Extensive diversity of malaria parasites circulating in Central African bats and monkeys

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Extensive diversity of malaria parasites circulating in

Central African bats and monkeys

Larson Boundenga, Barthélémy Ngoubangoye, Illich Manfred Mombo, Thierry

Audrey Tsoubmou, Francois Renaud, Virginie Rougeron, Franck Prugnolle

To cite this version:

Larson Boundenga, Barthélémy Ngoubangoye, Illich Manfred Mombo, Thierry Audrey Tsoubmou,

Francois Renaud, et al..

Extensive diversity of malaria parasites circulating in Central African

bats and monkeys.

Ecology and Evolution, Wiley Open Access, 2018, 8 (21), pp.10578-10586.

10578  

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1 | INTRODUCTION

The order of Haemosporidia gathers many protozoan parasites for which the most studied are the agents of human malaria. The hae‐ mosporidian parasites are known to infect many host species and groups, including birds, reptiles, and mammals (Boundenga, et al., 2017; Garnham, 1966). Currently, this order contains over 550 spe‐ cies that are classified in 17 extant genera (Martinsen & Perkins, 2013). However, the vast majority of described species have been assigned to the four genera Plasmodium, Hepatocystis, Haemoproteus and Leucocytozoon (Borner, et al., 2016; Boundenga, et al., 2016; Levine, 1980).

Up to now, the studies on haemosporidian parasites have pri‐ marily focused on the genus Plasmodium from a wide range of hosts. This interest in the genus Plasmodium was motivated by the desire to better understand the evolutionary history of human malaria agents

and more particularly Plasmodium falciparum (the major causative agent of human malaria) (Perkins, 2014). Other genera like the genus

Hepatocystis have historically received far less attention and only a

couple of strains were genetically characterized up to recently where several studies, using molecular tools, have explored the diversity of

Hepatocystis parasites infecting several groups of mammals (Ayouba,

et al., 2012; Schaer, et al., 2013; Thurber, et al., 2013). One study performed in West Africa revealed that a large and yet unknown di‐ versity of Hepatocystis parasites circulated in several species of bats (Schaer, et al., 2017). Others demonstrated a large diversity of this parasite genus in African primates (Ayouba, et al., 2012; Thurber, et al., 2013). Some bat, rodent, and monkey species in Asia were also shown to be infected with parasites of this genus (Hatta, Divis, & Singh, 2012; Olival, Stiner, & Perkins, 2007; Putaporntip, et al., 2010). Thus, all previous studies combined revealed that these par‐ asites circulate in several mammal groups and that they are widely

Received: 13 March 2018 

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O R I G I N A L R E S E A R C H

Extensive diversity of malaria parasites circulating in Central

African bats and monkeys

Larson Boundenga

_{| Barthélémy Ngoubangoye}

_{| Illich Manfred Mombo}

_| Thierry

Audrey Tsoubmou

_{| François Renaud}

_{| Virginie Rougeron}

_{| Franck Prugnolle}

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2018 The Authors Ecology and Evolution Published by John Wiley & Sons Ltd

1_{Centre International de Recherches}

Médicales de Franceville (CIRMF), Franceville, Gabon

2_{Laboratoire MIVEGEC, UMR 224‐5290}

CNRS‐IRD‐UM1‐UM2, Centre Hospitalier Régional Universitaire, Montpellier, France

Correspondence

Larson Boundenga, Groupe Evolution et Transmission Inter‐Espèces des Pathogènes (GETIP), CIRMF, Franceville, Gabon. Email: boundenga@gmail.com

Funding information

Centre International de Recherches Médicales de Franceville; Centre National de la Recherche Scientifique

Abstract

The order Haemosporidia gathers many protozoan parasites which are known to in‐ fect many host species and groups. Until recently, the studies on haemosporidian parasites primarily focused on the genus Plasmodium among a wide range of hosts. Genera, like the genus Hepatocystis, have received far less attention. In the present study, we present results of a survey of the diversity of Hepatocystis infecting bats and monkeys living in a same area in Gabon (Central Africa). Phylogenetic analyses revealed a large diversity of Hepatocystis lineages circulating among bats and mon‐ keys, among which certain were previously observed in other African areas. Both groups of hosts harbor parasites belonging to distinct genetic clades and no transfers of parasites were observed between bats and monkeys. Finally, within each host group, no host specificity or geographical clustering was observed for the bat or the primate Hepatocystis lineages.

K E Y W O R D S

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BOUNDENGA EtAl.

distributed (Ayouba, et al., 2012; Olival, et al., 2007; Putaporntip, et al., 2010; Schaer, et al., 2013, 2017 ). Nevertheless, all these studies dealt with the diversity in each group of hosts separately, without considering all hosts at the same time and the possibilities of trans‐ fers of these lineages among hosts.

In the present study, we analyzed the diversity of Hepatocystis parasites infecting different host groups (bats and monkeys) liv‐ ing in the same area. The study was performed at the primatology center of CIRMF (Centre International de Recherches Médicales de Franceville), which is located in the South‐East of Gabon and con‐ stitutes an interesting environment because different hosts (nonhu‐ man primates and bats) dwell in a small area. Here, we wish to verify host specificity of Hepatocystis in monkeys and bats and evaluate if potential switches of Hepatocystis between these different mammal groups may occur.

2 | MATERIALS AND METHODS

2.1 | Sampling of animals’ blood

Two hundred and sixty‐five blood samples were collected from ani‐ mals living in and around the primatology center of CIRMF (1°37’59" N/13°34’59 "E), located in the city of Franceville in the province of Haut‐Ogooué, South‐East Gabon (Figure 1). The samples were taken on animals belonging to two groups: monkeys and bats. Regarding monkeys, animals live in semi‐free ranging enclosures for man‐ drills (250 individuals) and Cercopithecus solatus (13 members in this group). The other monkeys of this study (Cercocebus torquatus,

Cercopithecus cephus, and Cercopithecus nictitans) live in different

aviaries. Except for a few monkeys that were born in this center, all other nonhuman primates were born in the wild. For monkeys, 76

blood samples were collected during routine annual sanitary con‐ trols in 2014. The criteria of animal well‐being were guaranteed by the CIRMF veterinarians who proceeded to the blood sampling on the nonhuman primates. Two to seven millilitre of blood were col‐ lected within EDTA tube for each animal according to the weight. Tubes were then preserved in an icebox and transported to labora‐ tories, where samples were conserved at −20°C.

The capture of bats occurred over a period of four months, be‐ tween March and June 2014 (rainy season), using harp traps or mist nets (12 × 2.4 m) placed close to the area of storage of the food of primates just before twilight. Bats were captured at the proximity of the monkey enclosures because frugivorous bats tend to feed on food and bananas leftovers in the feeding areas of monkeys. Animals were identified by trained field biologists as previously described in (Maganga et al, 2014). Blood samples were collected from the bra‐ chial vein by puncture with a 0.4 × 13 mm gauge needle. The animals were subsequently released and marked with a microchip implant. All blood samples obtained were stored at −20°C. This study was done in agreement with the recommendations of the Gabonese National Ethics Committee (Authorization N°PROT/0020/2013I/ SG/CNE). Animal handling by the CDP veterinarians complied with the criteria for animal care defined in the guidelines of the American Society of Mammalogists (www.mammalsociety.org/committees/ animal‐care‐and‐use).

2.2 | Extraction of DNA, PCR and

Phylogenetic analyses

All DNA samples were extracted from whole blood using the Qiagen DNeasy Blood & Tissue kit (Courteboeuf, France) according to manufacturer conditions and parasite infections were determined after amplification of a portion of parasite mitochondrial genome F I G U R E 1   Study area. This map

shows the location of Franceville in Gabon, the city in which the CIRMF is located. The box on the right is an aerial picture of the CIRMF in which are reported the areas of capture of bats (in yellow) and the enclosures in which the monkeys involved in the study live

Haut-Ogooue River

Franceville

Areas of capture of bats Monkeys enclosures

GABON

(cytochrome b: cyt‐b) as described in (Boundenga, et al., 2017; Prugnolle, et al., 2010). Around 10 µl of all PCR‐amplified products were send and used as templates for sequencing, after were con‐ firmed as positive on 1.5% agarose gels in TAE buffer. This sequenc‐ ing method did not allow us to highlight the co‐infections.

Phylogenetic analyses were performed using the cyt‐b sequences derived from chromatograms with no ambiguous base calls. To exam‐ ine the relationship of the cyt‐b sequences obtained with the different haemosporidian species known so far, a phylogenetic tree was con‐ structed using a set of reference sequences belonging to different species from Genbank (Supporting Information Table S1). Note that five of these sequences (the ones from Asian rodent Hepatocystis), although published by Genbank, have not been associated to any publication so far. Phylogenetic analyses were then performed after multiple alignments of the obtained partial cyt‐b sequences (696 nu‐ cleotides) and of the Genbank references sequences. We used both maximum likelihood (ML) (Figure 2 and Supporting Information Figure S1) and Bayesian (Supporting Information Figure S1) methods for tree construction. For the former, the best‐fitting model of sequence

evolution based on the Akaike Information Criterion was GTR (General Time Reversible) + Gamma, as determined using ModelTest (Posada & Crandall, 1998). The highest‐likelihood DNA tree and the correspond‐ ing bootstrap support values were then obtained by using PhyML software (Dereeper, et al., 2008; Guindon, Lethiec, Duroux, & Gascuel, 2005) (freely available at the ATGC bioinformatics platform https:// www.atgc‐montpellier.fr/). maximum likelihood tree was rooted using

Leucocytozoon species. For phylogenetic Bayesian inferences, the soft‐

ware MrBayes 3.2.6 under Geneious 11.0.5 was used (Huelsenbeck & Ronquist., 2001; Suchard et al., 2018). Bayesian posterior probabilities were computed under the same ML model by running two runs, four chains for 1,100,000 MCMC generations using the program default priors on model parameters. Mixing and convergence to stationary dis‐ tributions were evaluated by inspecting graphically the trace of the log likelihood or sampled parameter values against the generation num‐ bers. Trees were sampled from the posterior probability distribution every 200 generations and 100,000 trees were discarded as “burn‐in” to ensure that the chains have reached stationarity. A Majority‐rule consensus tree was retrieved from this study.

F I G U R E 2   Phylogenetic relationships between the cyt‐b sequences of haemosporidian parasites obtained in our study (in bold) and the reference cyt‐b sequences obtained from existing databases and classified (with color codes and icons) according to their host species. The tree was constructed using a maximum likelihood method based on 696 bp‐long cyt‐b sequences. One thousand bootstrap replications were performed to assess confidence in topology. Branch colors indicate different groups of host species among vertebrates (red for bats, green for monkeys, blue for rodents and black for other parasites of mammals). For parasites of the genus Plasmodium, the subgenus is also provided (in brackets)

GU929975-Hepatocystis_sp.Monkeys-Thailand GU929951-Hepatocystis_sp.Monkeys-Thailand n14GB-B281_Epomops_franqueti JQ070951-Hepatocystis_sp.Monkeys-Cameroun JX090244-Hepatocystis_sp._ex_Callosciurus_notatus_HNH-2013 JQ070929-Hepatocystis_sp.Monkeys-Cameroun EU400409-Hepatocystis_sp.Monkeys-Thailand KF159713-Hepatocystis_sp_Mic_pus_G1_4 JQ070914-Hepatocystis_sp.Monkeys-Cameroun KC262848-Hepatocystis_sp.Monkeys-Uganda KF159676-Hepatocystis_sp_Mic_pus_G5_2 n14GB-PNH15_Cercocebus_torquatus JX090243-Hepatocystis_sp._ex_Callosciurus_notatus n14GB-B253_Epomops_franqueti n14GB-B321_Epomops_franqueti n14GB-PHN3_Cercopithecus_cephus KF159689-Hepatocystis_sp_Hyp_mon_L2_1 JQ070933-Hepatocystis_sp.Monkeys-Cameroun KF159686-Hepatocystis_sp_Mic_pus_G1_8 KF159695-Hepatocystis_sp_Epom_gamb_G4_1 KF159670-Hepatocystis_sp_Mic_pus_G2_1 GU930067-Hepatocystis_sp.Monkeys-Thailand KF159698-Hepatocystis_sp_Nan_vel_L1_1 n14GB-B282_Epomops_franqueti KF159702-Hepatocystis_sp_Mic_pus_G1_9 AF069626-Hepatocystis_sp.Monkeys-Ethiopia n14GB-B337_Eidolon_helvum n14GB-PNH5_Cercopithecus_cephus n14GB-PHN30_Manddrillus_sphinx n14GB-B249_Epomops_franqueti DQ396529-Hepatocystis_sp._LB3_cyt n14GB-B274_Epomops_franqueti JF923758-Hepatocystis_sp.Monkyes-Gabon KF159679-Hepatocystis_sp_Mic_pus_G4_1 n14GB-B335_Epomops_franqueti KC262846-Hepatocystis_sp.Monkeys-Uganda JX090251-Hepatocystis_sp.Rodent-Asia_Southeast KF159701-Hepatocystis_sp_E_buett_G4_1 n14GB-B244_Epomops_franqueti n14GB-B280_Epomops_franqueti n14GB-B289_Epomops_franqueti n14GB-B326_Epomops_franqueti n14GB-B259_Epomops_franqueti n14GB-B245_Epomops_franqueti DQ396531-Hepatocystis_sp.Bat-SouthEast n14GB-B251_Epomops_franqueti n14GB-PHN27_Cercocebus_torquatus KC262809-Hepatocystis_sp._TLG-2012_isolate_BAB1810 JQ070920-Hepatocystis_sp.Monkeys-Cameroun JQ070916-Hepatocystis_sp.Monkeys-Cameroun n14GB-B258_Epomops_franqueti KF159684-Hepatocystis_sp_Nan_vel_C8_1 KC262865-Hepatocystis_sp.Monkeys-Uganda n14GB-319_Epomops_franqueti KF159715-Hepatocystis_sp_Nan_vel_C8_2 n14GB-B310_Epomops_franqueti JX090237-Hepatocystis_sp_Rodent-Asia_Southeast KF159705-Hepatocystis_sp_Myo_lep_L3_1 EU400408-Hepatocystis_sp.Monkeys-Thailand KF159717-Hepatocystis_sp_Mic_pus_G5_1 KC262866-Hepatocystis_sp-Monkeys-Uganda KF159709-Hepatocystis_sp_Mic_pus_G2_2 KF159693-Hepatocystis_sp_Mic_pus_G4_2 DQ396528-Hepatocystis_sp.Bat-Asia-SouthEast n14GB-B257_Epomops_franqueti JF923757-Hepatocystis_sp.Monkeys-Gabon n14GB-B292_Epomops_franqueti JF923760-Hepatocystis_sp.Monkeys-Gabon KF159712-Hepatocystis_sp_Hyp_mon_L4_1 n14GB-PHN1_Mandrillus_sphinx FJ168565-Hepatocystis_sp.Bat-Asie_Southeast KF188066-Hepatocystis_sp_Myo_lep_C7_1 n14GB-B303_Epomops_franqueti n14GB-B338_Hypsignathus_monstrosus n14GB-PHN9_Cercopithecus_cephus JF923759-Hepatocystis_sp.Monkeys-Gabon n14GB-B314_Epomops_franqueti JX090235-Hepatocystis_sp.Rodent-Asia_Southeast 0.84 0.85 0.76 0.980.97 0.78 0.84 0.98 0.61 0.96 0.9 1 0.91 0.78 0.93 0.9 0.740.82 0.76 0.87 0.91 0.75 0.86 1 1 0.99 0.71 0.9 0.92 0.75 0.54 0.94 1 0.96 0.94 0.82 0.5 0.7 0.83 0.99 0.96 0.93 0.790.98 0.75 0.95 0.92 0.95 Plasmodium (Vinckeia) Plasmodium (Laverania) Plasmodium (Plasmodium) Nycteria Polychromophilus Hepatocystis Parasites of primates African monkeys Asian monkeys Great apes Asian bats African bats Parasites of bats Parasites of rodents Asian rodents 0.94 0.79 0.07 n14GB-PHN19_Mandrillus sphinx KF159690-Nycteria sp. AB354571-Plasmodium ovale DQ414649-Plasmodium chabaudi KT824296-Plasmodium gaboni KU182368-Polychromophilus melanipherus NC_012450-Leucocytozoon majoris KP053763-Nycteria sp. EU400399-Plasmodium inui DQ414645-Plasmodium berghei JF923761-Plasmodium praefalciparum NC_009336-Leucocytizoon sabrazesi AF069614-Plasmodium simiovale HM235074-Plasmodium blacklocki KX090647-Nycteria gabonensis GU045322-Plasmodium adleri KF159671-Plasmodium voltaicum n14GB-PHN33_Mandrillus sphinx DQ414658-Plasmodium yoelii n14GB_PHN48_Mandrillus sphinx n14GB-PHN62_Mandrillus sphinx GU815511-Plasmodium billcollinsi AF069625-Plasmodium ovale KF159700-Polychromophilus sp. JF923753-Plasmodium sp._DAJ-2004l JF923751-Plasmodium gonderi GU045315-Plasmodium reichenowi JF923755-Plasmodium sp._DAJ-2004 DQ414654-Plasmodium vinckei n14GB-PNH54_Mandrillus sphinx JX444723-Plasmodium vivax n14GB-PHN42_Mandrillus sphinx AY099063-Leucocytozoon dubreuli 0.76 0.96 1 1 1 1 0.76 0.87 0.86 0.98 0.98 1 0.63 0.99 0.87 1 0.98 1 0.88 0.95 0.6 1 1 0.94 0.7 Leucocytozoon

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BOUNDENGA EtAl.

Finally, pairwise genetic distances (p‐distances) between different groups of sequences were estimated using MEGA 6.

3 | RESULTS

A total of 265 samples were screened: 189 from four species of bats and 76 from five different species of NHPs (For more details see Table 1). Individuals from the two groups of vertebrates were found infected by haemosporidian parasites and belonging to different

genera (Hepatocystis and Plasmodium). The infection rates are re‐ ported in Table 1.

Regarding bats, four species were collected inside the CIRMF:

Epomops franqueti, Hypsignathus monstrosus, Eidolon helvum, and Rousettus aegyptiacus. We found 12.2% of individuals sampled in‐

fected by haemosporidian parasites, belonging to three of the four bats species sampled. The infections rates varied from one spe‐ cies to another: E. franqueti 13.12% (21/160), H. monstrosus 4.8% (1/21) and E. helvum 16.67% (1/8) (Table 1). Phylogenetic analyses revealed that haemosporidian parasites identified among the bats TA B L E 1   Description of the species of bats and monkeys analyzed in this study. The number of samples collected per host species, the prevalence of infection with a hemosporidian parasite in each host species and the parasite genus identified are indicated

Animal group Family Host species Sample number Prevalence % Parasites genus

Bats Pteropodidae Epomops franqueti 160 13.12% (21/160) Hepatocystis

Pteropodidae Hypsignathus monstrosus 21 4.8% (1/21) Hepatocystis

Pteropodidae Eidolon helvum 6 16.67% (1/8) Hepatocystis

Pteropodidae Rousettus aegyptiacus 2 0 —

Monkeys Cercopithecidae Mandrillus sphinx 50 16% (8/50) Plasmodium and

Hepatocystis

Cercopithecidae Cercocebus torquatus 5 40% (2/5) Hepatocystis

Cercopithecidae Cercopithecus solatus 13 0 —

Cercopithecidae Cercopithecus cephus 4 75% (3/4) Hepatocystis

Cercopithecidae Cercopithecus nictitans 4 0 —

F I G U R E 3   Phylogenetic relationships between the cyt‐b sequences of Hepatocystis of African bats with some outgroups (e.g.

Hepatocystis of Asian bats, P. ovale…). The unrooted tree was inferred from 696 bp nucleotides. The tree was performed using a maximum likelihood method. One thousand bootstrap replications were performed to assess confidence in topology (only values above 80% are shown). Four genetic clusters supported by high bootstrap values are highlighted with colors. Cyt‐b sequences obtained in our study are in bold n14GB -B25 9_Gabo n KF 1596_86-H epatocyst is sp._Gu inea KF159709-Hepatocyst is sp._Guinea n14GB-B303-Gabon n14GB-B245_Gabo n n1 4GB-B257_Gabon n14GB-B244_Gabon KY753522 -He pa tocystis sp._South Suda n KF159713 -Hepatocysti s sp._Guinea n14GB-B 314_Gab on KF159670-He_patocystis sp._Guinea KY753510-Hepatocystis sp._South Sudan KY753514 -He pa_tocyst is sp._ Sou th _Suda n KY 7535 05 He pato cys_{tis sp} ._Sout h Suda n n14GB-B274_Gabon KF159717 -Hepatocyst is_sp ._Guine a n14GB-B280_ Gabon KY75350 7-He patocystis sp._South Sudan n14GB-B258_Gabon KF159 705-Hepa tocy stis sp._Liber ia KF159 702-Hep_atocys tis sp._Guine_a KY753516-_Hepa tocystis sp._So_{uth Suda}

n KY 753526-Hepatocystis sp._South Sudan n14GB-B321_Gab on KF159698 -Hepat ocysti s sp._Liber ia KY 753506-Hep atocystis sp._Sou th Sudan KY7 53503 -Hepa tocystis _sp ._Sout h Su dan n14 GB-B3₃₇ _G_abon n14 GB-B326_Gab on n14G B-B251_ Gabo n KF159689 -H_{epatocystis_sp._Liberia} n14GB-B3 35_Ga bon KY753504-Hepatocyst_{is sp._Sout} h Sudan KF159684-Hepatocysti s sp._C ote d’ivoir e KF188066 -Hepatocysti s sp._Cote d’ivoire KY753_511-Hepat ocystis sp._South Sudan n14GB-B310_Gabon KY753 521 -He patocysti s sp._South Sudan n14GB-B282_G abo n n14G B-B253_Gabon

KY753513-Hepato_{cystis sp._South Su} dan n14G B-B249_Gabon n14GB-292_Gabon KF15 9712-H_ep atocystis_sp. _Li_beri a n14GB-B281_Gabon n14GB-B 338_ Gab on KF159676-Hepatocystis sp._Guinea n14GB-B289_Gabon KF159693 -Hepato cystis sp._ Guine a KF1596 89-Hepatocys tis_sp._Li beri a n14GB-B319_G abon 0.79 0.87 0.78 0.97 0.97 0,85 0.98 0.75 0.71 0.87 0.85 0.94 0.93 0.97 0.92 0.7₇ 0.84 0.67 0.79 0.84 0.99 0.99 0.99 0.84 0.90 0.84 Cluster 1 Cluster 2 Cluster 3 Cluster 4 JX090237-Hepatocys

tis sp._Asian Rodent

KF159710 -P. cyclopsi

AF069625-P. ovale

FJ168565-Hepatocystis sp._Asian Bat DQ396531-Hepatocystis sp._Asian Bat

JX090251-Hepatocystis

species clustered with several lineages of the Hepatocystis genus identified previously in other African areas (Figure 2 and Supporting Information Figure S1). Thus, all lineages obtained in this study in‐ fecting African bats belonged to a monophyletic group including all lineages recently described in several species of West Central and East African bats (Figure 2). In our phylogeny, we can discriminate at least four distinct clades infecting the African bats (clade 1–4 in Figure 3). These clades are well supported (high bootstrap values) and the average divergences (p‐distance) measured between each pair of clade vary from 0.02 to 0.03, a range similar to the diver‐ gence measured between P. falciparum and P. reichenowi (0.03). As previously noted by Schaer et al. (2013), this observation suggests that the group of African Hepatocystis from bats actually comprises different Hepatocystis species. Phylogenetic analyses revealed that

all sequences of parasites identified during our study and all differ‐ ent lineages from diverse geographical zones (countries) are mixed throughout the tree for African chiropterans. Thus, analyses indicate no strict geographic distribution within Hepatocystis isolates identi‐ fied (Figure 4).

Beside the chiropteran hosts, five monkey species were included in the study: Mandrillus sphinx, Cercocebus torquatus, Cercophithecus

cephus, Cercopithecus nictitans, and Cercophithecus solatus. Three of

them (M. sphinx 16% [8/50], C. torquatus 40% [2/5] and C. cephus 75% [3/4]) were found infected with haemosporidian parasites (rep‐ resenting nearly 22.03% of individuals infected) belonging either to the Hepatocystis or the Plasmodium genus (Table 1). Regarding the Hepatocystis, they form a monophyletic group. This group com‐ prises parasites from both African and Asian monkeys and is closely

F I G U R E 4   Phylogeny of the cyt‐b sequences of haemosporidian parasites obtained in our study and from other studies and classified (with color codes) according to their geographical origin (map performed using Adobe illustrator CS6 software). Hepatocystis sequences are identified with a color code by country. At the bottom of the tree is represented the country in which the different bats were captured

n14GB-B249_Epomops franqueti-Gabon

n14GB-B244_Epomops franqueti-Gabon

KY753522-Micropteropus pusillus-South Sudan

KF159705-Myonycteris leptodon-Liberia

KF159684-Nanonycteris veldkampii-Ivory Coast KY753510-Epomophorus sp.-South Sudan

KF159712-Hypsignathus monstrosus-Liberia KC262809-Monkeys-Uganda n14GB-PNH15_Cercocebus torquatus-Gabon n14GB-B258_Epomops franqueti-Gabon n14GB-B281_Epomops franqueti-gabon n14GB-B335_Epomops franqueti-Gabon KC262866-Monkeys-Uganda DQ396531-Pteropus vampyrus-Malasia (Borneo)

JF923760-Cercopithecus cephus-Gabon

DQ396528-Cynopterus brachyotis-Malasia (Borneo) n14GB-B253_Epomops franqueti-Gabon

JQ070920-Cercopithecus nictitans-Cameroon n14GB-292_Epomops franqueti-Gabon

KY753516-Epomophorus sp.-South Sudan

JQ070929-Cercopithecus nictitans-Cameroon KY753511-Epomophorus sp.-South Sudan KF159670-Micropteropus pusillus-Guinea GU929975-Macaca sp.-Thailand KF159695-Epomophorus gambianus-Guinea EU400409-Macaca sp.-Thailand AB354571-Plasmodium ovale KF159702-Micropteropus pusillus-Guinea KF159686-Micropteropus pusillus-Guinea KF159693-Micropteropus pusillus-Guinea n14GB-282_Epomops franqueti-Gabon n14GB-289_Epomops franqueti-Gabon

JX090237-Callosciurus notatus-Malasia (Borneo) JQ070916-Cercopithecus nictitans-Cameroon

JX090251-Callosciurus notatus-Malasia (Borneo)

KY753513-Epomophorus sp.-South Sudan

KY753506-Epomophorus sp.-South Sudan KY753505-Epomophorus sp.-South Sudan

JX090244-Callosciurus notatus-Malasia (Borneo) JQ070933-Cercopithecus nictitans-Cameroon

n14GB-B245_Epomops franqueti-Gabon

GU929951-Macaca sp.-Thailand

KF159676-Micropteropus pusillus-Guinea

KF159715-Nanonycteris veldkampii-Ivory Coast

GU930067-Macaca sp.-Thailand

JQ070951-Cercopithecus nictitans-Cameroon

JX090235-Callosciurus notatus-Malasia (Borneo) n14GB-B321_Epomops franqueti-Gabon JQ070914-Cercopithecus nictitans-Cameroon n14GB-B310_Epomops franqueti-Gabon n14GB-PNH5_Cercopithecus cephus-Gabon n14GB-319_Epomops franqueti-Gabon KF159717-Micropteropus pusillus-Guinea

KY753503-Epomops franqueti-South Sudan

KC262846-Monkeys-Uganda

KF159701-Epomops buettikoferi-Guinea

n14GB-PNH3_Cercopithecus cephus-Gabon n14GB-B274_Epomops franqueti-Gabon

n14GB-PNH9_Cercopithecus cephus_gabon

JF923757-Miopithecus talapoin-GabonJF923759-Mandrillus sphinx-Gabon

DQ396529-Cynopterus brachyotis-Malasia (Borneo)

KY753514-Epomophorus sp.-South Sudan KY753504-Epomops franqueti-South Sudan

KF188066-Myonycteris leptodon-Ivory Coast

n14GB-B338_Hypsignatus monstrosus-Gabon

FJ168565-Pteropus hypomelanus-Asie Southeast n14GB-B257_Epomops franqueti-Gabon

KY753526-Micropteropus pusillus-South Sudan

KF159698-Nanonycteris veldkampii-Liberia

KY753507-Epomophorus sp.-South Sudan

JF923758-Cercopithecus cephus-Gabon KF159689-Hypsignathus monstrosus-Liberia EU400408-Macaca sp.-Thailand KF159679-Micropteropus pusillus-Guinea n14GB-PNH30_Mandrillus sphinx KF159689-Hypsignathus monstrosus-Liberia n14GB-B337_Eidolon helvum-Gabon KC262865-Monkeys-Uganda KF159709-Micropteropus pusillus-Guinea n14GB-B259_Epomops franqueti-Gabon KF159713-Micropteropus pusillus-Guinea

KY753521-Hypsignathus monstrosus-South Sudan

n14GB-B314_Epomops franqueti-Gabon KC262848-Monkeys-Uganda n14GB-PNH27_Cercocebus torquatus-Gabon n14GB-B280_Epomops franqueti-Gabon n14GB-PNH1_Mandrillus sphinx-Gabon AF069625-Plasmodium ovale AF069626-Papio nubensis-Ethiopia

JX090243-Callosciurus notatus-Malasia (Borneo) n14GB-B251_Epomops franqueti-Gabon n14GB-B303_Epomops franqueti-Gabon n14GB-B326_Epomops franqueti-Gabon 0.67 0.95 0.69 0.95 0.77 0.93 0.86 0.75 0.99 0.72 1 0.95 0.9 0.74 1 0.76 0.86 0.86 0.68 0.87 0.74 0.75 0.94 0.91 0.75 0.91 0.98 0.55 0.78 0.77 0.99 0.77 0.95 0.99 0.98 0.73 0.86 0.85 0.79 1 1 0.94 0.78 0.84 1 0.59 0.73 0.92 0.99 0.69 0.86 0.93 0.99 0.73 1 0.84 0.82 0.83 Hepatocystis Gabon Guinea South Sudan Liberia Ivory Coast Malasia (Borneo) Cameroon Uganda Ethiopia Thailand Lineages Hepatocystis Hepatocystis Host species-Country Hepatocystis Hepatocystis

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BOUNDENGA EtAl.

related to the Hepatocystis of Asian rodents and bats (Figure 2 and Supporting Information Figure S1). Among the African primates, phylogenetic analyses suggest the existence of at least four well‐ supported clades (Figure 5) which average divergence (p‐distance) range from 0.02 to 0.04, again estimates close to the divergence estimated between well‐defined species (e.g., 0.03 between P. fal‐

ciparum and P. reichenowi). Within the tree, we observe no grouping

of sequences by geographical areas, rather a mixed of sequences from different countries and no grouping by host species or genus (Figure 4). No host switch was observed between bats and primates (i.e., no primate Hepatocystis were found among bats or vice versa). For the Plasmodium species infected monkeys, we identified two parasites: Plasmodium sp.‐DAJ‐2004 and Plasmodium gonderi. These species were only found among mandrills.

4 | DISCUSSION

The discovery of new species in groups of parasites yet historically well studied because of their proximity to human malaria agents (e.g., the primate malaria) has revived the interest to explore the diversity

of extant malaria parasites in different host groups using molecular methods (Boundenga et al., 2015; Megali, Yannic, & Christe, 2011; Schaer, et al., 2015). In this study, we investigated the diversity of extant malaria parasites circulating among bats and monkeys living in the same area, using molecular tools to analyze the parasite con‐ tent of each host species collected and determine whether transfers of parasites may occur among host groups.

Molecular analyses revealed that the individuals of different groups of mammals (primates and bats) are infected by haemospo‐ ridian parasites belonging to at least two distinct genera: Plasmodium and Hepatocystis (the focus of this study) (Figure 2 and Table 1). No parasites of Polychromophilus and Nycteria genera which were pre‐ viously reported among bats (Duval, et al., 2012; Karadjian, et al., 2016) were found during this study.

Regarding the genus Hepatocystis, they were found in bats and primates in our study. In the Gabonese bats, we observed a large diversity of lineages belonging to distinct clades. Most of these lin‐ eages were genetically close or similar to lineages recently described in West and East African bats (Schaer, et al., 2013, 2017 ). Such di‐ versity of sequences may reflect the presence of different species of

Hepatocystis or a single species with a large geographical distribution

F I G U R E 5   Phylogenetic relationships between the Cyt‐b sequences of Hepatocystis of African monkeys with some outgroups (e.g.

Hepatocystis of Asian bats and rodents…). The unrooted tree was inferred from 696 bp nucleotides. The tree was performed using a

maximum likelihood method. One thousand bootstrap replications were performed to assess the confidence in topology (only values above 80% are shown). Four genetic clusters supported by high bootstrap values are highlighted with colors. Cyt‐b sequences obtained in our study are in bold

JX090251 -Hepatocystis sp._Asian Rodent n14GB-PNH2 7_Gab on JF923757-Hepatocysti s sp._Ga bon n14GB-PN H5_Gabo n AF069626-Hepatocysti s sp._Ethiopi a KC2628 65-Hepatoc ysti s sp._Uganda GU930067-Hepatocystis sp.__Thailand KC262866 -H_epatocystis sp._Ugand a JX090237-Hepatocystis sp._Asian Rodent JQ070929-Hepatocystis sp_{._Cameroun} DQ396531-He patocystis sp._Asian Ba t

FJ168565-Hepatocystis sp._Asian Ba

t EU400409-Hep atocys tis sp._Thaila nd n14GB-PNH30_Gabo n KC262846-Hepatocys tis sp._Uganda GU9299 75-H_epato cystis sp._ Thailand JF923759-Hepatocystis sp._Gabo n n14GB-PNH9_Gabon JQ070916 -Hepatocystis sp._ C am eroun JF923758-Hepatocystis sp._Gabon JQ070933 -Hepatocystis sp_Camerou n n14GB-PNH1_Gabon KC262848-Hepat ocystis _{sp._} Uganda JQ070 951-Hepatocys tis sp._Cameroun EU400408-Hepat ocy stis sp._Thailand JQ070914-Hepa tocystis sp._Camerou n JF923760-Hepa tocystis sp._Gabo n GU929951-H epatocysti s sp._Thaila nd JQ 070920-Hepatocystis sp._Came roun n14GB -_PNH3_Gabon n14G B-PNH15_Gabon 0.77 0.96 0.98 1 0.9 0.94 0.74 0.74 0.78 0.95 0.75 0.7 8 0.93 0.78 0.97 0.96 0.95 0.92 0. 91 Cluster 3 Cluster 1 Cluster 4 Cluster 2

and a complex evolutionary history that led to the maintenance of divergent lineages within this species. The divergences observed be‐ tween clades (which are close or higher than the divergences mea‐ sured between well‐described species of Plasmodium) are rather in favor of the presence of distinct species.

As previously noted by Schaer et al. (2017), no clear geographic patterns nor grouping according to the bat species or the bat genus could be observed in the distribution of these lineages in the phy‐ logeny. They generally display a large geographical range but also a large host range, infecting different species and even different gen‐ era (Figure 4). Thus, certain lineages observed in Epomops franqueti from our study area in Gabon were also described from the same host species in Uganda or South Sudan or from a different bat genus (e.g., Micropteropus pusillus). This lack of clustering according to host and geography may be of multiple origins and future studies explor‐ ing this in more details should be done. This will necessitate more systematic data on the diversity of Hepatocystis in the African bats all over the continent and in more host species. This variability could, for instance, be related to the historical expansion and contraction of bat distribution ranges and their secondary or primary contacts.

The diversity of lineages observed in bats varies from one species to another. In our study, most lineages were for instance observed from only one species of bats, E. franqueti. This could obviously be linked to sample size that was far higher for this species than for the others but it has been demonstrated for other pathogen groups like viruses that ecological factors such as the home range, the size of the animals, the roosting patterns, or the physiological character‐ istics may also explain these variations in the diversity of pathogen communities among species (Maganga, et al., 2014). Again more sys‐ tematic data on the diversity of Hepatocystis in bats will be needed to explore such aspects in details.

To our knowledge, our study is the first to report infections with haemosporidian parasites in Eidolon helvum. This result needs nev‐ ertheless to be considered with caution. PCR methods are known to be very sensitive and may, in certain cases, detect DNA of para‐ sites in the blood of a wrong host after the parasites have died (Vafa Homann et al., 2017). The only valid proof of an infection would be the observation of the parasites in a blood smear or, at least, if only molecular analyses are available, confirmation of infection in multi‐ ple individuals of the same species.

Regarding the Hepatocystis found in the African monkeys, they form a monophyletic clade with the Hepatocystis from the Asian monkeys, distinct from that of the African bats (Putaporntip, et al., 2010; Seethamchai, Putaporntip, Malaivijitnond, Cui, & Jongwutiwes, 2008). The entire clade is a sister group of the clade formed by the Hepatocystis of Asian bats and rodents (Cox‐Singh et al., 2006) (Figure 2). As in bats, the Hepatocystis from African monkeys form a diverse clade and it is yet unclear whether these

Hepatocystis lineages are the image of multiple taxa or a single

widely dispersed species (Ayouba, et al., 2012). Nevertheless, as previously noted for the bat Hepatocystis, the level of diver‐ gence observed between clades is of similar order of magnitude as the one observed between well‐defined species of Plasmodium

parasites. So it is likely that these different lineages correspond to different species. Our study and previous ones (Ayouba, et al., 2012; Prugnolle, et al., 2011; Thurber, et al., 2013) suggest that several African monkey species can be infected with these differ‐ ent parasite lineages, which do not seem to cluster according to host species or genus.

No transfers of Hepatocystis have been observed in our study between bats and primates despite the fact that they were captured in the same area and that all hosts share the same habitat. This ob‐ servation is somehow in contrast with the history of this genus that was marked by multiple and recurrent host switches between bats, rodents, and primates. Several explanations may be given to explain the absence of transfers between sympatric populations of hosts: (a) the parasites of each host species do not encounter the other host species or (b) there is a strong host specificity that prevent the transfers. Regarding the first hypothesis, this could be linked to the absence of vectors playing the role of bridge between the different host species. Very few are known regarding the vectors of Hepatocystis except that they might be Culicoides (Thurber, et al., 2013). For the second hypothesis, nothing is known about the fac‐ tors involved in the invasion of the host by species of this genus and this again should be an area of future researches.

From an evolutionary point of view, the sequences of Hepatocystis available so far suggest that the history of this group of parasites has been complex, characterized by repeated host shifts between different host groups associated to repeated events of geographic expansion from Africa to Asia and vice versa. Nevertheless, data are still too scarce to draw robust scenarios to explain the observed re‐ lationships between the parasites of different host and geographic areas. A more systematic analysis of the diversity of these parasites in a wider range of hosts covering larger geographic areas would be needed to understand this history. The analysis of the genetic rela‐ tionships between these different hosts would certainly also be a plus. However, parasites of this lineage are genetically closely re‐ lated and completely included in the group of parasites belonging to the genus Plasmodium (Supporting Information Figure S2). Thus, as previously suggested, we think that a complete revision of this genus should be performed in light of these recent results.

To finish, a couple of words regarding the Plasmodium species found in monkeys. They belonged to two species. P. gonderi and

P. sp._DAJ‐2004. If P. gonderi is well known and well described for

a long time, P. sp._DAJ‐2004 was only molecularly described for the first time in 2004, isolated from a drill (Manrdrillus leucopheaus). Since then, infections were observed mainly in mandrills (Mandrillus

sphinx) and in only a few occasions (in particular unnatural settings)

in some Cercopithecus species (Faust & Dobson, 2015; Prugnolle, et al., 2011). Until a formal description of this new species is written, we now propose to name this species in reference to the genus of primates (genus Mandrillus) that seems to represent its principal host: Plasmodium mandrilli. Although we acknowledge that naming this species does not follow the taxonomical standards, we never‐ theless think that providing a name for this lineage should ease fu‐ ture discussion on it.

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BOUNDENGA EtAl.

ACKNOWLEDGMENTS

We thank the two reviewers for their very constructive comments on the manuscript that really helped improve the manuscript. We thank all the people of primatology center and veterinarians for tech‐ nical assistance who were involved in capture and blood sampling. This work was supported by Centre International de Recherches Médicales de Franceville (CIRMF, Gabon) and Centre National de la Recherche Scientifique (CNRS, France: ANR JCJC SVSE 7‐2012 ORIGIN).

CONFLIC T OF INTEREST

The authors declare that they have no competing interests. AUTHORS’ CONTRIBUTION

LB, BN, and FP designed this study; LB, BN, TAT and IMM contrib‐ uted to the acquisition of samples in fieldwork; LB, FP, VR, IMM, and FP contributed to data interpretation, FP, FR and LB conducted and supervised this work, LB wrote manuscript, FP, VR and FR, revised. All authors read and approved the final manuscript.

DATA ACCESSIBILIT Y

All sequences obtained (accession numbers: MG602631– MG602666) and used are accessible in GenBank.

ORCID

Larson Boundenga http://orcid.org/0000‐0001‐9551‐3017

Virginie Rougeron http://orcid.org/0000‐0001‐5873‐5681

Franck Prugnolle http://orcid.org/0000‐0001‐8519‐1253

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SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of the article.

How to cite this article: Boundenga L, Ngoubangoye B, Mombo IM, et al. Extensive diversity of malaria parasites circulating in Central African bats and monkeys. Ecol Evol. 2018;8:10578–10586. https://doi.org/10.1002/ece3.4539