The chiropteran haemosporidian Polychromophilus melanipherus: A worldwide species complex restricted to the family Miniopteridae

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Abstract

This paper attempts to expand on the current knowledge regarding the evolutionary history of bat haemosporidian parasites. Using modern molecular tools as adjuncts to existing morphological descriptions, our understanding of the diversity of these parasites is discussed. The biogeography and host range distribution together with possible host-parasite interactions remain to be evaluated in more detail. Using a nested-PCR cytochrome b mitochondrial gene approach, we established a screening programme and survey of several months duration for haemosporidian parasites in four central African bat species living in an ecological community. The aim of the study was to describe parasites morphologically and molecularly, together with parasite prevalence variations over time, and evaluate parasite host-specificity in these sympatric cave bats. Over the survey period, Polychromophilus melanipherus was the only haemosporidian parasite identified in Miniopterus inflatus, with a continuous molecular prevalence of at least 60%. Molecular phylogenetic analyses show that P. melanipherus is a monophyletic group infecting Miniopterus bats which is, a sister group to P. murinus and Polychromophilus spp. This monophyletic group is composed of different cyt b haplotypes molecularly distantly related (but morphologically similar), circulating without geographic or host species distinction. This suggests that P. melanipherus is a species complex restricted to the family Miniopteridae. The phylogenetic analysis confirms that Polychromophilus parasites are distributed worldwide and supports the view that they are more closely related to avian haemosporidian parasites.

Introduction

Haemosporidian parasites, belonging to the phylum Apicomplexa and transmitted by haematophagous dipterans, are intracellular blood parasites infecting a wide variety of vertebrates (Garnham, 1966). Recent studies have revealed an unsuspected but remarkable molecular diversity of Plasmodium parasites in great apes (chimpanzees, gorillas and bonobos) in Africa (Krief et al., 2010, Ollomo et al., 2009, Prugnolle et al., 2010, Rich et al., 2009), as well as parasitic exchange events between these primates and humans in the past leading to the divergence of Plasmodium falciparum in humans from gorillas (Liu et al., 2010), thus resulting in the potential reservoir role of great apes for human malaria parasites (Duval et al., 2010, Rayner et al., 2011).

The biology and ecology of haemosporidian parasites remain to be fully documented in order to better understand the interactions between parasites and their natural hosts and the potential adaptability of specific haemosporidians to new non-natural hosts (Garamszegi, 2009). An essential first step is an exploration of the biogeography, distribution and dynamics with morphological, molecular and genomic descriptions of these blood parasites in animal populations or a specific mammalian group, i.e. great apes and other non-human primates, prosimians, rodents, bats etc., as well as in other vertebrate hosts such as birds, squamates and chelonians, living in diverse natural habitats (Jenkins and Owens, 2011, Kaiser et al., 2010, Martinsen et al., 2006, Martinsen et al., 2008).

Bats, belong to the order Chiroptera, the second highest order of the mammalian class, and represent, with over thousand species, twenty percent of all mammals (Teeling et al., 2005, Venditti et al., 2011). These are the only natural flying mammals, distributed worldwide and colonising very diverse ecological habitats ranging from temperate and tropical forests to caves and in human dwellings (Kunz and Fenton, 2003). Due to their wide diversity of species and habitats, bats are a unique model in which to explore co-evolution of pathogens within their hosts, their ability to jump species barriers and thus their potential role as reservoirs for emerging diseases (Calisher et al., 2006).

Four Haemosporidia genera are reported to infect bats; Plasmodium, Hepatocystis, Nycteria and Polychromophilus (Garnham and Heisch, 1953, Garnham, 1966, Rosin, 1978). The Polychromophilus parasites, whose vectors are species of nycteribiid flies, appear to be widespread among insectivorous bats both in temperate and in tropical regions (Dionisi, 1898a, Dionisi, 1898b, Garnham, 1973a, Garnham, 1973b). This genus was classified by some authors as comprising two subgenera, Polychromophilus and Bioccala (Landau et al., 1980). This latter subgenus was subsequently raised to generic status (Landau et al., 1984). Five Polychromophilus species were described principally based on the morphology of their exoerythrocytic schizogony. Polychromophilus (Polychromophilus) melanipherus and Polychromophilus (Bioccala) murinus, the two type species, with the assumption that P. (P.) melanipherus is restricted to the Miniopteridae and P. (B.) murinus to Vespertilionidae (Garnham, 1966). Additionally, P. (P.) corradetti and P. (P.) adami have been described from the Greater Long-fingered bat, Miniopterus inflatus, in Gabon and from the Least Long-fingered bat, Miniopterus minor minor in Republic of Congo respectively (Landau et al., 1980). These latter two species are close to P. melanipherus. Finally, P. (B.) deanei was reported from vespertilionid bats in America (Garnham et al., 1971).

Two recent studies, with a combined approach based on morphological, molecular, phylogenic and/or vector data, reported Polychromophilus parasites in European, Malagasy and South-East Asian Miniopterus spp. (Duval et al., 2007, Megali et al., 2011). These two studies, resulting from molecular data approaches, support the previous hypotheses in the older literature based on morphological descriptions and parasites life cycle that (a) Polychromophilus parasites are divided into at least two distinct groups/species; P. melanipherus infecting bats of the family Miniopteridae and P. murinus infecting Vespertilionidae, a closely related family (Miller-Butterworth et al., 2007), (b) bat parasites belonging to the genus Polychromophilus are more closely related to avian malaria parasites than with other mammalian malaria parasites. In the context of epidemiological surveillance, the Centre International de Recherches Médicales de Franceville (CIRMF) in Gabon, targeted bat populations for virological and parasitological investigations amongst those living in Belinga Mountain caves located about 100 km north of Makokou city in the north-east of Gabon (Ogooué-Invindo province), where Ebola outbreaks occurred in 1994–1997 and 2001–2002 (Georges et al., 1999, Milleliri et al., 2004, Leroy et al., 2005). The aim of the study was to screen and survey, for a period of a few months, haemosporidian infections in an ecological community of four sympatric central African cave bat species; the Giant Roundleaf bat (Hipposideros gigas), Sundevall’s Round-leaf bat (Hipposideros caffer), African Sheath-tailed bat (Coleura afra) and the Greater Long-fingered bat (M. inflatus), living in the Faucon cave on Belinga Mountain.

Section snippets

Sampling

A total of 164 bats, 20 H. gigas, 13 H. caffer, 47 C. afra and 84 M. inflatus, was captured and sampled from the Faucon cave located on the Belinga Mountain in the north east of Gabon (N1.07287; E13.20739) near the border of the Republic of Congo in an equatorial forest with a wide variety of caves (Fig. 1) (Brosset, 1966). Faucon cave is an iron rock cave, housing a saprophytic flora. Stagnant water is maintained inside the cave during the dry season and flows out of the cave during the rainy

Results

From a total of 164 bats sampled, thin blood smears and blood spots, examined by microscopy and screened using molecular cyt b detection method for Haemosporidia infection respectively, only M. inflatus samples were positive for haemosporidian parasites (Table 1) over the seven months of the survey.

Haemosporidian microscopic prevalence in M. inflatus ranged from 66.7 % in December 2010 (highest value) to 17.6 % in April 2011 (lowest value) while molecular prevalence was at least 60% for all of

Discussion

This study represents the first molecular report, associated with morphological data from circulating blood forms, of Polychromophilus parasites infecting M. inflatus bats in Central Africa (Gabon). The results confirm that haemosporidian Polychromophilus parasites are widespread among insectivorous bats (Miniopteridae and Vespertilionidae) in temperate (Europe) (Megali et al., 2011), tropical (Madagascar, Central Africa and South-East Asia) (Adam and Landau, 1973, Adam and Le Pont, 1974,

Acknowledgements

Fieldwork was funded by the Centre International de Recherches Médicales de Franceville (CIRMF), Ministère des Affaires Etrangères et Européennes (MAEE) of France and the Centre de Coopération Internationale en RechercheAgronomique pour le Développement (CIRAD). We thank the Centre National de Recherches Scientifiques et Techniques (CENAREST) of Gabon for the accommodation provided during field missions. We thank all the persons involved in sample collection, and especially André Délicat,

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