozoon caimani (Carini, 1909) Pessôa, De Biasi & De Souza, 1972 in the caiman Caiman c. crocodilus, the frog Rana catesbeiana and the mosquito Culex fatigans. Mem Inst Oswaldo Cruz 98: 103-113., Paperna & Lainson 2004PAPERNA I & LAINSON R. 2004. Hepatozoon cf. terzii (Sambon & Seligman, 1907) infection in the snake Boa constrictor constrictor from north Brazil: transmission to the mosquito Culex quinquefasciatus and the lizard Tropidurus torquatus. Parasite 11: 175-181., Perles et al. 2019PERLES L ET AL. 2019. Genetic diversity of Hepatozoon spp. in rodents from Brazil. Sci Rep 9: 10122.), ophiophagy reveals other possible routes of infection, with snakes also potentially acting in the trophic transmission of the parasite. This hypothesis requires testing, but may be plausible, considering the high diversity and low specificity of hemogregarines (Smith 1996SMITH TG. 1996. The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol 82: 565-585., Paula et al. 2021PAULA FR, PICELLI AM, SILVA MRL, CORREA JKC & VIANA LA. 2021. Hepatozoon cevapii (Apicomplexa: Hepatozoidae) in the Thamnodynastes lanei snake (Colubridae, Tachymenini) from the Eastern Amazon, Brazil. Parasitol Res 120: 2981-2987.), the lack of elucidated life cycles of these parasites, and also due to the various ophiophagus vertebrate species (Pelegrini et al. 2019PELEGRINI SJS, VENÂNCIO NM & KUNIY AA. 2019. Note on an event of double predation: Leptodeira annulata (Linnaeus, 1758) (Serpentes: Dipsadidae) being predated by Drymarchon corais Boie, 1827 (Serpentes: Colubridae), being predated in turn by Bothrops atrox (Linnaeus, 1758) (Serpentes: Viperidae). Herpetol Notes 12: 1193-1195.), including other snakes recorded as being parasitized by Hepatozoon in Brazil (Pessôa et al. 1974PESSÔA SB, DE BIASI P & PUORTO G. 1974. Nota sobre a frequência de hemoparasitas em serpentes no Brasil. Mem Inst Butantan 38: 69-117., Borges-Nojosa et al. 2017BORGES-NOJOSA DM, BORGES-LEITE MJ, MAIA JP, ZANCHI-SILVA D, BRAGA RR & HARRIS DJ. 2017. A new species of Hepatozoon Miller, 1908 (Apicomplexa: Adeleina) from the snake Philodryas nattereri Steindacher (Squamata: Dipsadidae) in northeastern Brazil. Syst Parasitol 94: 65-72.), such as Erythrolamprus aesculapii (Linnaeus, 1758), Hydrodynastes gigas (Dúmeril, Bibron and Dúmeril, 1854), O. trigeminus and Philodryas nattereri (Steindachner, 1870) (Marques & Puorto 1994MARQUES OAV & PUORTO G. 1994. Dieta e comportamento alimentar de Erythrolamprus aesculapii, uma serpente ofiófaga. Rev Brasil Biol 54: 253-259., López & Giraudo 2004LÓPEZ MS & GIRAUDO A. 2004. Diet of the large water snake Hydrodynastes gigas (Colubridae) from northeast Argentina. Amphib-Reptil 25: 178-184., Coelho-Lima et al. 2020COELHO-LIMA AD, OLIVEIRA RAMOS G, MARTINS RBX & CASTRO MEIRA LPD. 2020. Fist record of ophiophagy in the false coral snake Oxyrhopus trigeminus Duméril, Bibron & Duméril, 1854. Cuad Herpetol 34: 89-91., Sales et al. 2020SALES RFD, SOUSA JD, LISBOA CMC, MARINHO PH, FREIRE EMX & KOKUBUM MNDC. 2020. New dietary records and geographic variation in the diet composition of the snake Philodryas nattereri in Brazil. Cuad herpetol 34: 285-293.).

The phylogenetic assessment in the present study found that sequences of H. odwyerae sp. nov. and H. trigeminum were clustered into the “non-carnivorous” major clade along with other Hepatozoon lineages from herpetofauna (Zechmeisterová et al. 2021ZECHMEISTEROVÁ K, JAVANBAKHT H, KVIČEROVÁ J & ŠIROKÝ P. 2021. Against growing synonymy: Identification pitfalls of Hepatozoon and Schellackia demonstrated on North Iranian reptiles. Eur J Protistol 79: 125780.). These new species were positioned in a well-supported monophyletic group composed exclusively of lineages isolated from Squamata sampled in different Brazilian biomes. In our study this clade maintained a similar topology to previous analyzes (Paula et al. 2021PAULA FR, PICELLI AM, SILVA MRL, CORREA JKC & VIANA LA. 2021. Hepatozoon cevapii (Apicomplexa: Hepatozoidae) in the Thamnodynastes lanei snake (Colubridae, Tachymenini) from the Eastern Amazon, Brazil. Parasitol Res 120: 2981-2987., Úngari et al. 2022ÚNGARI LP, NETHERLANDS EC, SANTOS ALQ, ALCANTARA EP, EMMERICH E, SILVA RS & O’DWYER LH. 2022. 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). Parasitol Int 89: 102587.) with the formation of three subclades: (i) a small cluster formed by two lineages, the sequences of H. cuestensis in C. durissus from the Cerrado (O’Dwyer et al. 2013O’DWYER LH, MOÇO TC, PADUAN KS, SPENASSATTO C, SILVA RJ & RIBOLLA PEM. 2013. Descriptions of three new species of Hepatozoon (Apicomplexa, Hepatozoidae) from rattlesnakes (Crotalus durissus terrificus) based on molecular, morphometric and morphologic characters. Exp Parasitol 135: 200-207., Úngari et al. 2018ÚNGARI LP, SANTOS ALQ, O’DWYER LH, SILVA MRL, SANTOS TCR, CUNHA MJR, PINTO RMC & CURY MC. 2018. Molecular characterization and identification of Hepatozoon species Miller, 1908 (Apicomplexa: Adeleina: Hepatozoidae) in captive snakes from Brazil. Parasitol Res 117: 3857-3865.), plus H. musa in C. durissus and P. nattereri from the Cerrado and Caatinga, respectively (Borges-Nojosa et al. 2017BORGES-NOJOSA DM, BORGES-LEITE MJ, MAIA JP, ZANCHI-SILVA D, BRAGA RR & HARRIS DJ. 2017. A new species of Hepatozoon Miller, 1908 (Apicomplexa: Adeleina) from the snake Philodryas nattereri Steindacher (Squamata: Dipsadidae) in northeastern Brazil. Syst Parasitol 94: 65-72., Úngari et al. 2018ÚNGARI LP, SANTOS ALQ, O’DWYER LH, SILVA MRL, SANTOS TCR, CUNHA MJR, PINTO RMC & CURY MC. 2018. Molecular characterization and identification of Hepatozoon species Miller, 1908 (Apicomplexa: Adeleina: Hepatozoidae) in captive snakes from Brazil. Parasitol Res 117: 3857-3865.); (ii) a subclade consisting of Hepatozoon sp. in B. constrictor from the Cerrado (Úngari et al. 2018ÚNGARI LP, SANTOS ALQ, O’DWYER LH, SILVA MRL, SANTOS TCR, CUNHA MJR, PINTO RMC & CURY MC. 2018. Molecular characterization and identification of Hepatozoon species Miller, 1908 (Apicomplexa: Adeleina: Hepatozoidae) in captive snakes from Brazil. Parasitol Res 117: 3857-3865.) plus H. odwyerae sp. nov. in D. corais from the Amazonia (the present study), and, closely related to them, the lineage of H. ameivae in A. ameiva from the Amazonia (Picelli et al. 2020PICELLI AM, SILVA MRL, RAMIRES AC, SILVA TRR, PESSOA FAC, VIANA LA & KAEFER IL. 2020. Redescription of Hepatozoon ameivae (Carini and Rudolph, 1912) from the lizard Ameiva ameiva (Linnaeus, 1758). Parasitol Res 119: 2659-2666.); and (iii) the small clade containing the sequences of H. trigeminum in C. clelia and O. trigeminus from the Amazonia (the present study) and Cerrado (Úngari et al. 2022ÚNGARI LP, NETHERLANDS EC, SANTOS ALQ, ALCANTARA EP, EMMERICH E, SILVA RS & O’DWYER LH. 2022. 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). Parasitol Int 89: 102587.), respectively, which comprises a sister taxon to all other lineages within this group. This may be the result of a possible biogeographic pattern (Harris et al. 2015HARRIS DJ, BORGES-NOJOSA DM & MAIA JP. 2015. Prevalence and diversity of Hepatozoon in native and exotic geckos from Brazil. J Parasitol 101: 80-85., Perles et al. 2019PERLES L ET AL. 2019. Genetic diversity of Hepatozoon spp. in rodents from Brazil. Sci Rep 9: 10122.); however, a larger sampling of hemogregarines is needed to test such a hypothesis. Furthermore, phylogenetic relationships among reptilian hemogregarines remain poorly understood.

With respect to second subclade, in addition to being phylogenetically close, H. odwyerae sp. nov. and Hepatozoon sp. (MF497767) exhibited low nucleotide divergence (0.24%), which indicates that this new sequence may be a haplotype of the lineage detected by Úngari et al. (2018)ÚNGARI LP, SANTOS ALQ, O’DWYER LH, SILVA MRL, SANTOS TCR, CUNHA MJR, PINTO RMC & CURY MC. 2018. Molecular characterization and identification of Hepatozoon species Miller, 1908 (Apicomplexa: Adeleina: Hepatozoidae) in captive snakes from Brazil. Parasitol Res 117: 3857-3865.. These authors suggest that Hepatozoon sp. (MF497767) from B. constrictor may be considered a new putative species, although despite presenting morphological and genetic data, they did not describe it taxonomically. However, this small p distance is similar to the divergence observed between H. cevapii and H. massardii, which are closely related and considered two distinct species (O’Dwyer et al. 2013O’DWYER LH, MOÇO TC, PADUAN KS, SPENASSATTO C, SILVA RJ & RIBOLLA PEM. 2013. Descriptions of three new species of Hepatozoon (Apicomplexa, Hepatozoidae) from rattlesnakes (Crotalus durissus terrificus) based on molecular, morphometric and morphologic characters. Exp Parasitol 135: 200-207., Úngari et al. 2018ÚNGARI LP, SANTOS ALQ, O’DWYER LH, SILVA MRL, SANTOS TCR, CUNHA MJR, PINTO RMC & CURY MC. 2018. Molecular characterization and identification of Hepatozoon species Miller, 1908 (Apicomplexa: Adeleina: Hepatozoidae) in captive snakes from Brazil. Parasitol Res 117: 3857-3865., 2021, Paula et al. 2021PAULA FR, PICELLI AM, SILVA MRL, CORREA JKC & VIANA LA. 2021. Hepatozoon cevapii (Apicomplexa: Hepatozoidae) in the Thamnodynastes lanei snake (Colubridae, Tachymenini) from the Eastern Amazon, Brazil. Parasitol Res 120: 2981-2987.). In addition to the morphological differences between H. odwyerae sp. nov. and Hepatozoon sp. (MF497767), highlighted in the remarks section, there are recognized limitations to the use of the 18S rRNA genetic marker to distinguish different species, especially when there is low molecular divergence (Gutiérrez-Liberato et al. 2021GUTIÉRREZ-LIBERATO GA, LOTTA-ARÉVALO IA, RODRÍGUEZ-ALMONACID CC, VARGAS-RAMÍREZ M & MATTA NE. 2021. Molecular and morphological description of the first Hepatozoon (Apicomplexa: Hepatozoidae) species infecting a neotropical turtle, with an approach to its phylogenetic relationships. Parasitol 148: 747-759., Hrazdilová et al. 2021HRAZDILOVÁ K, ČERVENÁ B, BLANVILLAIN C, FORONDA P & MODRÝ D. 2021. Quest for the type species of the genus Hepatozoon–phylogenetic position of hemogregarines of rats and consequences for taxonomy. Syst Biodivers 19: 1-10., Léveillé et al. 2021LÉVEILLÉ AN, ZELDENRUST EG & BARTA JR. 2021. Multilocus genotyping of sympatric Hepatozoon species infecting the blood of Ontario ranid frogs reinforces species differentiation and identifies an unnamed Hepatozoon species. J Parasitol 107: 246-261.). At the present time, then, we cannot confirm that the parasite found by Úngari et al. (2018)ÚNGARI LP, SANTOS ALQ, O’DWYER LH, SILVA MRL, SANTOS TCR, CUNHA MJR, PINTO RMC & CURY MC. 2018. Molecular characterization and identification of Hepatozoon species Miller, 1908 (Apicomplexa: Adeleina: Hepatozoidae) in captive snakes from Brazil. Parasitol Res 117: 3857-3865. belongs to the same species as that described in the present study.

In conclusion, the diversity of hemogregarines from snake hosts in Brazil is still greatly underestimated, especially in the Amazon region. The present study provides the first molecular detection of Hepatozoon spp. from C. clelia and D. corais, and the combination of this data with morphological traits allowed us to describe a new species and register a new host species from a new geographic location. Further studies with H. trigeminum and H. odwyerae sp. nov. should include other molecular markers to disentangle the puzzle of their phylogenetic relationships with other hemogregarines, especially in relation to the closeness between H. odwyerae sp. nov. and Hepatozoon sp. from B. constrictor. It is also important to try to identify vectors and explore hypotheses about transmission routes in ophiophagus snakes.

ACKNOWLEDGMENTS

We are grateful to the Programa de Desenvolvimento da Pós-Graduação (PDPG - Amazônia Legal / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and to the One Health Project in Areas of Urban and Peri-Urban Streams from Porto Velho (SUIg_PVH - Saúde Única nas Áreas de Igarapés Urbanos e Periurbanos de Porto. Velho / UNIR – Universidade Federal de Rondônia / CAPES) for the Postdoctoral Fellowship to AMP; to the Laboratório Temático de Microscopia Óptica e Eletrônica (Thematic Laboratory of Optical and Electronic Microscopy, or LTMO/CPAAF/INPA) for allowing the use of the equipment and imaging system; and to Adriane C. Ramires for editing the figures. This study was supported by the Brazilian National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq Universal 429.132/2016-6 to LAV).

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Publication Dates

  • Publication in this collection
    29 May 2023
  • Date of issue
    2023

History

  • Received
    4 Feb 2022
  • Accepted
    22 Dec 2022
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