
==== Front
Int J Parasitol Parasites Wildl
Int J Parasitol Parasites Wildl
International Journal for Parasitology: Parasites and Wildlife
2213-2244
Elsevier

S2213-2244(24)00082-8
10.1016/j.ijppaw.2024.100986
100986
Article
Polychromophilus (Haemosporida: Plasmodiidae): A review of association with bats (Mammalia, Chiroptera) and the first record in the Neotropical bat, Myotis albescens (Chiroptera, Vespertilionidae) from Colombia
Ceballos-Pérez Diego Fernando ab
Alvarez-Londoño Johnathan bc
Ramírez-Chaves Héctor E. bd
Rivera-Páez Fredy A. fredy.rivera@ucaldas.edu.co
b⁎
a Programa de Biología, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Calle 65 No. 26-10 A.A 275, 170004, Manizales, Caldas, Colombia
b Grupo de Investigación en Genética, Biodiversidad y Manejo de Ecosistemas (GEBIOME), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Calle 65 No. 26-10 A.A 275, 170004, Manizales, Caldas, Colombia
c Maestría en Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Calle 65 No. 26-10 A.A 275, 170004, Manizales, Caldas, Colombia
d Centro de Museos, Museo de Historia Natural, Universidad de Caldas, Calle 58 No. 21-50, 170004, Manizales, Caldas, Colombia
⁎ Corresponding author. fredy.rivera@ucaldas.edu.co
07 9 2024
12 2024
07 9 2024
25 1009861 8 2024
4 9 2024
5 9 2024
© 2024 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Some species within the family Plasmodiidae (Haemosporida) have been extensively studied due to their implications for human health. However, for other haemosporidians that infect wild animals the knowledge is limited. Species within the genus Polychromophilus have thus far been documented exclusively as hemoparasites of bats. Records of Polychromophilus are primarily from Africa, Europe, and Southeast Asia, with limited information available for the Americas. Here, we assessed the state of knowledge on Polychromophilus species infecting bats worldwide and searched for the presence of Polychromophilus in blood samples of neotropical bats from Colombia. We found a total of 65 records of Polychromophilus in 46 bat species belonging to the families Emballonuridae, Hipposideridae, Miniopteridae, Rhinolophidae, Rhinonycteridae, and Vespertilionidae worldwide, except for Antarctica. In the Americas, records of the genus Polychromophilus are exclusively from Vespertilionidae bats in Brazil, Colombia, the United States, and Panama. The morphological and molecular analyses of blood from 125 bats, belonging to 39 species and captured in seven localities within the departments of Arauca and Caldas (Colombia), confirmed the presence of Polychromophilus deanei in a silver-tipped myotis, Myotis albescens (Vespertilionidae). This finding represents the first morphological and molecular confirmation of P. deanei in the Americas. Additionally, it expands the knowledge on the diversity and distribution of Polychromophilus in Neotropical bats.

Graphical abstract

Image 1

Highlights

• Global Diversity: Forty-six different species from six bat families have reported cases of Polychromophilus worldwide.

• First Report: First morphological and molecular report of Polychromophilus deanei.

• Main Reservoir: The genus Myotis is the primary reservoir of Polychromophilus in the Americas.

• Phylogenetic Link: Bats are a crucial informative link in the phylogeny of Haemosporida.

Keywords

Chiroptera
Hemoparasites
Malaria
Neotropics
Vespertilionidae
==== Body
pmc1 Introduction

Haemosporidians of the family Plasmodiidae (Haemosporida: Apicomplexa) represent a diverse group of blood parasites transmitted by vectors that infect a wide range of vertebrate hosts worldwide (Liu et al., 2010; Galen et al., 2018; Harris et al., 2019). These haemosporidians exhibit complex and similar life cycles, involving sexual reproduction stages in the arthropod vectors and asexual stages in vertebrate hosts (Garnham, 1966; Valkiunas, 2004; Perkins, 2014). Over the past decade, the implementation of morphological and molecular tools has facilitated the identification of a wide diversity of morphospecies and lineages of haemosporidians (Perkins and Schaer, 2016; Lutz et al., 2016; Arnuphapprasert et al., 2020). However, the true diversity and phylogenetic relationships are not well-established due to descriptions based either only on morphology or isolated molecular data (Valkiunas, 2004; Perkins, 2014; Sándor et al., 2021).

In mammals, haemosporidian infections within Plasmodiidae are primarily known in primates and rodents due to their significance in human health research (e.g., Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax) (Schaer et al., 2013; Lalremruata et al., 2015; Borner et al., 2016; Minozzo et al., 2021). Currently, Plasmodiidae includes six genera that infect mammals, i.e., Dionisia, Hepatocystis, Nycteria, Plasmodium, Rayella, and Polychromophilus (including Biguetiella), all of which have been detected in bats (Chiroptera), except Rayella, and three exclusively infecting these hosts (i.e., Dionisia, Nycteria and Polychromophilus) (Perkins and Schaer, 2016). In this context, bats are a crucial group for the taxonomic, systematic, and evolutionary study of haemosporidians in mammals (Lutz et al., 2016). Polychromophilus is the genus with the widest global distribution, primarily associated with insectivorous bats of the families Miniopteridae and Vespertilionidae in temperate and tropical regions (Garnham, 1973; Perkins and Schaer, 2016). These haemosporidians are transmitted to bats by highly specialized dipterans of the family Nycteribiidae (Megali et al., 2011; Ramasindrazana et al., 2018). There are five known species of Polychromophilus: Polychromophilus adami and Polychromophilus corradetti, described from Africa; Polychromophilus melanipherus and Polychromophilus murinus, described from Europe; and Polychromophilus deanei, described from South America (Garnham et al., 1971; Perkins and Schaer, 2016).

In the Americas, the initial reports of Polychromophilus were based solely on morphological traits of records coming from the USA, Brazil, and Colombia (Wood, 1952; Garnham, 1966; 1970; 1971; Foster, 1979; Marinkelle, 1995). The first molecular record of Polychromophilus sp. corresponds to a DNA sequence in Myotis nigricans from Panama (Borner et al., 2016). Subsequently, eight additional sequences of Polychromophilus sp. were reported in Neoeptesicus diminutus, Myotis ruber, Myotis riparius, and Myotis sp. in Brazil (Minozzo et al., 2021; Mathias et al., 2023). However, there are no studies that combine both morphological and molecular methods for identifying haemosporidians of the genus Polychromophilus infecting bats across the continent. Given the existing information gaps on Polychromophilus species, and the fact that up to 222 bat species have been recorded in Neotropical countries like Colombia (Ramírez-Chaves et al., 2024), this research aimed to gather available information of Polychromophilus infecting bats worldwide and provide morphological and molecular information to enhance the understanding of the biology of Polychromophilus.

2 Materials and methods

2.1 Literature review

To compile the available information on Polychromophilus associated with bats on a global scale, we conducted a comprehensive literature search in the Web of Science and Scopus databases (from January 1960 to May 2024) using the search term “Polychromophilus”. To ensure the quality and replicability of the studies, we limited the search to peer-reviewed articles (Bohada-Murillo et al., 2021). To gather the most comprehensive set of Polychromophilus reports, we included only studies presenting novel and non-duplicated information. Additionally, we included a report not part of the initial search, concerning the species Bioccala deanei, now known as P. deanei (Marinkelle, 1995). The review followed the methodology proposed in the PRISMA statement (Page et al., 2021).

The initial search yielded 75 articles, which were reduced to 48 after removing duplicates. Subsequently, 27 articles were retained after excluding those that did not contain relevant information for the study. The selected articles were thoroughly reviewed to determine if they met the following inclusion criteria: (1) morphological and/or molecular detection method, (2) detection in bats, (3) georeferenced location or a detailed description of the sampling area, and (4) reports of infections in wild environments. The inclusion of articles was performed by the same person (DFCP) to avoid potential reviewer bias. As a result, we excluded four articles that did not meet the inclusion criteria, resulting in a total of 23 articles that provided 65 reports of Polychromophilus (Fig. 1). With this information, we generated a table with the extracted information from the selected studies, indicating the bat family, country record, detected Polychromophilus species, and bibliographic reference (Table 1).Fig. 1 Flow diagram of elements of reporting for systematic reviews and Meta-Analysis (PRISMA; 2020 version) summarizing the selection of articles included in this review.

Fig. 1

Table 1 Records of species of Polychromophilus (Plasmodiidae) in bats (Chiroptera) around the world. The information of the family and bat species, and country of the record is included.

Table 1Host family	Bat species	Locality	Polychromophilus species	References	
Rhinonycteridae	Paratriaenops furculus	Madagascar	Polychromophilus melanipherus	Ramasindrazana et al. (2018)	
Hipposideridae	Hipposideros semoni	Australia	Polychromophilus melanipherus	Perkins and Schaer (2016)	
Hipposideros caffer	Congo	Polychromophilus murinus	Perkins and Schaer (2016)	
Hipposideros gentilis	Thailand	Polychromophilus sp.	Poofery et al. (2023)	
Rhinolophidae	Rhinolophus ferrumequinum	Romania	Polychromophilus murinus	Sándor et al. (2021)	
Rhinolophus hipposideros	Romania	Polychromophilus murinus	Sándor et al. (2021)	
Rhinolophus mehelyi	Romania	Polychromophilus murinus	Sándor et al. (2021)	
Miniopteridae	Miniopterus schreibersii	Romania, Hungary, Switzerland, Italy, Turkey, Russia, Israel, Congo, Portugal, Spain, France, Croatia, Slovakia	Polychromophilus melanipherus	Perkins and Schaer (2016); Sándor et al. (2021); Szentiványi et al. (2020)	
Miniopterus inflatus	Gabon	Polychromophilus melanipherus and Polychromophilus corradetti	Duval et al. (2012); Perkins and Schaer (2016)	
Miniopterus orianae	Australia	Polychromophilus melanipherus	Holz et al. (2019); Holz et al. (2020)	
Miniopterus africanus	Kenya	Polychromophilus melanipherus	Lutz et al. (2016)	
Miniopterus cf. fraterculus	Kenya	Polychromophilus melanipherus	Lutz et al. (2016)	
Miniopterus natalensis	Kenya	Polychromophilus melanipherus	Lutz et al. (2016)	
South Africa	Polychromophilus sp.	Szentiványi et al. (2020)	
Miniopterus rufus	Kenya	Polychromophilus melanipherus	Lutz et al. (2016)	
Miniopterus minor	Tanzania	Polychromophilus melanipherus	Lutz et al. (2016)	
Gabon and Congo	Polychromophilus adami	Perkins and Schaer (2016); Rosskopf et al. (2019)	
Miniopterus fuliginosus	Japan	Polychromophilus melanipherus	Rosyadi et al. (2022)	
Miniopterus villiersi	Guinea	Polychromophilus sp.	Schaer et al. (2013)	
Miniopterus gleni	Madagascar	Polychromophilus sp.	Rasoanoro et al. (2021); Perkins and Schaer (2016)	
Miniopterus ambohitrensis	Madagascar	Polychromophilus sp.	Rasoanoro et al. (2021)	
Miniopterus egeri	Madagascar	Polychromophilus sp.	Rasoanoro et al. (2021)	
Miniopterus griveaudi	Madagascar	Polychromophilus sp.	Rasoanoro et al. (2021)	
Miniopterus manavi	Madagascar	Polychromophilus sp.	Perkins and Schaer (2016)	
Miniopterus sp.	Madagascar, Vanuatu, Australia	Polychromophilus melanipherus	Ramasindrazana et al. (2018); Perkins and Schaer (2016)	
Vespertillionidae	Myotis silogorensis	Thailand	Polychromophilus murinus	Arnuphapprasert et al. (2020)	
Myotis daubentonii	Romania, Switzerland	Polychromophilus murinus	Witsenburg et al. (2012), (2014); Sándor et al. (2021)	
Myotis myotis	Bulgaria	Polychromophilus murinus	Sándor et al. (2021)	
Myotis macrodactylus	Japan	Polychromophilus murinus	Rosyadi et al. (2022)	
Myotis blythii	Romania	Polychromophilus murinus	Sándor et al. (2021)	
Myotis albescensa	Colombia	Polychromophilus deanei	This study	
Myotis nigricans	Brazil	Polychromophilus deanei	Garnham et al., (1971)	
Panama	Polychromophilus sp.	Borner et al. (2016)	
Myotis tricolor	Kenia	Polychromophilus sp.	Perkins and Schaer (2016)	
Myotis austroriparius	United States	Polychromophilus sp.	Foster (1979)	
Myotis goudoti	Madagascar	Polychromophilus murinus and Polychromophilus sp.	Perkins and Schaer (2016); Ramasindrazana et al. (2018); Rasoanoro et al. (2021)	
Myotis riparius	Brazil	Polychromophilus sp.	Minozzo et al. (2021); Mathias et al. (2023)	
Myotis ruber	Brazil	Polychromophilus sp.	Minozzo et al. (2021); Mathias et al. (2023)	
Myotis sp.	Israel, England Russia, Switzerland	Polychromophilus murinus	Perkins and Schaer (2016)	
Brazil	Polychromophilus sp.	Mathias et al. (2023)	
Vespertilio murinus	Italy	Polychromophilus murinus	Perkins and Schaer (2016)	
Pipistrellus aff. grandidieri	Guinea	Polychromophilus sp.	Schaer et al. (2013)	
Neoromicia capensis	Guinea	Polychromophilus sp.	Schaer et al. (2013)	
Eptesicus fuscus	Colombia	Polychromophilus (Bioccala) deanei*	Marinkelle (1995)	
Neoeptesicus diminutus	Brazil	Polychromophilus sp.	Minozzo et al. (2021)	
Nyctophilus bifax	Australia	Polychromophilus sp.	Perkins and Schaer (2016)	
Scotophilus kuhlii	Thailand	Polychromophilus sp.	Chumnandee et al. (2021)	
Scotophilus robustus	Madagascar	Polychromophilus sp.	Rasoanoro et al. (2021)	
Kerivoula hardwickii	Cambodia	Polychromophilus sp.	Perkins and Schaer (2016)	
Vespadelus pumilus	Australia	Polychromophilus melanipherus	Perkins and Schaer (2016)	
Emballonuridae	Taphozous melanopogon	Thailand	Polychromophilus melanipherus	Arnuphapprasert et al., (2020)	
Bioccala is a subgenus of Polychromophilus (discussed by Witsenburg et al., 2012).

a New association reported in this study.

2.2 Searching for Polychromophilus in Neotropical bats

To explore the presence of Polychromophilus in Neotropical bats, we conducted field work between October and November 2021, and November 2022, in six localities across four municipalities (La Dorada, Manizales, Palestina, and Villamaría) in the Department of Caldas and one locality in the municipality of Arauca in the Department of Arauca, Colombia (Table 2, Fig. 2). The Department of Caldas is situated in the central-western region of Colombia (5°17′N and 75°21′W; central point), covering an approximate area of 7888 km2. Caldas encompasses a wide diversity of ecosystems along the Central and Western Andes and the inter-Andean valleys of the Cauca and Magdalena rivers, with elevations ranging from 140 to 5350 m a.s.l. (Cardona-Salazar et al., 2020). The average monthly temperature varies from 13 °C to 27 °C, and monthly precipitation ranges from 150 to 400 mm, with peak rainfall occurring during April–May and August–November (Escobar-Lasso et al., 2013; Ruiz and Melo, 2022). The Department of Arauca is in the northern part of the Colombian Orinoquia (6°37′ N, 70°59′ W; central point) and covers a total area of 23,818 km2. It is situated within the warm thermal floor, with an average annual temperature of 27 °C and an average annual precipitation of 1500 mm, following a monomodal precipitation regime (lower rainfall from December to February) known as the dry season (McNish, 2007). The landscape of the Orinoquia alluvial plain, dominated by savanna, is characterized by herbaceous vegetation associations with scattered trees and seasonal water availability patterns dictated by a markedly dry climatic season (Rippstein, 2001).Table 2 Information on the department, municipality, and locality of the bats captured and analyzed in this study. Locality numbers are shown in Fig. 2.

Table 2Department	Municipality	Locality	Locality number	Latitude	Longitude	Elevation (meters above sea level)	Habitat type	
Caldas	Palestina	Vereda El Retiro	1	5.136722	−75.678944	823	Agroforestry matrix in dry forest	
	Kilómetro 35	2	5.099806	−75.698583	847	National road edge in dry forest	
Manizales	Jardín Botánico Universidad de Caldas	3	5.055833	−75.495278	2131	Botanical garden in urban matrix	
	Vereda Rosario	4	5.027472	−75.581917	1241	Agroforestry matrix in tropical submontane forest	
Villamaría	Reserva Forestal Protectora Bosques de la CHEC	5	5.024611	−75.395778	2635	Private peri-urban forest reserve	
La Dorada	Vereda La Atarraya	6	5.710056	−74.727444	178	Agroforestry and livestock matrix in dry forest	
Arauca	Arauca	Los Trompillos	7	6.779556	−70.716667	120	Cattle grazing area in floodable savanna	

Fig. 2 Study area. (a) America with Colombia (black), (b) map of Colombia with the departments of Arauca and Caldas (black), (c) Department of Caldas, and (d) Department of Arauca. Sampled localities in the municipalities of Palestina (1, vereda El Retiro; 2, Kilómetro 35), Manizales (3, Jardín Botánico de la Universidad de Caldas; 4, vereda Rosario) Villamaría (5, Reserva Forestal Protectora Bosques de la CHEC) La Dorada (6, vereda La Atarraya) and Arauca (7, Los Trompillos).

Fig. 2

To capture bats, we installed between two and six mist nets at each locality and operated from 17:30 to 20:00 h. To confirm the taxonomic identity, captured bats were euthanized and collected following the animal care recommendations proposed by Sikes and Animal Care and Use Committee of the American Society of Mammalogists (2016). Tissue samples from the sacrificed specimens were also collected to search for other pathogens (e.g., Anaplasma, Babesia, Borrelia, Coxiella, Rickettsia). Some of these data have already been published and are available in the literature (Mancilla-Agrono et al., 2022; Velásquez-Guarín et al., 2024; Ossa-López et al., 2024a, 2024b). The morphological identification of the collected bats was based on taxonomic keys (e.g., Gardner, 2008). We took blood samples of each bat via cardiac puncture, and we prepared at least two blood smears per individual and fixed in absolute methanol for 3 min. The smears were transported to the laboratory and stained with 4% Giemsa solution, pH 7.2, for 45 min at 17 °C (Alvarez-Londoño et al., 2022). The remaining blood was stored in absolute ethanol for subsequent molecular analyses.

Sample collection was conducted under the framework of the permit granted by the National Environmental Licenses Authority (ANLA) to the 10.13039/501100007626 Universidad de Caldas , as stipulated in resolution No. 02497 of December 31, 2018, updated by resolution No. 000026 of January 9, 2024. Additionally, no species registered in the red list of threatened species of Colombia, as stated in resolution No. 1912 of 2017, updated by resolution No. 0126 of February 6, 2024, were collected. The capture and collection of wild mammals were approved by the Comité de Bioética of the Facultad de Ciencias Exactas y Naturales of the Universidad de Caldas (June 2, 2017, and September 20, 2019). All collected samples and specimens were deposited in the mammals and ectoparasites collections of the Museo de Historia Natural of the Universidad de Caldas (MHN-UCa). Additionally, we determined the percent prevalence of Polychromophilus in the captured bats using the equation [(number of infected individuals/number of examined individuals) * 100] (Bush et al., 1997).

2.3 Identification

We performed the morphological identification of haemosporidians of the genus Polychromophilus by observing blood smears using an Olympus BX43 light microscope at 40X and 100× magnification, examining approximately 100–150 fields per objective (Valkiunas, 2004; Campbell, 2015). We morphologically identified the parasites using the original descriptions of the genus Polychromophilus (Garnham, 1966, Garnham et al., 1971; Landau et al., 1984; Marinkelle, 1995). We captured images of the parasites with an Olympus DP28 digital camera and edited using Olympus CellSens Standard software v3.22.11. We also identified the ectoparasites of bats infected by Polychromophilus following Brown (2016).

To molecularly determine Polychromophilus infection in bat blood samples, we extracted DNA using the Promega Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, USA), following the manufacturer's suggested instructions. To detect Polychromophilus DNA, nested Polymerase Chain Reaction (PCR) assays were conducted on a fragment of the cytochrome b (cyt b) gene. For the initial PCR, primers AE064/AE066, which amplify a 1109 bp fragment for the major haemosporidians genera, were used (Pacheco et al., 2018). The second PCR used the initial PCR amplicon with primers HaemF/HaemR2, which amplify a 525 bp fragment specific to Plasmodiidae (Hellgren et al., 2004). All PCR reactions included negative controls (H2Odd) and positive controls (Plasmodium homopolare).

Additionally, to confirm the taxonomic identity of the bat and its ectoparasites, we conducted PCR assays targeting the mammalian cyt b gene using primers L14816/H15173 (Parson et al., 2000) and the invertebrate cytochrome oxidase 1 (COI) gene using primers LCO1490/HCO2198 (Folmer et al., 1994). Following the confirmation of the hemoparasite, to search for haemosporidian DNA in the liver tissue of the individual and its ectoparasites, we conducted a PCR assay All PCR products were visualized by horizontal electrophoresis on 1% agarose gels stained with SYBR SAFE (Thermo Fisher Scientific, Waltham, USA) and viewed using a UVP GelDoc-It® 2310 photodocumenter (Thermo Fisher Scientific, Waltham, MA, USA). We sent the positive samples to Macrogen (Seoul, South Korea) for purification and Sanger sequencing, and we edited the sequence in Geneious Prime (2023.0.4. https://www.geneious.com/) and compared with sequences deposited in the GenBank database. All sequences obtained were deposited in GenBank.

2.4 Phylogenetic analysis

We inferred phylogenetic relationships among haemosporidians using Bayesian Inference (BI) and Maximum Likelihood (ML) methods, incorporating our sequence along with partial cyt b gene sequences obtained from GenBank (Table S1), and a sequence from Leucocytozoon buteonis (HF543630) as an outgroup. Sequences were aligned using MEGA11 software (Tamura et al., 2021), resulting in a total alignment length of 490 bp. The General Time Reversible (GTR) + I + G model was selected based on the corrected Akaike Information Criterion using jModeltest v.2.1.6 (Darriba et al., 2012).

We performed the BI analysis using MrBayes 3.2.7a (Ronquist and Huelsenbeck, 2003) via the CIPRES Science Gateway v3.3 (Miller et al., 2010). We estimated posterior probability distributions (PP) using two independent Markov Chain Monte Carlo (MCMC) chains, run for 15 million generations with four chains sampled every 1000 generations. Convergence was assessed using Tracer (Rambaut and Drummond, 2007), with 25% of the trees discarded as burn-in and the remaining trees used to construct a consensus tree based on a 50% majority rule. We conducted the ML analysis using PhyML version 3.0 (Guindon and Gascuel, 2003), with nodal support values calculated via Bootstrap (BS) analysis using 1000 replicates. The phylogeny was visualized using FigTree v1.3.1 (Rambaut, 2009).

3 Results

From the 23 studies analyzed, we found 65 records of Polychromophilus in 46 bat species distributed across all continents except Antarctica (Table 1). Most Polychromophilus reports were detected in bats from the families Vespertilionidae (22 species), Miniopteridae (16 species), Hipposideridae (3 species), Rhinolophidae (3 species), with Rhinonycteridae and Emballonuridae having one species each (Table 1). The records were obtained in 30 countries and the most detected species was P. melanipherus (n = 14) and P. murinus (n = 12), P. adami and P. corradetti had a single record each, as did the species P. deanei, to which the record from this study is added and Polychromophilus sp. had 22 records.

Specifically, in America, Polychromophilus infections have been recorded exclusively in bats of the family Vespertilionidae, including Eptesicus fuscus, Myotis austroriparius, M. nigricans, M. riparius, M. ruber, Myotis sp., and N. diminutus in Brazil, Colombia, the United States, and Panama (Table 1). The searched for new records of Polychromophilus in bats from Colombia included 125 individuals belonging to 39 species and 5 families captured in seven localities (Table 3). The families with the highest number of bat individuals were Phyllostomidae (n = 52), Molossidae (n = 43), Vespertilionidae (n = 23), and Noctilionidae (n = 6), while the family Emballonuridae was represented by only one individual (Table 3). The species with the highest number of individuals captured were Molossus molossus (n = 14), Cynomops planirostris (n = 9), Neoeptesicus orinocensis (n = 9), Molossus pretiosus (n = 6), and Noctilio albiventris (n = 6) (Table 3). We detected only one individual of Myotis albescens (Vespertilionidae) infected with Polychromophilus through morphological analysis of blood smears and through molecular analysis of the blood, since it was not possible to detect it molecularly in the liver samples. The M. albescens captured in the municipality of Palestina was infected with P. deanei (prevalence = 0.8%). In this individual, we detected morphologically intraerythrocytic sexual forms (gametocytes) in blood smears, matching the original description of P. deanei (Fig. 3). The mature macrogametocytes measured between 5.5 and 7.5 μm in length and 4.0–5.0 μm in width (Fig. 3h–l, n, o). Microgametocytes were less abundant, measuring approximately 7 μm in length and 4.5 μm in width (Fig. 3 m). The number of hemozoin granules of gametocytes varied between 24 and 35 (Fig. 3h–o). Molecular analyses of the hemoparasite cyt b gene showed 100% identity with Polychromophilus sp. sequences (MW984522 and MW984518) from Brazil in M. ruber and M. riparius. The GenBank accession code for the P. deanei sequence obtained in this study is (PP971136). Additionally, we molecularly confirmed the taxonomic identity of the infected bat (M. albescens) (GenBank accession: PP975259). During capture, this bat was parasitized by mites, identified morphologically and molecularly as Spinturnix americanus (Fig. 4). The GenBank accession codes for the (COI) gene of S. americanus are (PP967722 and PP967723).Table 3 Bat species examined for Polychromophilus detection in the departments of Arauca and Caldas, Colombia. Localities are shown in Fig. 2.

Table 3Host family	Species	Number of bats examined	Number of bats per locality	
1	2	3	4	5	6	7	
Emballonuridae	Saccopteryx leptura	1							1	
Molossidae	Cynomops greenhalli	1						1		
Cynomops milleri	1							1	
Cynomops planirostris	9							9	
Eumops auripendulus	1		1						
Eumops glaucinus	2							2	
Molossops griseiventer	1						1		
Molossops temminckii	3							3	
Molossus coibensis	1							1	
Molossus molossus	14	10			3			1	
Molossus pretiosus	6							6	
Molossus rufus	4							4	
Noctilionidae	Noctilio albiventris	6							6	
Phyllostomidae	Artibeus lituratus	4			3			1		
Artibeus obscurus	1						1		
Carollia brevicauda	5		1	1			2	1	
Carollia castanea	3		1				2		
Carollia perspicillata	4						4		
Carollia sp.	4			4					
Dermanura anderseni	5						5		
Desmodus rotundus	1							1	
Glossophaga soricina	4			2			2		
Lophostoma nicaraguae	3						3		
Phyllostomus discolor	2						1	1	
Phyllostomus hastatus	1						1		
Platyrrhinus helleri	2						2		
Sturnira cf. parvidens	1						1		
Sturnira erythromos	3					3			
Sturnira giannae	3						3		
Sturnira luisi	2						2		
Sturnira parvidens	1					1			
Trinycteris nicefori	1						1		
Uroderma convexum	1						1		
Uroderma magnirostrum	1							1	
Vespertilionidae	Myotis albescensa	5	5							
Myotis handleyi	5							5	
Myotis keaysi	1					1			
Myotis sp.	3							3	
Neoeptesicus orinocensis	9							9	
Total		125	15	3	10	3	5	34	55	
a Polychromophilus deanei positive species.

Fig. 3 Erythrocytic forms of Polychromophilus deanei found in Myotis albescens. (a–d) trophozoites, (e–g) immature gametocytes, (h-l, n, o) mature macrogametocytes, (m) mature microgametocyte. Short white arrow indicates healthy erythrocytes. White arrowhead indicates parasite nucleus. Black arrowhead indicates hemozoin granules. Short black arrow indicates infected erythrocyte cytoplasm. Scale bar = 10 μm.

Fig. 3

Fig. 4 Female Spinturnix americanus collected on a Myotis albescens in Colombia. (A) dorsal view (B) ventral view. PD = dorsal plate, PV = ventral plate. Scale bar = 500 μm.

Fig. 4

Our phylogenetic reconstruction of the haemosporidians grouped all Polychromophilus sequences into a well-supported clade (PP = 1 and BS = 100%; Fig. 5). Our sequence formed a clade with sequences identified as Polychromophilus sp. found in Myotis bats from Brazil and Panama (genetic distances ≤0.8%), with a segregated sequence of Polychromophilus sp. detected in N. diminutus in Brazil (Table S2; Fig. 5). Furthermore, these sequences formed a clade with sequences of P. murinus reported in bats and flies of the family Nycteribiidae from the Old World (Madagascar and Switzerland) (genetic distance = 2%; Table S2; Fig. 5).Fig. 5 Bayesian phylogeny based on 71 partial sequences of the mitochondrial cytochrome b (cyt b) gene of parasites of the order Haemosporida (Table S1). Leucocytozoon buteonis (HF543630) was used as an outgroup. Posterior probability (PP)/bootstrap (BS) values are shown above the nodes. Scale bar = 0.04 substitutions per site.

Fig. 5

4 Discussion

The results of our review indicate that Polychromophilus has been recorded infecting bats worldwide, except in Antarctica, with a higher concentration of cases in Africa, Europe, and Asia. Most of these reports have been in bats of the family Vespertilionidae, with Myotis being the most frequently host globally (Megali et al., 2011; Witsenburg et al., 2014; Ramasindrazana et al., 2018; Arnuphapprasert et al., 2020; Rosyadi et al., 2022). Similarly, P. melanipherus and P. murinus are the most documented species, primarily in bats of the families Miniopteridae and Vespertilionidae, respectively (Witsenburg et al., 2012; Perkins and Schaer, 2016; Lutz et al., 2016; Sándor et al., 2021). In contrast, P. adami and P. corradetti have been recorded only in miniopterids from Africa (Congo and Gabon) (Perkins and Schaer, 2016). Similarly, P. deanei has only been reported in vespertilionids from South America (Brazil, Colombia) (Garnham et al., 1971; Marinkelle, 1995). Additionally, many of the yet undescribed species of Polychromophilus are found in the tropical regions, which are known for the high diversity of bats (Schaer et al., 2013; Borner et al., 2016; Arnuphapprasert et al., 2020; Chumnandee et al., 2021; Mathias et al., 2023). this underscores the need to expand research on Polychromophilus in these regions.

Our study represents the first report of P. deanei in M. albescens for the Americas. Morphologically, the immature intraerythrocytic forms (trophozoites) and sexual forms (gametocytes) of Polychromophilus match the original description of P. deanei in M. nigricans proposed by Garnham et al. (1971). Furthermore, our observations were similar to reports of Polychromophilus sp. in Myotis austroriparius from the United States (Foster, 1979) and P. deanei (as Bioccala deanei) in Eptesicus fuscus miradorensis from Colombia (Marinkelle, 1995). Additionally, our sequence of cyt b of Polychromophilus (PP971136) grouped with sequences identified as Polychromophilus sp. reported in Vespertilionidae bats in America in three previous studies (Borner et al., 2016; Minozzo et al., 2021; Mathias et al., 2023). However, these studies only used molecular techniques for detecting Polychromophilus, so there is no morphospecies associated with these sequences (Borner et al., 2016; Minozzo et al., 2021; Mathias et al., 2023). We recommend integrating morphological and molecular tools for understanding the interactions between Polychromophilus and bats, considering that both sources of information are key to resolve deeper phylogenetic discrepancies in Haemosporida (Outlaw and Ricklefs, 2011). Phylogenetic relationships evidenced a separation between Polychromophilus species in bats within Vespertilionidae from the Americas and the old World P. murinus. These results align with the differentiation of Polychromophilus in vespertilionid bats from different zoogeographic regions (Garnham et al., 1971; Perkins and Schaer, 2016). In this context, bats appear to be a crucial link in the circulation of haemosporidian of the Plasmodiidae in vertebrates, particularly parasites of the genus Polychromophilus (Perkins and Schaer, 2016). Therefore, understanding the interactions between bats, Polychromophilus, and their vectors is crucial to understand their zoonotic potential (Witsenburg et al., 2012).

In this study, we reported a prevalence of 0.8%, similar to values obtained in Brazil (Minozzo et al., 2021; Mathias et al., 2023). However, the prevalence reported in the study by Mathias et al. (2023), considering only individuals of the genus Myotis, was approximately 21.7%, compared to the ∼7.1% found in this study. This discrepancy could be explained by the greater representation of Myotis in the Brazilian samples, as well as by ecological variables still unexplored in the Neotropics (e.g., seasonality and age of the bats) (Dew and McMillan, 1970; Witsenburg et al., 2014). These results contrast with high absolute prevalences (>40%) found in studies conducted in bats in countries in Africa and Europe (Schaer et al., 2013; Witsenburg et al., 2014; Lutz et al., 2016). It has been suggested that in temperate regions, the infection cycles of Polychromophilus might respond to habitat characteristics (e.g., seasonality) or to ecological traits of their hosts (e.g., age or reproductive periods) (Dew and McMillan, 1970; Witsenburg et al., 2014). Therefore, future research should gather information to contrast the unimodal seasonal profile of the tropics with the ecological and reproductive aspects of Polychromophilus reservoirs. This is particularly relevant in the Neotropical context, where seasonal dynamics may differ from those in temperate regions. Additionally, a high diversity of non-competent hosts reduces the probability of infection by vector-borne parasites (Garrido et al., 2021), and this could particularly occur in the Neotropics, where a higher diversity of bats has been recorded compared to temperate zones in Europe or tropical regions in Africa (Mickleburgh et al., 2002; Mena et al., 2011; Ramírez-Chaves et al., 2024).

Additionally, we identified wing mites of the species S. americanus on an individual of M. albescens infected with P. deanei. This mite species is exclusive to bats and is distributed from southern Canada to southern Brazil (Rudnick 1960; Dick et al., 2003). However, reports of its presence on M. albescens in Colombia are scarce and have been limited to morphological confirmations (Tarquino-Carbonell et al., 2015). In this study, we report for the first time COI gene sequences for the mite S. americanus. Since mites can act as vectors of pathogens, it is crucial to further investigate the possible role of S. americanus in disease transmission in bats, as this relationship could have important implications for the ecology and health of bats in areas where these mites are present.

Finally, this study represents the first morphological and molecular report of P. deanei in M. albescens in America. Additionally, we confirmed the presence of Polychromophilus in bats in Colombia, almost 30 years after the first and only report by Marinkelle (1995). Given these findings, it is necessary to expand the diversity of examined bat hosts, as well as use additional molecular markers to perform more detailed phylogenetic analyses. New studies should include morphological observations and molecular identification in other tissues of bat hosts, as well as perform these same analyses for their infective forms in the vector. This will provide a comprehensive picture of the biology of Polychromophilus species.

Funding

This work was funded by the Program: “Relación, distribución, taxonomía de especies de garrapatas asociadas a mamíferos silvestres en zonas endémicas de rickettsiosis en Colombia. Un acercamiento a la comprensión de la relación vectores patógenos-reservorios”, granted by the Ministerio de Ciencia, Tecnología e Innovación - Minciencias (Code: 120385270267 and CTO 80740-200-2021) - Project: Garrapatas asociadas a mamíferos silvestres en el departamento de Caldas: Diversidad, detección de patógenos y distribución. Funded by Minciencias (Code: 71717). and also by the Project: Garrapatas (Acari: Ixodidae) de pequeños mamíferos en Arauca (Arauca, Colombia): una aproximación al ciclo de transmisión de borreliosis o enfermedad de Lyme. Funded by Vicerrectoría de investigaciones y Posgrados - Universidad de Caldas; Code: 0277620. Additionally, we received support from the Call for Economic Resources Support to Research Groups of the 10.13039/501100007626 University of Caldas. - 2023 - Vicerrectoría de Investigaciones y posgrados - Universidad de Caldas .

CRediT authorship contribution statement

Diego Fernando Ceballos-Pérez: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Johnathan Alvarez-Londoño: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Héctor E. Ramírez-Chaves: Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. Fredy A. Rivera-Páez: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Multimedia component 2

Multimedia component 2

Acknowledgments

We thank all members of the GEBIOME group, particularly Dr. Paula A. Ossa-López and the researchers Erika M. Ospina-Pérez, María José Narváez-Moreno, Estefani T. Martínez-Sánchez, the members of the Natural History Laboratory at the Museo de Historia Natural de la Universidad de Caldas (IZBD), Alison Guerrero-Diaz, and José J. Henao-Osorio, Wendy Juliana Muñoz-Márquez as well as all members of the Semillero de Investigación en Genética Molecular (SeGeM) for their valuable collaboration and support during the development of this research.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijppaw.2024.100986.
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