Genetic diversity of Hepatozoon spp. in rodents from Chile

This study aimed to investigate the genetic diversity of Hepatozoon spp. in rodents from Valdivia, Chile. A total of 74 rodents (synanthropic n=38; wild n=36) were trapped in Valdivia. We performed conventional PCR assays for Apicomplexa organisms targeting two overlapping 18S rDNA gene fragments (600 bp and 900 bp) followed by sequencing of selected amplicons. Hepatozoon spp. occurrence was 82.43% (61/74). Twelve sequences obtained from the 600 bp and ten from the 900 bp 18S rDNA fragments were identified as Hepatozoon sp. Six sequences obtained from 18S rDNA-based overlapping PCR protocols were used for concatenated (1,400 bp) phylogenetic, haplotype and distance analyses. Hepatozoon spp. 18S rDNA concatenated sequences from the present study were detected in Oligoryzomys longicaudatus , Rattus norvegicus , Mus musculus , and Abrothrix longipilis grouped with Hepatozoon species earlier described in rodents and reptiles from Chile and Brazil. Nucleotide polymorphism of the six 18S rDNA sequences (1,400 bp) from this study, and other Chilean sequences from rodents and rodent’s ticks, showed high diversity with a total of nine Chilean haplotypes. Three haplotypes from Valdivia were identified for the first time in this study, suggesting the circulation of novel haplotypes in rodents from southern Chile.


Introduction
The genus Hepatozoon (Adeleorina; Hepatozoidae) comprises apicomplexan parasites that were first detected in India (Bentley, 1905).Since then, many species have been described, from various vertebrate hosts, such as mammals, birds, reptiles, and amphibians.Although not considered a primary zoonotic pathogen, zoonotic potential has been described, as Hepatozoon sp. was detected in Russian and Philippine patients (Craig, 2006;Lappin, 2010;Shuĭkina et al., 2004).
In Chile, although the presence of Hepatozoon was confirmed in rodents, marsupials, and associated ectoparasites, little is known about the genetic diversity of this group of tick-borne pathogens.Hepatozoon sp. was detected in 54.5% (6/11) of Olive gray mouse (Abrothrix olivaceus) and 50% (2/4) of long-haired akodont (Abrothrix sanborni) sampled in Senda Darwin Biological Station and forest in Chiloé Island, southern Chile (Merino et al., 2009).Recently, Hepatozoon spp. was detected in Ixodes sp. and Ornithodoros atacamensis ticks collected from wild rodents (Phyllotis darwini) in national parks of Pan de Azúcar and Bosque Fray Jorge, in Northern Chile (Muñoz-Leal et al., 2019).Phylogenetic analysis of Hepatozoon sp.detected in ectoparasites from Chile showed its relatedness to Hepatozoon spp.detected in the marsupial Monito del monte (Dromiciops gliroides) and Olive gray mouse (A.olivaceus) from Chile (Merino et al., 2009).
This study aimed to investigate the genetic diversity of Hepatozoon spp. in wild and synanthropic rodents from the Valdivia province, southern Chile.

Rodent trapping and sampling
The trapping occurred from November 2016 and November 2017, on dairy farms from the Valdivia province.Traps (20 cm~ 20 cm~ 60 cm Tomahawk cages) were baited with oatmeal and vanilla flavoring, placed in areas where rodents were usually seen and reviewed daily, during the morning for the period of four days.Any endangered, threatened, or protected species were immediately released.
The captured rodents were euthanized in the Pathology building of the Universidad Austral de Chile (UACh).Euthanasia was performed using a lethal dose, equivalent to 5 times the anesthetic dose (inhaled Isoflurane, followed by an intraperitoneal injection of a combination of Xylazine-Ketamine) (Hedenqvist & Hellebrekers, 2003).Each rodent's spleen was aseptically removed after euthanasia, stored and preserved in 70% Ethanol (Merck © , USA) at -20°C, until further analyses.
The capture, management, and euthanasia of rodents followed the specifications of the American Society of Mammologists and the protocols and norms established by the funding agency (CONICYT, 2008).Biological protocols for dangerous material were used for carcasses disposal.

DNA Extraction from Rodent Spleen and PCR for Mammals' endogenous gene
The frozen rodent spleens were thawed at room temperature and 15 mg portions were refrozen with liquid nitrogen and manually macerated with a plastic pestle.DNA extraction of the macerated spleen suspension was performed with the "Tissue DNA Kit" (E.Z.N.A. Omega BioTek ® , Norcross, GA, USA), as per the manufacturer's instructions (100 µL elution).A spectrophotometer (NanoDrop ND-1000 Thermo Scientific © , Waltham, MA, USA) was used for measuring the DNA concentration and absorbance ratio (260/280nm).Nuclease-free water (Thermo Scientific © , USA) was used as a template to verify cross-contamination, every 20 extractions.DNA was stored at −20 °C before performing PCR assays.
To verify the presence of amplifiable DNA and check the integrity of the DNA template, the irbp ("interphotoreceptor retinoid-binding protein") endogenous mammalian gene was used (Ferreira et al., 2010).Positive samples for the irbp gene were submitted to a conventional PCR protocol to amplify a fragment (600 bp) of the 18S rDNA of Apicomplexa organisms, as previously described (Vilcins et al., 2009).All PCR runs were performed with nuclease-free water (Thermo Scientific © , Waltham, MA, USA) as a negative control and Taq DNA polymerase (Life technologies © , Carlsbad, CA, USA) for amplification.Hepatozoon caimani DNA obtained from naturally infected crocodiles was used as a positive control (Bouer et al., 2017).
All the samples were further tested for a second 18S rDNA-based PCR protocol targeting a complimentary (900 bp) fragment of Apicomplexan organisms (Perkins & Keller, 2001), aiming at obtaining a larger portion of the 18S rDNA gene (1,400 bp) for phylogenetic and haplotype analyses.
Both 600 bp and 900 bp 18S rDNA amplicons showing high intensity bands in agarose gel electrophoresis were purified using "Silica Bead DNA Gel Extraction Kit" (Fermentas, São Paulo-SP), following the manufacturer's instructions, and sent to the Center of Biological Resources and Genomic Biology (CREBIO, Jaboticabal, SP, Brazil) for sequencing by Sanger's method with ABI PRISM 3700 DNA Analyzer (Applied Biosystems © , Foster city, CA, USA).Only sequences obtained for both overlapping 18S rDNA-based PCR protocols were used for concatenated (1,400 bp) phylogenetic and haplotype analyses.

BLAST Analysis
Electropherograms were submitted to PhredPhrap analysis (Ewing et al., 1998), with the Phred quality score (peaks around each base call) established as higher than 20 (99% accuracy of the base call), to determine the nucleotide composition.We performed a BLAST analysis to evaluate the identity percentage of our sequences with those deposited in the GenBank database (Altschul et al., 1990;Benson et al., 2004).

Splits network analysis
Splits tree v4.11.3 (Huson, 1998) was used to generate a phylogenetic distance network with sequences obtained from the present study and sequences from Genbank (Supplementary Table S1).Final trees and haplotype network design were created with Biorender.com(Biorender, 2020).

Phylogenetic analysis
According to the phylogenetic inference, five different clades were observed, two of them represented by Hepatozoon spp.(Figure 2).Both subclades containing the concatenated sequences from Chile shared a common ancestor.Hepatozoon 18S rDNA sequences from rodents in Valdivia were closely related to those previously detected in rodents and reptiles, while distant from carnivore related Hepatozoon species.
Four haplotypes were found within the Corral locality (haplotypes #1, #2, #3 and #4): while haplotypes #1 and #2 were detected in R. norvegicus, haplotype #3 was identified in A. longipilis, and haplotype #4 in M. musculus.Additionally, Reumén showed only one haplotype (#5).For the other localities reported in previously published studies (northern Chile) one haplotype was observed in a rodent tick from Pan de Azúcar (northern Chile) (#7), two (haplotypes #6 and #8) were detected in rodents' ticks from National Park Bosque Fray Jorge (northern Chile) and three haplotypes were found in rodents from Chiloé (southern Chile) (haplotypes #1, #3 and #9).The only localities that shared haplotypes were Corral (this study, southern Chile) and Chiloé (haplotypes #1 and #3).Table S1 summarizes the polymorphism and genetic diversity of 18S rDNA sequences of Hepatozoon species detected in rodents from Valdivia.The haplotype network is presented (Figure 3).
Hepatozoon species are widely distributed geographically (Baneth et al., 2000;Latrofa et al., 2014), and have been detected by PCR screening in a broad variety of hosts from America, Africa, Europe, and Asia (Criado-Fornelio et al., 2006;Moustafa et al., 2017).Although Hepatozoon spp.were previously reported in rodents (A.olivaceus and A. sanborni) and in a marsupial (D. gliroides), both from Chiloé Island, southern Chile (Merino et al., 2009) as well as in ticks collected from rodents in northern Chile (Muñoz-Leal et al., 2019), little is known about their genetic diversity.To the best of the authors' knowledge, this is the first report of Hepatozoon spp. in rodents belonging to the following species: M. musculus, R. norvegicus, R. rattus, A. longipilis, and O. longicaudatus from Chile.Additionally, the nucleotide diversity and the haplotype structure of Hepatozoon species were evaluated for the first time in biological samples of rodents and rodents' ticks from Chile, using the 18S rDNA gene.
According to the phylogenetic inference of the 18S rDNA gene, Hepatozoon spp.sequences detected in rodents from the Valdivia province were grouped into two clades, and separated from carnivore Hepatozoon spp.by the genus Karyolysus, corroborating previous studies (Maia et al., 2016a;Karadjian et al., 2015).The clustering patterns observed in our results were similar to those described by Maia et al. (2016a) and Karadjian et al. (2015).As such, Hepatozoon sequences were separated between carnivores (lineages M-P) and rodents, reptiles, amphibian, ticks, marsupials and birds (lineages D-H).Hepatozoon spp.sequences from synanthropic and wild rodents from the present study were positioned with other Hepatozoon sequences from lineage H.
The nucleotide polymorphism analysis of Hepatozoon concatenated 18S rDNA sequences were diverse with a high number of haplotypes (n=5) among the population of sampled rodents, with some of the haplotypes (n=3) only identified in the present study, suggesting that novel haplotypes occur in rodents from the Valdivia province, southern Chile.Haplotype diversity is influenced by multiple processes, such as mutation, recombination, and demography (Stumpf, 2004).The haplotype diversity of Hepatozoon spp.found in rodents in the present study [(Hd) = 0.933] was higher than the one described [(Hd) = 0.426] by Perles et al. (2019) in rodents from Brazil.The former study covered a much broader geographic area, whereas the Chilean samples were collected only within the Valdivia province (southern Chile).Other studies, based on 18S rDNA sequence data, found four 18S rDNA Hepatozoon haplotypes in capybaras (Hydrochoerus hydrochaeris) from northern Brazil (de Azevedo Gomes et al., 2018), and three Hepatozoon haplotypes in rodents (Thrichomys fosteri) from the Brazilian Pantanal (de Sousa et al., 2017).
The haplotype analysis network showed a possible haplotype affinity to certain rodent groups, disregarding the geographic location.For instance, the Corral locality presented a variety of haplotypes associated with R. rattus (synanthropic, haplotypes #1 and #2), A. longipilis (wild, haplotype #3), and M. musculus (synanthropic, haplotype #4); while, Reumén showed only one haplotype, found in O. longicaudatus (wild, haplotype #5).Different rodent groups (synanthropic versus sylvatic) and genera may harbor different haplotypes of Hepatozoon spp.However, Hepatozoon spp.are known to have low host specificity.Host preference for Hepatozoon haplotypes in rodents was previously described in Finland, Estonia, Russia, Poland, and Nigeria (Kamani et al., 2018;Karbowiak et al., 2005;Laakkonen et al., 2001), and thus the structure of the rodent populations may play a role in the occurrence of certain Hepatozoon spp.haplotypes.Further molecular characterization based on fast evolving genes is required to confirm this hypothesis.
Interestingly, in our study Corral was the locality with the highest number of Hepatozoon spp.haplotypes (n=3) in rodents.Corral also shared haplotypes with the previous study in Chiloé island (#1 and #3) (Merino et al., 2009), albeit geographically distant.The distribution and sharing of some haplotypes might result from the versatility of synanthropic and wild rodents, the microclimate conditions, and the topography of each sampling site (Muñoz-Zanzi et al., 2014).
A higher number of haplotypes was observed from southern (n=6) compared to northern (n=3) Chile, and they did not share any Hepatozoon spp.haplotypes.This could be due to the distance and the biomes' specific characteristics from which rodents were sampled.While Pan de Azúcar (northern Chile) is characterized by coastal desert weather (Squeo et al., 1998) Chile), which is classified as a temperate rain forest (Carmona et al., 2010;Villagran, 1991;Villagrán et al., 2004), and also shares similar elevations to the coastal region, which varies from 0-700 meters (Carmona et al., 2010;Instituto Nacional de Estadísticas, 2007).Also, different Hepatozoon spp.host adaptability (vertebrate vs invertebrate) could be related to the divergence of haplotypes, as the northern Chile samples only included rodent ticks and the southern samples included rodents.The variability in the southern samples may be due to a variety of ticks involved or other arthropods that play a role as vectors and remain unknown at the time.To the best of our knowledge there are no studies of ticks associated to rodents in Southern Chile (Valdivia or Chiloé).Ticks previously described in rodents from other regions in Chile include Ixodes spp., Ixodes abrocomae, Ixodes sigelos, Rhipicephalus sanguineus and Amblyomma tigrinum (González-Acuña & Guglielmone, 2005;Landaeta-Aqueveque et al., 2021).
According to the Splits tree analysis, the Hepatozoon 18S rDNA sequences obtained from reptiles and rodents clustered together in a major clade.On the other hand, minor clades grouped Hepatozoon sequences from rodents and ixodid ticks reported in Chile by Muñoz-Leal et al. (2019).These findings are similar to the results reported by de Sousa et al. (2017), by Hamšíková et al. (2016) and Perles et al. (2019), and validated by Karadjian et al. (2015) and Maia et al. (2016a), confirming that Hepatozoon spp.from rodents were closely related to Hepatozoon spp.from reptiles, but distant form Hepatozoon spp.described in canids and felids.As previously reported with rodent-associated Hepatozoon from Brazil (Perles et al., 2019), Hepatozoon in rodents from Chile did not seem to participate in epidemiological cycles of Hepatozoon species infecting domestic and wild canids and felids.Those results suggest that rodents from Chile might play a role as intermediate hosts for Hepatozoon infections in reptiles and future studies should explore this hypothesis.
The results from Chile are preliminary and based on the 18S rDNA gene.Future studies in South America should explore mitochondrial genes for further Hepatozoon spp.diversity characterization, as recently described (Léveillé et al., 2019).

Conclusions
The findings of this study revealed Hepatozoon spp. in synanthropic and wild rodents in the province of Valdivia.This is the first molecular detection of Hepatozoon in M. musculus, R. norvegicus, R. rattus, A. longipilis and O. longicaudatus rodents from Chile.The 18S rDNA sequences from this study were closely related to those previously detected in rodents and reptiles from Chile and Brazil, but distant form Hepatozoon spp.described in canids and felids.Different Hepatozoon haplotypes were observed in southern and northern Chile.Finally, Hepatozoon haplotypes from rodents sampled in Valdivia were genetically diverse, and novel haplotypes were described in rodents from southern Chile.The preliminary results from this study warrant for additional investigation on the genetic diversity of Hepatozoon spp., including in a broader population of rodents from Chile and the analysis of mitochondrial genes.

Figure 1 .
Figure 1.Rodent sampling sites within the Valdivia Province, Southern Chile.

Figure 2 .
Figure 2. Phylogenetic tree based on an alignment of concatenated Hepatozoon 18S rDNA sequences, using Maximum likelihood method and TVM+I+G4+F as an evolutionary model.Numbers at nodes correspond to bootstrap.Hepatozoon sp.sequences detected in the present study are in bold letters.Dactylosoma ranarum was used as outgroups.Squared colors identify each clade.

Figure 3 .
Figure 3. Hepatozoon spp.18S rDNA haplotype network with northern (ticks from Pan de Azucar and Bosque Fray) (Muñoz-Leal et al., 2019) and southern Chile (rodents from Chiloe Island) (Merino et al., 2009) sequences, including the concatenated sequences from the present study in rodents from the Valdivia province (Corral, Reumen).Each dash line represents a mutational event.Present study's rodents are in bold letters.

Figure 4 .
Figure 4. SplitsTree analysis generated by Neighbor-net and uncorrected P distance of Hepatozoon spp.18S rDNA sequences obtained from rodents sampled in the present study.Present study's sequences are in bold letters.