First Molecular Detection of Polychromophilus Parasites in Brazilian Bat Species

Blood parasites of the Haemosporida order, such as the Plasmodium spp. responsible for malaria, have become the focus of many studies in evolutionary biology. However, there is a lack of molecular investigation of haemosporidian parasites of wildlife, such as the genus Polychromophilus. Species of this neglected genus exclusively have been described in bats, mainly in Europe, Asia, and Africa, but little is known about its presence and genetic diversity on the American continent. Here, we investigated 406 bats from sites inserted in remnant fragments of the Atlantic Forest and Cerrado biomes and urbanized areas from southern Brazil for the presence of Polychromophilus species by PCR of the mitochondrial cytochrome b encoding gene. A total of 1.2% of bats was positive for Polychromophilus, providing the first molecular information of these parasites in Myotis riparius and Eptesicus diminutus, common vespertilionid bats widely distributed in different Brazilian biomes, and Myotis ruber, an endangered species. A Bayesian analysis was conducted to reconstruct the phylogenetic relationships between Polychromophilus recovered from Brazilian bats and those identified elsewhere. Sequences of Brazilian Polychromophilus lineages were placed with P. murinus and in a clade distinct from P. melanipherus, mainly restricted to bats in the family Vespertilionidae. However, the sequences were split into two minor clades, according to the genus of hosts, indicating that P. murinus and a distinct species may be circulating in Brazil. Morphological observations combined with additional molecular studies are needed to conclude and describe these Polychromophilus species.


Introduction
The phylum Apicomplexa forms one of the most diverse groups of unicellular protists with a wide environmental distribution. They are classified as mandatory intracellular parasites and they have mobile invasive stages. They are characterized by the presence of an evolutionarily unique structure called the apical complex, used to adhere and invade host cells. Many of the species that are part of this group are considered pathogens in humans and other vertebrates. All animal species are believed to host at least one species of apicomplexan parasites [1][2][3]. Apicomplexa are divided into two orders: Eucoccidiorida (coccidian parasites) and Haemosporida (haemosporidian parasites). Haemosporida are Microorganisms 2021, 9,1240 2 of 14 organized into four families: Garniidae, Haemoproteidae, Leucocytozoidae, and Plasmodiidae, which include malaria parasites that infect various vertebrates and invertebrate hosts [4].
The hosts of the order Chiroptera have the greatest diversity of haemosporidian parasites among mammals, including nine genera. In addition to the well-known genera (Plasmodium and Hepatocystis), seven genera exclusively infect chiropterans: Polychromophilus, Nycteria, Bioccala, Biguetiella, Dionisia, Johnsprentia, and Sprattiella [5,6], clearly highlighting this group of mammals as a vital tool in the taxonomic, systematic, and evolutionary study of haemosporidians in mammals. Although Bioccala was elevated to a genus in 1984 [7], many studies, as well as this work, still use it as a subgenus of Polychromophilus, since its species present similar morphological characteristics and its genetics have not been studied [8].
The genus Polychromophilus has been found in insectivorous bats in tropical and temperate regions [9][10][11][12]. Only five species of Polychromophilus are known. Although they can be distinguished by slight differences in ultrastructure, they are classified mainly based on the type of host [13]. Of the five species of Polychromophilus described, Polychromophilus (Polychromophilus) melanipherus and Polychromophilus (Bioccala) murinus are mainly linked to two bat families: Miniopteridae and Vespertilionidae, respectively [14]. However, occasionally, P. melanipherus has been reported in Hipposideridae and Vespertilionidae and P. murinus in Rhinolophidae, Hipposideridae, and Miniopteridae [6]. In addition, the species P. (P.) corradetti and P. (P.) adami have been described in bats from the African region: Miniopterus inflatus in Gabon and Miniopterus minor in the Republic of Congo [13].
Recent studies have demonstrated a greater concentration of molecular studies aimed at African and European bats, e.g., [8,[15][16][17]. In contrast, our knowledge about haemosporidian parasites of Brazilian bats is still restricted to morphological investigations, such as the case of Polychromophilus (Bioccala) deanei found in Myotis nigricans (Vespertilionidae). Myotis nigricans is an evening bat from Brazil, and is the first chiropteran host in which this group of parasites was found in the New World [18,19]. Nevertheless, no molecular data is available for this parasite in Brazil, and the only sequence of Polychromophilus sp. of bats from the American continent is from Myotis nigricans, from the Vespertilionidae family, found in Panama [20].

Sampling
Brain tissue samples of bats with no identified species (n = 406) were acquired from the Parana State Reference Laboratory (LACEN) program for monitoring rabies virus circulation. They were collected between September 2019 and August 2020 in 67 different municipalities in the State of Paraná, most of them inserted in remnant fragments of Atlantic Forest and Cerrado biomes, as well as in urbanized areas ( Figure 1).
All tissue samples and bats were collected and handled under appropriate authorizations by the Brazilian government. The project was approved by the Ethics in Use of Animals Committee, CEUA/SESA, of the Centro de Produção e Pesquisa de Imunobiológicos-CPPI/PR (approval number 01/2019 and date of approval 3 March 2020). All tissue samples and bats were collected and handled under appropriate authorizations by the Brazilian government. The project was approved by the Ethics in Use of Animals Committee, CEUA/SESA, of the Centro de Produção e Pesquisa de Imunobiológicos-CPPI/PR (approval number 01/2019 and date of approval 3 March 2020).

Polychromophilus Detection
The extraction of total nucleic acid (DNA and RNA) from collected samples was performed using the BioGene Extraction kit (K204-4, Bioclin, Belo Horizonte, MG, Brazil), following the manufacturer's instructions.
A fragment of ~1.1 kb (approximately 92% of the gene) from the mitochondrial cytochrome b gene (cytb) was amplified using a nested polymerase chain reaction (PCR), taking standard precautions to prevent cross-contamination of samples. The PCR reactions were conducted as previously described [21] using primers DW2 and DW4 and 5 uL of genomic DNA in the first reaction, and 1 uL aliquot of this product was used as a template for a nested reaction with primers DW1 and DW6.
PCR products were sequenced using BigDye ® Terminator v3.1 Cycle Sequencing Kit in ABI PRISM ® 3500 Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA) using nested PCR primers. The cytb sequences were obtained and aligned with the sequences available at the GenBank ® database.
The phylogenetic relationship among reported parasites was inferred using partial cytb gene sequences (1116 bp). GenBank ® accessions of the used sequences are shown in the phylogenetic trees. The phylogenetic reconstruction was performed using the Bayesian inference method implemented in MrBayes v3.2.0 [22]. Bayesian inference was executed with two Markov Chain Monte Carlo searches of 3 million generations, with each sampling 1 of 300 trees. After a burn-in of 25%, the remaining 15,002 trees were used to calculate the 50% majority-rule consensus tree. The phylogeny was visualized using FigTree version 1.4.0 [23].

Polychromophilus Detection
The extraction of total nucleic acid (DNA and RNA) from collected samples was performed using the BioGene Extraction kit (K204-4, Bioclin, Belo Horizonte, MG, Brazil), following the manufacturer's instructions.
A fragment of~1.1 kb (approximately 92% of the gene) from the mitochondrial cytochrome b gene (cytb) was amplified using a nested polymerase chain reaction (PCR), taking standard precautions to prevent cross-contamination of samples. The PCR reactions were conducted as previously described [21] using primers DW2 and DW4 and 5 uL of genomic DNA in the first reaction, and 1 uL aliquot of this product was used as a template for a nested reaction with primers DW1 and DW6.
PCR products were sequenced using BigDye ® Terminator v3.1 Cycle Sequencing Kit in ABI PRISM ® 3500 Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA) using nested PCR primers. The cytb sequences were obtained and aligned with the sequences available at the GenBank ® database.
The phylogenetic relationship among reported parasites was inferred using partial cytb gene sequences (1116 bp). GenBank ® accessions of the used sequences are shown in the phylogenetic trees. The phylogenetic reconstruction was performed using the Bayesian inference method implemented in MrBayes v3.2.0 [22]. Bayesian inference was executed with two Markov Chain Monte Carlo searches of 3 million generations, with each sampling 1 of 300 trees. After a burn-in of 25%, the remaining 15,002 trees were used to calculate the 50% majority-rule consensus tree. The phylogeny was visualized using FigTree version 1.4.0 [23].

Host Species Identification
The positive samples were processed using a PCR protocol that amplifies host DNA with primers L14841 and H15149 that were designed to amplify fragments with~390 bp of the mitochondrial cytb gene from a wide range of animals, including mammals, birds, amphibians, reptiles, and fish [24]. Amplified fragments were sequenced directly using the Microorganisms 2021, 9, 1240 4 of 14 corresponding flanking primers. Obtained sequences were compared to other sequences deposited in the GenBank ® database (www.ncbi.nlm.nih.gov/blast/Blast.cgi accessed on 19 March 2021). The best close match (BCM) algorithm was used to identify the best barcode matches of a query, and the species name of that barcode was assigned to the query if the barcode was sufficiently similar [25]. Positive identification and host species assignment were made when sequences presented a match of >97%.

Results
This study detected five samples that were positive for Polychromophilus sp. The positive samples were processed using a PCR protocol that amplifies host DNA with primers L14841 and H15149 that were designed to amplify fragments with ~390 bp of the mitochondrial cytb gene from a wide range of animals, including mammals, birds, amphibians, reptiles, and fish [24]. Amplified fragments were sequenced directly using the corresponding flanking primers. Obtained sequences were compared to other sequences deposited in the GenBank ® database (www.ncbi.nlm.nih.gov/blast/Blast.cgi, accessed on 19 March 2021). The best close match (BCM) algorithm was used to identify the best barcode matches of a query, and the species name of that barcode was assigned to the query if the barcode was sufficiently similar [25]. Positive identification and host species assignment were made when sequences presented a match of >97%.

Results
This study detected five samples that were positive for Polychromophilus sp.  The nucleic acid polymorphism in mitochondrial cytb sequences (1116 bp) of Polychromophilus sp. isolates from Brazil compared to the best match sequence from GenBank ® (#LN483038 of Myotis nigricans from Panama with 595 bp) is shown in Table 1. Thirteen sites were polymorphic among Brazilian sequences ( Table 1). The Panamanian sequence, The nucleic acid polymorphism in mitochondrial cytb sequences (1116 bp) of Polychromophilus sp. isolates from Brazil compared to the best match sequence from GenBank ® (#LN483038 of Myotis nigricans from Panama with 595 bp) is shown in Table 1. Thirteen sites were polymorphic among Brazilian sequences ( Table 1). The Panamanian sequence, the only available one obtained from bats from the American continent, showed two nucleic acid substitutions found only in this isolate (gray columns) ( Table 1).  Isolate  219  247  261  273  339  405  512  789  792  810 811 853 885 945 1086   116  C  T  A  T  T  T  T  T  C  C  T  C  A  T  A   198  C  T  A  T  G  T  T  T  T  C  T  C  A  T  G   335  C  T  A  T  G  T  T  T  T  C  T  C  A  T  G   650  T  T  C  A  A  C  T  C  T  T  C  T  T  T  A   69642  C  T  A  T  T  T  T  T  The sequence obtained from bat 650 was the most divergent, with 98-99% of identity with the others (with 11 or 12 nucleic acid substitutions) ( Table 2). The Panamanian sequence presented two to eight nucleic acid substitutions compared to Brazilian sequences (98-99% of identity) ( Table 2). The phylogenetic tree in Figure 3 was generated with reference sequences found in the Genbank ® database, covering different haemosporidian genera obtained from different hosts (Table A1, Appendix A). The Polychromophilus sequences found in this study and all sequences of the genus available in the Genbank ® database (Table A2, Appendix A) were included. The clade of the genus Polychromophilus is shown in evidence, and the remaining haemosporidian from other genera were collapsed.
Phylogenetic analysis based on cytb did not produce conflict in any of the main nodes. All the main genera and subgenera were recovered and represented in the phylogenetic tree by separate monophyletic clades. The results show the existence of four clades within the Haemosporida order analyzed here. Phylogeny also showed Polychromophilus as a sister clade of a group that contains Plasmodium species of ungulates, but with a distant relationship between Plasmodium and Hepatocystis from other mammals, such as primates and rodents.  Tables A1 and A2 in Appendix A. * Sequence HM055583 has also been reported in P. murinus from Eptesicus serotinus, Nyctalus noctule, and Myotis myotis (Table A2, Appendix A). Eimeria spp. were used as an external group. The support values of the nodes (in percentage) indicate posterior probabilities. The red branches highlight the haemosporidian sequences found in mammals. The yellow branches highlight the haemosporidian sequences found in birds. The green branches highlight the haemosporidian sequences found in reptiles. The sequences found in the present study are highlighted in bold. The remaining reference sequences are collapsed to highlight the branch of the Polychromophilus genus.
All Polychromophilus sequences from bats of different parts of the world were grouped into a monophyletic clade (posterior probability of 100) composed of four subclades, with all Polychromophilus found in Brazilian bats segregated in only one of them. The first distinct subclade comprised all sequences of P. melanipherus from Miniopterus bat hosts, and the second subclade exclusively included sequences of Polychromophilus from vespertilionids (including Brazilian ones), confirming a clear separation of parasites from miniopterid and vespertilionid hosts. The other subclade that was separated contained the Polychromophilus sequences from Scotophilus kuhlii from Thailand (MT750305-MT750309). Two samples of parasites of Pipistrellus aff. grandidieri and Laephotis capensis from Guinea (KF159700 and KF159714) formed a separate group.
The subclade of Polychromophilus from vespertilionids was divided into two branches: one contained sequences of P. murinus from bats in Europe (Switzerland, Bulgaria), Madagascar, and Thailand, and a sequence of Eptesicus diminutus (650) from Brazil, and the other

Discussion
Based on the results presented herein, although the total number of bat families tested is unknown, Polychromophilus infection in Brazilian bats appears to be limited to just one family (Vespertilionidae). This finding is in accordance with the only previous report of Polychromophilus from Brazil, described as P. deanei, found in Myotis nigricans, also a Vespertilionidae bat [18,19].
According to one study, Paraná state has poor fauna regarding the number of bat species, with only 53 species from five families recorded [31]. The Phyllostomidae family has the highest species richness (25; 47% of the total), followed by Molossidae (13; 24%), Vespertilionidae (12; 22%), Noctilionidae (2; 4%), and Emballonuridae (1; 2.5%) [31]. Miretzki also showed the occurrence of only 55% of the species of the Atlantic Forest biome and the relative predominance of vespertilionids and molossids over phyllostomids. Herein, we analyzed samples obtained from much of the state's area, with great sampling opportunities for other families. However, we were unable to find Polychromophilus in bat species that were not vespertilionids, suggesting that this parasite may be restricted to this group of bats in Brazil.
Regarding the frequency, we found the lowest positivity rate reported to date, although the total number of samples analyzed herein is one of the highest among published studies (Table 3). This could be related to the sample type analyzed in this study. This was the first time that Polychromophilus DNA was obtained from brain tissue, probably from parasites in the blood vessels that irrigate the organ. Thus, the direct comparison of the prevalence data with published studies that used blood samples is impaired.  Three different Brazilian bats species were found to be positive for Polychromophilus sp.: two Myotis species (M. ruber and M. riparius) and one species from the Eptesicus genus (E. diminutus). There are reports of Myotis species infections in Africa (M. tricolor in Kenya and M. goudoti in Madagascar) [17,36,37], Europe (M. daubentonii and M. myotis in Switzerland) [38], and Asia (M. siligorensis in Thailand) [40]. However, the only record of Polychromophilus infection in Eptesicus comes from Europe (E. serotinus in Switzerland) [38].
It is important to note that in our molecular identification of the host species using cytb and sequence comparisons, Eptesicus furinalis was the species with the best close match with the sequence obtained from bat 650. However, the percentage of identity was low (89%) compared to sequences available in the GenBank ® database, making it impossible to identify the species. Thus, alternatively, we used the coi gene and the BOLD database (https://www.boldsystems.org/ (accessed on 31 March 2021), finding 98% of identity with an Eptesicus diminutus sequence, a reliable value for the species identification using the BCM method. Eptesicus diminutus has a distribution in the north and east regions of Paraná state [31]. It is from the Vespertilionidae family, and it is absent from the GenBank ® database, which explains the first finding. Thus, we considered specimen 650 to be Eptesicus diminutus.
Our phylogenetic analysis showed a strongly defined clade represented by Plasmodium infecting rodents and primate hosts, which also included Hepatocystis isolated from bats. Similar data were obtained by other authors [38,50]. Haemoproteus and Leucocytozoon species were grouped separately in individual clades, as previously shown [51,52].
Regarding Polychromophilus sequences, a similar topology in the phylogenetic tree was obtained by Chumnandee et al. [39], where they grouped into a monophyletic clade with a clear separation of parasites from miniopterid and vespertilionid hosts. Four Brazilian sequences (GenBank ® MW984519, MW984520, MW984522 from Polychromophilus sp. isolated of Myotis riparius, and MW984518 from Polychromophilus sp. isolated of Myotis ruber) were positioned close to the sequence of Polychromophilus sp. of bats of the species Myotis nigricans, Vespertilionidae family, from the Latin American region (Panama) (GenBank ® #LN483038) [20]. One Brazilian sequence (GenBank ® #MW984521, from Polychromophilus isolated from Eptesicus diminutus) was grouped with all P. murinus sequences in a sister clade. The latter, likely P. murinus, presented 1% divergence in the cytb sequence compared to the other Brazilian or Panamanian sequences, and was obtained from a different genus of bats. Thus, the possibility of most Brazilian sequences being a different Polychromophilus species must be investigated.
The present study provides the first molecular description of Polychromophilus parasites in Myotis ruber, Myotis riparius, and Eptesicus diminutus from Brazil and confirms the presence of this parasite 50 years after its first and only report in Brazilian territory. Moreover, our results suggest the occurrence of two distinct Polychromophilus species infecting two different genera of hosts, improving the current knowledge on blood parasites infecting Brazilian bats. However, it is crucial to add additional molecular markers to the phylogenetic analysis for an in-depth investigation. A three-genome phylogenetic analysis for robust haemosporidian phylogenies has been recommended [53] and must be properly included as part of a follow-up paper. Moreover, additional studies including morphological observations of these parasites combined with molecular data are needed to resolve its taxonomy. Furthermore, due to the great Brazilian extensions and the immense diversity of species and biomes, new bat populations should be investigated to provide a complete portrait of the biology of host-parasite interactions.

Data Availability Statement:
The data presented in this study are available in Appendix A and also in the GenBank ® database (https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 19 March 2021)) (accession numbers MW984518-MW984522).

Conflicts of Interest:
The authors declare no conflict of interest.