Molecular Investigation Confirms Myotis Genus Bats as Common Hosts of Polychromophilus in Brazil.

Plasmodium spp. and some other blood parasites belonging to the order Haemosporida are the focus of many epidemiological studies worldwide. However, haemosporidian parasites from wild animals are largely neglected in scientific research. For example, Polychromophilus parasites, which are exclusive to bats, are described in Europe, Asia, Africa, and Oceania, but little is known about their presence and genetic diversity in the New World. In this study, 224 samples of bats from remaining fragments of the Atlantic Forest and Pantanal biomes, as well as urbanized areas in southern and southeastern Brazil, were analyzed for the presence of haemosporidian parasites by PCR of the mitochondrial gene that encodes cytochrome b (cytb). The PCR fragments of the positive samples were sequenced and analyzed by the Bayesian inference method to reconstruct the phylogenetic relationships between Polychromophilus parasites from bats in Brazil and other countries. Sequences from Brazilian lineages of Polychromophilus were recovered in a clade with sequences from Polychromophilus murinus and close to the one Polychromophilus sequence obtained in Panama, the only available sequence for the American continent. This clade was restricted to bats of the family Vespertilionidae and distinct from Polychromophilus melanipherus, a parasite species mainly found in bats of the family Miniopteridae. The detection of Polychromophilus and the genetic proximity to P. murinus were further confirmed with the amplification of two other genes (clpc and asl). We also found a Haemosporida parasite sequence in a sample of Noctilio albiventris collected in the Pantanal biome, which presents phylogenetic proximity with avian Haemoproteus sequences. Morphological and molecular studies are still needed to conclude and describe the Polychromophilus species in Brazilian Myotis bats in more detail and to confirm Haemoproteus parasites in bats. Nevertheless, these molecular results in Brazilian bats confirm the importance of studying these neglected genera.

Non-invasive investigation of Polychromophilus parasite infections in bat populations in Serbia using bat flies.

BACKGROUND: Haemosporidian parasites of the genus Polychromophilus infect bats worldwide. They are vectored by obligate ectoparasitic bat flies of the family Nycteribiidae. Despite their global distribution, only five Polychromophilus morphospecies have been described to date. The two predominant species, Polychromophilus melanipherus and Polychromophilus murinus, are broadly distributed and mainly infect miniopterid and vespertilionid bats, respectively. In areas where species from different bat families aggregate together, the infection dynamics and ability of either Polychromophilus species to infect other host families is poorly characterized. METHODS: We collected 215 bat flies from two bat species, Miniopterus schreibersii and Rhinolophus ferrumequinum, which sometimes form mixed clusters in Serbia. Miniopterus schreibersii is known to be frequently infected with P. melanipherus, whereas R. ferrumequinum has been observed to be incidentally infected with both Polychromophilus species. All flies were screened for Polychromophilus infections using a PCR targeting the haemosporidian cytb gene. Positive samples were subsequently sequenced for 579 bp of cytochrome b (cytb) and 945 bp of cytochrome oxidase subunit 1 (cox1). RESULTS: Polychromophilus melanipherus DNA was detected at six out of nine sampling locations and in all three examined bat fly species collected from M. schreibersii (Nycteribia schmidlii, n = 21; Penicillidia conspicua, n = 8; Penicillidia dufourii, n = 3). Four and five haplotypes were found for cytb and cox1, respectively. Evidence for multiple Polychromophilus haplotypes was found in 15 individual flies. These results point to a high diversity of P. melanipherus parasites in Miniopterus hosts and efficient transmission throughout the study area. A single Phthiridium biarticulatum bat fly collected from R. ferrumequinum screened positive for P. melanipherus, but only yielded a partial cox1 sequence fragment. Nevertheless, this result suggests that secondary hosts (both bat and fly species) are regularly confronted with this parasite. CONCLUSIONS: The results of this study provide new insights into the prevalence and distribution of Polychromophilus parasites in European bats and their nycteribiid vectors. The use of bat flies for the non-invasive investigation of Polychromophilus infections in bat populations has proven to be efficient and thus represents an alternative for large-scale studies of infections in bat populations without the need to invasively collect blood from bats.

Isolation and molecular characterization of Polychromophilus spp. (Haemosporida: Plasmodiidae) from the Asian long-fingered bat (Miniopterus fuliginosus) and Japanese large-footed bat (Myotis macrodactylus) in Japan.

Bats (order, Chiroptera) account for more than one-fifth of all mammalian species in the world and are infected by various intra-erythrocytic parasites of the family Plasmodiidae (Apicomplexa: Haemosporida), including Polychromophilus Dionisi, 1899. Recent advance in the molecular characterization of haemosporidian isolates has enabled their accurate identification, particularly in the last decade. Studies are actively conducted in tropical regions, Europe, and Australia; however, data on haemosporidian infection in bats in Asian temperate areas, including Japan, remain limited. In this study, 75 bats of 4 species (Miniopterus fuliginosus, Myotis macrodactylus, Rhinolophus nippon, and Rhinolophus cornutus) were captured at three sites in western Japan (Yamaguchi Prefecture), and haemosporidian parasites were screened microscopically and molecularly via nested polymerase chain reaction (PCR) targeting the cytochrome b (cytb), cytochrome c oxidase subunit I (cox-1), apicoplast caseinolytic protease C (clpc), and nuclear elongation factor 2 (EF2) genes. The survey detected Polychromophilus melanipherus in 15 (40.5%) miniopterid bats (M. fuliginosus) and Polychromophilus murinus in 6 (46.2%) vespertilionid bats (M. macrodactylus), whereas none of the 25 rhinolophid bats (R. nippon and R. cornutus) was infected, indicating the robust host specificity for miniopterid (P. melanipherus) and vespertilionid (P. murinus) bats regardless of orthotopic nesting. The 15 Polychromophilus cytb sequences obtained from 11 miniopterid and 4 vespertilionid bats were classified into six cytb haplotypes (three for each species), showing no region-specific variation in a phylogenetic tree of Polychromophilus isolates in the Old World. Similarly, multiple haplotypes (seven for cox-1 and nine for clpc) and genotypes (three for EF2) were characterized for the Japanese isolates of Polychromophilus, and the results were consistent with those based on a haemosporidian cytb analysis. Bat flies (Nycteribia allotopa and another undetermined Nycteribia sp.) collected from the body surface of bats harbored Polychromophilus oocysts on the external surface of the midgut. This is the first study to report the isolation and molecular characterization of Polychromophilus spp. in miniopterid and vespertilionid bats in the temperate area of Asia (western Japan). Future studies should evaluate the global prevalence of haemosporidian infections in bats.

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.

Diversity, distribution, and drivers of Polychromophilus infection in Malagasy bats.

BACKGROUND: Numerous studies have been undertaken to advance knowledge of apicomplexan parasites infecting vertebrates, including humans. Of these parasites, the genus Plasmodium has been most extensively studied because of the socio-economic and public health impacts of malaria. In non-human vertebrates, studies on malaria or malaria-like parasite groups have been conducted but information is far from complete. In Madagascar, recent studies on bat blood parasites indicate that three chiropteran families (Miniopteridae, Rhinonycteridae, and Vespertilionidae) are infected by the genus Polychromophilus with pronounced host specificity: Miniopterus spp. (Miniopteridae) harbour Polychromophilus melanipherus and Myotis goudoti (Vespertilionidae) is infected by Polychromophilus murinus. However, most of the individuals analysed in previous studies were sampled on the western and central portions of the island. The aims of this study are (1) to add new information on bat blood parasites in eastern Madagascar, and (2) to highlight biotic and abiotic variables driving prevalence across the island. METHODS: Fieldworks were undertaken from 2014 to 2016 in four sites in the eastern portion of Madagascar to capture bats and collect biological samples. Morphological and molecular techniques were used to identify the presence of haemosporidian parasites. Further, a MaxEnt modelling was undertaken using data from Polychromophilus melanipherus to identify variables influencing the presence of this parasite RESULTS: In total, 222 individual bats belonging to 17 species and seven families were analysed. Polychromophilus infections were identified in two families: Miniopteridae and Vespertilionidae. Molecular data showed that Polychromophilus spp. parasitizing Malagasy bats form a monophyletic group composed of three distinct clades displaying marked host specificity. In addition to P. melanipherus and P. murinus, hosted by Miniopterus spp. and Myotis goudoti, respectively, a novel Polychromophilus lineage was identified from a single individual of Scotophilus robustus. Based on the present study and the literature, different biotic and abiotic factors are shown to influence Polychromophilus infection in bats, which are correlated based on MaxEnt modelling. CONCLUSIONS: The present study improves current knowledge on Polychromophilus blood parasites infecting Malagasy bats and confirms the existence of a novel Polychromophilus lineage in Scotophilus bats. Additional studies are needed to obtain additional material of this novel lineage to resolve its taxonomic relationship with known members of the genus. Further, the transmission mode of Polychromophilus in bats as well as its potential effect on bat populations should be investigated to complement the results provided by MaxEnt modelling and eventually provide a comprehensive picture of the biology of host-parasite interactions.

Molecular characterization of Polychromophilus parasites of Scotophilus kuhlii bats in Thailand.

Parasites of the haemosporidian genus Polychromophilus have exclusively been described in bats. These parasites belong to the diverse group of malaria parasites, and Polychromophilus presents the only haemosporidian taxon that infects mammalian hosts in tropical as well as in temperate climate zones. This study provides the first information of Polychromophilus parasites in the lesser Asiatic yellow bat (Scotophilus kuhlii) in Thailand, a common vespertilionid bat species distributed in South and Southeast Asia. The gametocyte blood stages of the parasites could not be assigned to a described morphospecies and molecular analysis revealed that these parasites might represent a distinct Polychromophilus species. In contrast to Plasmodium species, Polychromophilus parasites do not multiply in red blood cells and, thus, do not cause the clinical symptoms of malaria. Parasitological and molecular investigation of haemosporidian parasites of wildlife, such as the neglected genus Polychromophilus, will contribute to a better understanding of the evolution of malaria parasites.

Host conservation through their parasites: molecular surveillance of vector-borne microorganisms in bats using ectoparasitic bat flies.

Most vertebrates host a wide variety of haematophagous parasites, which may play an important role in the transmission of vector-borne microorganisms to hosts. Surveillance is usually performed by collecting blood and/or tissue samples from vertebrate hosts. There are multiple methods to obtain samples, which can be stored for decades if properly kept. However, blood sampling is considered an invasive method and may possibly be harmful to the sampled individual. In this study, we investigated the use of ectoparasites as a tool to acquire molecular information about the presence and diversity of infectious microorganism in host populations. We tested the presence of three distinct vector-borne microorganisms in both bat blood and bat flies: Bartonella bacteria, malaria-like Polychromophilus sp. (Apicomplexa), and Trypanosoma sp. (Kinetoplastea). We detected the presence of these microorganisms both in bats and in their bat flies, with the exception of Trypanosoma sp. in South African bat flies. Additionally, we found Bartonella sp. in bat flies from one population in Spain, suggesting its presence in the host population even if not detected in bats. Bartonella and Polychromophilus infection showed the highest prevalence in both bat and bat fly populations. Single, co- and triple infections were also frequently present in both. We highlight the use of haematophagous ectoparasites to study the presence of infectious microorganism in host blood and its use as an alternative, less invasive sampling method.

TITLE: Conservation des hôtes grâce à leurs parasites : surveillance moléculaire des microorganismes à transmission vectorielle chez les chauves-souris à l'aide de mouches ectoparasites. ABSTRACT: La plupart des vertébrés hébergent une grande variété de parasites hématophages, qui peuvent jouer un rôle important dans la transmission de microorganismes à transmission vectorielle à leurs hôtes. La surveillance est généralement effectuée en prélevant des échantillons de sang et/ou de tissus sur des hôtes vertébrés. Il existe plusieurs méthodes pour obtenir des échantillons, qui peuvent être conservés pendant des décennies dans des bonnes conditions. Cependant, le prélèvement sanguin est considéré comme une méthode invasive et peut éventuellement être nocif pour l'individu prélevé. Dans cette étude, nous avons étudié l'utilisation d'ectoparasites comme outil pour acquérir des informations moléculaires sur la présence et la diversité des microorganismes infectieux dans les populations hôtes. Nous avons testé la présence de trois microorganismes distincts, transmis par des vecteurs, dans le sang et les mouches des chauves-souris : les bactéries Bartonella, Polychromophilus sp. (Apicomplexa) et Trypanosoma sp. (Kinetoplastea). Nous avons détecté la présence de ces microorganismes à la fois chez les chauves-souris et chez leurs mouches des chauves-souris, à l'exception de Trypanosoma sp. chez les chauves-souris sud-africaines. De plus, nous avons trouvé Bartonella sp. chez les mouches des chauves-souris d'une population en Espagne, ce qui suggère sa présence dans la population hôte même si elle n'est pas détectée chez les chauves-souris elles-mêmes. Les infections à Bartonella et Polychromophilus ont montré la prévalence la plus élevée dans les populations de chauves-souris et de mouches des chauves-souris. Des infections simples, doubles et triples étaient également fréquemment présentes dans les deux cas. Nous mettons en évidence l'utilisation d'ectoparasites hématophages pour étudier la présence de microorganismes infectieux dans le sang de l'hôte et son utilisation comme méthode alternative et moins invasive d'échantillonnage.

First molecular investigation of haemosporidian parasites in Thai bat species.

Malaria parasites in the phylum Apicomplexa (Order: Haemosporida) infect diverse vertebrates and invertebrate hosts. At least seven genera of haemosporidian parasites have been described to exclusively infect bats. Most of these parasites remain enigmatic with a poorly known host range. Here, we investigated 271 bats belonging to 21 species and seven families from six provinces of Thailand. Overall, 124 out of 271 bats (45.8%) were positive for haemosporidian parasites, while none had Plasmodium, based on microscopic examination of blood smears and PCR amplification. We obtained 19 distinct cytochrome b (cytb) nucleotide haplotypes of Hepatocystis from seven bat species (families: Craseonycteridae, Hipposideridae, Pteropodidae, and Rhinolophidae). Nycteria was found in four bat species (Craseonycteridae, Emballonuridae, Megadermatidae, and Pteropodidae) and Polychromophilus in two species (Emballonuridae, Vespertilionidae). Phylogenetic analysis inferred from cytb sequences placed Hepatocystis into 2 different clades. Most Hepatocystis infections were found in insectivorous bats and clustered together with a sequence from Hipposideros larvatus in Cambodia (in subclade 1a). A single sequence of Hepatocystis obtained from a frugivorous bat, Cynopterus brachyotis, was placed in the same clade with Hepatocystis from the same bat species previously reported in Malaysia (clade 2). Nycteria in these Thai bats were clearly separated from the African isolates previously reported in bats in the family Rhinolophidae. Polychromophilus murinus from Myotis siligorensis was placed in a distinct clade (clade 2) from Polychromophilus melanipherus isolated from Taphozous melanopogon (clade 1). These results confirmed that at least two distinct species of Polychromophilus are found in Thailand. Collectively, Hepatocystis presented no host specificity. Although Megaderma spasma seemed to be infected by only Nycteria, its respective parasite does not show specificity to only a single bat host. Polychromophilus murinus and P. melanipherus seem to infect a narrower host range or are somehow restricted to bats in the families Vespertilionidae and Emballonuridae, respectively.

Polychromophilus melanipherus and haemoplasma infections not associated with clinical signs in southern bent-winged bats (Miniopterus orianae bassanii) and eastern bent-winged bats (Miniopterus orianae oceanensis).

While bats are often viewed as carriers of infectious disease agents, little research has been conducted on the effects these pathogens may have on the bat populations themselves. The southern bent-winged bat (Miniopterus orianae bassanii) is a critically endangered subspecies endemic to south-eastern Australia. Population numbers of this bat have declined over the past 50 years, but the reasons for this are unclear. As part of a larger study to determine if disease could be a contributing factor to this decline, southern bent-winged bats from several locations in Victoria and South Australia were captured and examined for the presence of the blood parasite, Polychromophilus melanipherus, and haemoplasmas (Mycoplasma sp.). Results were compared with those obtained from populations of the more common, partially sympatric, eastern bent-winged bat (Miniopterus orianae oceanensis) from three different locations in Victoria. Both organisms were found in both subspecies (prevalence of P. melanipherus 60% by PCR for southern bent-winged bats compared with 46% for eastern bent-winged bats; prevalence of haemoplasmas 10% for southern bent-winged bats compared with 8% for eastern bent-winged bats), with no association between the probability of infection, body weight, abnormal blood parameters or any other indicators of ill health. However, Victorian southern bent-winged bats had heavier burdens of P. melanipherus than both the South Australian southern bent-winged bats and eastern bent-winged bats. Further investigations are required to determine if these differences are impacting population health.

Nycteria and Polychromophilus parasite infections of bats in Central Gabon.

Haemosporida are arthropod-borne blood parasites that infect a wide range of vertebrate hosts, including numerous species of bats. Here, we present data of haemosporidian infections in different bat species that were surveyed in Ngounié province, Gabon. We detected Nycteria parasites in Rhinolophus bats and Polychromophilus in Miniopterus minor, a rare and poorly known bat species. Strikingly, no Hepatocystis parasites, which are abundant in epauletted fruit bats elsewhere in Africa, were detected. Our findings suggest that Hepatocystis infections in bats display diverse regional patterns of distribution and transmission dynamics, that cannot be predicted from host abundance. Nycteria parasites are widely distributed in several African rhinolophid species and Polychromophilus parasites of diverse Miniopterus species worldwide belong to the same parasite species.

Polychromophilus spp. (Haemosporida) in Malagasy bats: host specificity and insights on invertebrate vectors.

BACKGROUND: Bats are home to diverse haemosporidian parasites namely Plasmodium and Plasmodium-related. While information is available at a worldwide level, haemosporidian infection in bats from Madagascar is still scarce and recent changes in the taxonomy of the island's bat fauna, particularly the description of several new species, require a reassessment of previously described patterns, including blood parasite ecology and vectorial transmission. METHODS: A sample representing seven of the nine known bat families and 31 of the 46 currently recognized taxa from Madagascar and collected in the western and central portions of the island were screened by PCR for the presence of Polychromophilus. In addition, Nycteribiidae flies parasitizing Miniopteridae and Vespertilionidae were screened for parasites with the aim to better understand aspects of vector transmission. Phylogenetic reconstruction using the mitochondrial cytochrome b encoding gene was used in a Bayesian analysis to examine the relationship between Polychromophilus recovered from Malagasy bats and those identified elsewhere. RESULTS: Polychromophilus infection was restricted to Miniopterus spp. (Miniopteridae), Myotis goudoti (Vespertilionidae), and Paratriaenops furculus (Rhinonycteridae), with an overall infection rate of 13.5%. Polychromophilus melanipherus was found infecting Miniopterus spp. and P. furculus, whereas Polychromophilus murinus was only recovered from M. goudoti. These two protozoan parasites species were also detected in bat flies species known to parasitize Miniopterus spp. and M. goudoti, respectively. Generalized linear model analyses were conducted to elucidate the effect of species and sex on haemoparasites infection in Miniopterus spp., which revealed that males have higher risk of infection than females and prevalence differed according to the considered Miniopterus host. Molecular screening of nycteribiid flies revealed three positive species for Polychromophilus spp., including Penicillidia sp. (cf. fulvida), Penicillidia leptothrinax, and Nycteribia stylidiopsis. These three fly species are known to parasitize Miniopterus spp. and M. goudoti and should be considered as potential vectors of Polychromophilus spp. CONCLUSION: Phylogenetic analyses demonstrated the existence of at least four distinct clades within the genus Polychromophilus, two of which were documented in the present study. The screening of nycteribiid flies overlaid on the highly diversified genus Miniopterus, provides considerable insight into parasite transmission, with bat infection being associated with their roosting behaviour and the occurrence of specific arthropod vectors.

A Modern Menagerie of Mammalian Malaria.

Malaria parasites belong to the diverse apicomplexan order Haemospororida and use a variety of vertebrate and dipteran hosts worldwide. Recently, the utilization of molecular methods has resulted in a burst of newly discovered and rediscovered taxa infecting mammalian hosts, particularly in apes, ungulates, and bats. Additional study of these diverse mammal-infecting taxa is crucial for better understanding the evolutionary history of malaria parasites, especially given that most previous comparative phylogenetic analyses have tended to use both limited taxon sampling and a small set of genetic loci, resulting in weakly supported (and sometimes hotly contested) hypotheses. The ability to generate genomic data from these mammalian parasites, even from subpatent infections, will open up exciting prospects for research on malaria parasites.

Bat flies (Diptera: Nycteribiidae and Streblidae) infesting cave-dwelling bats in Gabon: diversity, dynamics and potential role in Polychromophilus melanipherus transmission.

BACKGROUND: Evidence of haemosporidian infections in bats and bat flies has motivated a growing interest in characterizing their transmission cycles. In Gabon (Central Africa), many caves house massive colonies of bats that are known hosts of Polychromophilus Dionisi parasites, presumably transmitted by blood-sucking bat flies. However, the role of bat flies in bat malaria transmission remains under-documented. METHODS: An entomological survey was carried out in four caves in Gabon to investigate bat fly diversity, infestation rates and host preferences and to determine their role in Polychromophilus parasite transmission. Bat flies were sampled for 2-4 consecutive nights each month from February to April 2011 (Faucon and Zadie caves) and from May 2012 to April 2013 (Kessipoughou and Djibilong caves). Bat flies isolated from the fur of each captured bat were morphologically identified and screened for infection by haemosporidian parasites using primers targeting the mitochondrial cytochrome b gene. RESULTS: Among the 1,154 bats captured and identified as Miniopterus inflatus Thomas (n = 354), Hipposideros caffer Sundevall complex (n = 285), Hipposideros gigas Wagner (n = 317), Rousettus aegyptiacus Geoffroy (n = 157, and Coleura afra Peters (n = 41), 439 (38.0 %) were infested by bat flies. The 1,063 bat flies recovered from bats belonged to five taxa: Nycteribia schmidlii scotti Falcoz, Eucampsipoda africana Theodor, Penicillidia fulvida Bigot, Brachytarsina allaudi Falcoz and Raymondia huberi Frauenfeld group. The mean infestation rate varied significantly according to the bat species (ANOVA, F (4,75) = 13.15, P < 0.001) and a strong association effect between bat fly species and host bat species was observed. Polychromophilus melanipherus Dionisi was mainly detected in N. s. scotti and P. fulvida and less frequently in E. africana, R. huberi group and B. allaudi bat flies. These results suggest that N. s. scotti and P. fulvida could potentially be involved in P. melanipherus transmission among cave-dwelling bats. Sequence analysis revealed eight haplotypes of P. melanipherus. CONCLUSIONS: This work represents the first documented record of the cave-dwelling bat fly fauna in Gabon and significantly contributes to our understanding of bat fly host-feeding behavior and their respective roles in Polychromophilus transmission.