Bonobos are naturally Laverania infected

Bonobos are found in the rain forests of the Congo Basin in the Democratic Republic of the Congo (DRC). Separated from eastern chimpanzees (P. t. schweinfurthii) and eastern lowland gorillas (G. b. graueri) by the Congo River, their range extends from the Lualaba River in the east, to the Kasai and Sankuru Rivers in the south, and the Lake Tumba and Lake Mai-Ndombe regions in the west (Fig. 1). Initial studies failed to identify Plasmodium infections in wild bonobos, but were conducted at only two locations (LK and KR)1. Although subsequent surveys included additional bonobo field sites (ML, LA, IK, BN, BJ, TL2), faecal samples were only tested for P. vivax-like parasites2. Here, we screened these (n = 646) as well as newly collected (n = 803) faecal samples from the same (LA, IK) and additional (LG, BX, MZ) study sites for Laverania infection (Fig. 1). Using conventional (diagnostic) PCR to amplify a 956 bp mitochondrial cytochrome B (cytB) fragment1, we failed to detect parasite sequences in 1418 samples from 10 of these 11 locations (Table 1). Surprisingly, however, 16 of 138 faecal specimens from the Tshuapa-Lomami-Lualaba (TL2) project site were Laverania positive as determined by direct amplicon sequencing (Table 1).

Fig. 1 Plasmodium infections of wild-living bonobos. Ape study sites are shown in relation to the ranges of the bonobo (P. paniscus, dashed red) and the eastern chimpanzee (P. t. schweinfurthii, dashed blue), with white dots indicating sites where no Plasmodium infection was found (see Table 1 and Supplementary Table 3 for a list of all field sites and their code designation). The Tshuapa–Lomami–Lualaba (TL2) site where bonobos are endemically infected with multiple Plasmodium species, including a newly discovered Laverania species (B1), is shown in red with two dots indicating sampling on both sides of the Lomami River. Eastern chimpanzee field sites with endemic P. reichenowi, P. gaboni and/or P. billcollinsi infections are shown in yellow. A red circle highlights one bonobo (KR) and one chimpanzee (PA) field site where B1 parasite sequences were detected in a single faecal sample. Forested areas are shown in dark green, while arid or semiarid areas are depicted in brown. Major lakes and rivers are shown in blue. Dashed yellow lines indicate national boundaries. The scale bar indicates 200 km Full size image

Table 1 Noninvasive screening of wild-living bonobo communities for Laverania infections Full size table

Reasoning that conventional PCR screening may have missed low-level Laverania infection, we retested all available cytB-negative faecal specimens by subjecting them to an intensified PCR protocol. Since most ape faecal samples contain limited quantities of parasite DNA, we reasoned that testing multiple aliquots of the same DNA preparation would increase the likelihood of parasite detection. To avoid PCR contamination, only initially negative samples were re-tested using the intensified approach. Performing 8 to 10 independent PCR reactions for each DNA sample, we identified 17 additional faecal samples from TL2 to contain cytB sequences, resulting in a total of 33 positive specimens from 24 different apes (Table 1). Although in most cases only one or a few replicates yielded an amplification product (Supplementary Table 1), the intensified PCR approach more than doubled the number of positives at the TL2 site, revealing an overall Laverania prevalence of 38% (Table 1). However, this was not observed for other bonobo field sites. Intensified PCR of the remaining 1105 samples identified only a single additional positive specimen from the Kokolopori Reserve (KR). Thus, malaria parasites are either absent or below the limits of faecal detection at the vast majority of bonobo field sites.

A new bonobo-specific Laverania species

Having identified Laverania-positive bonobo samples, we next sought to molecularly characterise the infecting parasites. Since apes are frequently co-infected with multiple Plasmodium species, we used limiting dilution PCR, also called single genome amplification (SGA), to generate mitochondrial cytB sequences (956 bp) devoid of Taq polymerase-induced artefacts such as in vitro recombination18. Using this approach, we generated 166 limiting dilution-derived cytB sequences from 34 Laverania-positive bonobo samples, including a unique haplotype from the single positive KR specimen (Supplementary Table 2). Phylogenetic analysis showed that these bonobo parasites fell into two well-supported clades within the Laverania subgenus (Fig. 2). One of these comprised a sublineage of P. gaboni (C2E) previously found in eastern chimpanzees (P. t. schweinfurthii) in the DRC3. Within this sublineage, bonobo and chimpanzee parasite sequences were completely interspersed, indicating that P. gaboni productively infects both of these Pan species (Fig. 2 and Supplementary Fig. 1). The other clade represented a distinct Laverania lineage (B1) that included only bonobo parasites, except for a single cytB sequence previously identified3 in an eastern chimpanzee sample (PApts368) east of the Congo/Lualaba River (Fig. 1).

Fig. 2 Relationship of bonobo parasites to ape Laverania species. A maximum likelihood tree of mitochondrial cytochrome B (cytB) sequences (956 bp) depicting the phylogenetic position of newly derived bonobo parasite sequences (magenta) is shown. Only distinct cytB haplotypes are depicted (the full set of SGA-derived bonobo parasite sequences is shown in Supplementary Fig. 1). Sequences are colour-coded, with capital letters indicating their field site of origin (see Fig. 1 for location of field sites) and lowercase letters denoting their host species and subspecies origin (ptt: P. t. troglodytes, red; pte: P. t. ellioti, orange; pts: P. t. schweinfurthii, blue; ggg: G. g. gorilla, green; pp: Pan paniscus, magenta). C1, C2 and C3 represent the chimpanzee parasites P. reichenowi, P. gaboni and P. billcollinsi; G1, G2 and G3 represent the gorilla parasites P. praefalciparum, P. adleri and P. blacklocki (the P. falciparum 3D7 reference sequence is shown in black). P. reichenowi (C1) and P. gaboni (C2) mitochondrial sequences are known to segregate into two geographically defined subclades according to their collection site in 'western' (W) or 'eastern' (E) Africa3. Bonobo parasite sequences (magenta) cluster with P. gaboni from eastern chimpanzees (C2E), but also form a new clade, termed B1. The tree was constructed using PhyML58 with TIM2+I+G as the evolutionary model. Bootstrap values are shown for major nodes only (the scale bar represents 0.01 substitutions per site) Full size image

To determine whether B1 parasites were more widespread among eastern chimpanzees than previously recognised, we used regular and intensified PCR to screen faecal samples (n = 562) from nine study sites located closest to the bonobo range (Fig. 1). Although this analysis yielded twice as many Laverania positive samples as conventional PCR (Supplementary Table 3), none of the newly derived cytB sequences fell within the B1 clade (Supplementary Table 4). Instead, eastern chimpanzees were exclusively infected with P. reichenowi (C1), P. gaboni (C2) and P. billcollinsi (C3) (Supplementary Fig. 1). These data indicate that TL2 bonobos harbour a form of P. gaboni that is highly prevalent in neighbouring eastern chimpanzees as well as a second Laverania species that seems unique to bonobos.

To characterise the newly identified bonobo parasites in other regions of their genomes, we used SGA to target additional organelle and nuclear loci for analysis (Supplementary Table 2). These included 3.4 and 3.3 kb mitochondrial DNA (mtDNA) fragments, which together span the entire mitochondrial genome; a 390 bp caseinolytic protease M (clpM) gene fragment from the apicoplast genome; and three nuclear loci, including portions of genes encoding the erythrocyte binding antigens 165 (eba165; 790 bp) and 175 (eba175; 394 bp), and the gametocyte surface protein P47 (p47; 800 bp). Phylogenetic analyses of 134 newly derived parasite sequences yielded very similar results (with respect to the clustering of parasites into major clades) in all genomic regions (Fig. 3 and Supplementary Fig. 2). Except for a single C1 eba175 sequence indicative of a rare P. reichenowi infection (Supplementary Fig. 2d), all other bonobo-derived sequences fell either within P. gaboni or the B1 clade (Supplementary Table 2). This new clade was supported by high bootstrap values in all genomic regions analysed, except for the short eba175 fragment. It also consistently grouped as a sister clade to P. reichenowi. These findings, along with the extent of genetic divergence between P. reichenowi and the newly identified bonobo parasite clade, argue strongly for the existence of an additional Laverania species that is specific for bonobos (Figs. 2 and 3 and Supplementary Figs. 1 and 2). The finding of B1 cytB (Fig. 2) and eba165 (Fig. 3a) parasite sequences in a single chimpanzee faecal sample collected 280 km east of TL2 does not argue against this, since it shows that B1 parasites reached this geographic region, but failed to spread in the resident chimpanzee population (Supplementary Table 4). We propose to name the new bonobo parasite species Plasmodium lomamiensis sp. nov. to highlight its discovery in Lomami National Park, using faecal-derived mitochondrial, apicoplast and nuclear parasite sequences as the type material (Supplementary Data 1)19. Although classifying ape Laverania species solely on the basis of genetic information has been controversial3,17,20, there are no obvious alternatives given the endangered status of wild apes, the prevalence of mixed Laverania species infections (Supplementary Table 2) and the cryptic nature of these parasites3,9,21.

Fig. 3 A new Laverania species specific for bonobos. a, b Maximum likelihood phylogenetic trees are shown for nuclear gene fragments of the a erythrocyte-binding antigen 165 (eba165; 790 bp) and b the gametocyte surface protein P47 (p47; 800 bp) of Laverania parasites. Sequences are labelled and coloured as in Fig. 2 (identical sequences from different samples are shown; identical sequences from the same sample are excluded). C1, C2 and C3 represent the chimpanzee parasites P. reichenowi, P. gaboni and P. billcollinsi; G1, G2 and G3 represent the gorilla parasites P. praefalciparum, P. adleri and P. blacklocki (PrCDC and Pf3D7 reference sequences are shown in black). Bonobo parasite sequences cluster within P. gaboni (C2) or form a new distinct clade (B1), indicating a new Laverania species (see text for information on the single eba165 B1 sequence from an eastern chimpanzee). The trees were constructed using PhyML58 with TPM3uf+G (a) and GTR+G (b) as evolutionary models. Bootstrap values are shown for major nodes only (the scale bar represents 0.01 substitutions per site) Full size image

TL2 bonobos also harbour non-Laverania parasites

SGA of bonobo faecal DNA also yielded rare sequences from non-Laverania parasites that resulted from primer cross-reactivity (Supplementary Table 2). One such cytB sequence clustered with a previously characterised parasite sequence from a chimpanzee sample (DGptt540), forming a well-supported lineage that was only distantly related to human and ape P. malariae (Fig. 4a), while two other clpM sequences clustered with ape and human P. vivax parasites (Fig. 4b). To search for additional non-Laverania infections, we used P. vivax- and P. malariae-specific primers to rescreen bonobo faecal samples from the BX (n = 1), KR (n = 69), LA (n = 199) and TL2 (n = 138) field sites using intensified PCR. This analysis confirmed P. vivax infection in one bonobo sample, and identified P. vivax and P. ovale curtisi sequences in two additional samples, all from the TL2 site (Fig. 4c). Further characterisation revealed that the P. ovale curtisi-positive sample also contained ape P. vivax sequences (Fig. 4d). Thus, of the 24 Laverania-positive bonobos at the TL2 site, 3 also harboured P. malariae-, P. vivax- and/or P. ovale-related parasites, while an additional bonobo exhibited a P. vivax monoinfection (Supplementary Table 2). Although the recovered sequences were too short to differentiate human- and ape-specific parasite lineages, the results show that bonobos, like chimpanzees and gorillas, are frequently infected with multiple Laverania and non-Laverania species1,5,7. However, unlike chimpanzees and gorillas, bonobos harbour these parasites in only one particular part of their range.

Fig. 4 Bonobo infections with non-Laverania parasites. Maximum likelihood phylogenetic trees are shown for mitochondrial and apicoplast gene sequences of non-Laverania parasites. Ape-derived a cytB (956 bp), b clpM (327 bp), c cox1 (296 bp) and d clpM (574 bp) sequences are labelled and coloured as in Fig. 2 (identical sequences from different samples are shown; identical sequences from the same sample are excluded). Human and monkey parasite reference sequences from the database are labelled by black squares and circles, respectively. Brackets indicate non-Laverania species, including P. malariae, P. vivax, P. ovale curtisi and P. ovale wallikeri (available sequences are too short to differentiate ape- and human-specific lineages) as well as the monkey parasites P. inui and P. hylobati. Newly identified bonobo parasite sequences are indicated by arrows, all of which are from the TL2 site. One TL2 cytB sequence clusters with a previously reported parasite sequence from a chimpanzee sample (DGptt540), forming a well-supported lineage that is only distantly related to human and ape P. malariae, and thus likely represents a new P. malariae-related species. The trees were constructed using PhyML58 with GTR+G (a), TRN+I (b, d) and TIM2+I (c) as evolutionary models. Bootstrap values over 70% are shown for major nodes only (the scale bar represents 0.01 substitutions per site) Full size image

The Lomami River is not a barrier to malaria transmission

Analysing mtDNA sequences to determine the population structure of wild bonobo populations, two previous studies reported that the Lomami River, but not other tributaries of the Congo River, represents a geographical barrier to bonobo gene flow22,23. We thus considered the possibility that bonobos in the western and central regions of the DRC had acquired a malaria protective trait that had not spread to bonobo populations east of the Lomami River. To investigate this, we subjected Plasmodium-positive and -negative samples from TL2 to the same host mtDNA analysis (Supplementary Data 2) and compared the resulting haplotypes to all previously reported bonobo mtDNA sequences (Fig. 5a and Supplementary Fig. 3). Phylogenetic analysis showed that most of the newly derived mtDNA sequences from TL2 (blue) fell into two clades that were exclusively comprised of sequences from bonobos sampled east of the Lomami River (Supplementary Fig. 3b)22,23. However, 4 new TL2 haplotypes representing 15 faecal samples, including 4 Laverania-positive specimens, did not fall within these two 'eastern' clades (arrows in Fig. 5a and Supplementary Fig. 3a). Analysis of their GPS coordinates revealed that they were all collected west (TL2-W) of the Lomami River (Fig. 5b). These results thus confirm and extend previous findings showing that bonobos east of the Lomami River represent a genetically (at least matrilineally) isolated population22,23. However, this isolation does not explain the geographic restriction of bonobo malaria, since Laverania-positive individuals were found on both sides of the Lomami River. Although it remains unknown how far the Plasmodium endemic area extends beyond TL2 in the eastern Congo, it seems clear that the Lomami River itself does not represent a barrier to malaria transmission.

Fig. 5 The Lomami River is not a barrier to Laverania parasite transmission. a Maximum likelihood phylogenetic tree of bonobo mitochondrial (D-loop) sequences. Haplotypes are labelled by field site (see Fig. 1 and refs. 7,22,23,77, for their geographic location and code designation), with those identified at multiple field sites indicated (e.g., C/Wamba/KR/BN/IK/LA). Newly derived haplotypes from the TL2 site are shown in blue (previously reported mtDNA sequences are shown in black)22,23,77. Brackets highlight two clades that are exclusively comprised of mtDNA sequences from bonobos sampled east of the Lomami River. TL2 haplotypes that do not fall within these clades (denoted by arrows) were all sampled west of the Lomami River (TL2-W). The tree was constructed using PhyML58 with HKY+G as the evolutionary model. Bayesian posterior probability values ≥ 0.6 are shown (the scale bar represents 0.01 substitutions per site). b Locations of individual bonobo faecal samples collected at the TL2 site. Sampling locations west (TL2-W) and east (TL2-E and TL2-NE) of the Lomami River were plotted using GPS coordinates, with red and white dots indicating Laverania parasite positive and negative specimens, respectively. Samples that contained P. reichenowi, P. malariae-like, P. vivax-like and P. ovale-like parasites are also indicated. Forested areas are shown in green, while savannas are depicted in brown. The Lomami River is shown in blue. Local villages are denoted by black squares. The scale bar indicates 2 km Full size image

Climate does not explain the distribution of bonobo malaria

Because climatic factors such as ambient temperature and rainfall are known to influence malaria transmission in humans24,25,26, we asked whether seasonal differences in Plasmodium prevalence could explain the lack of parasite detection at the majority of bonobo study sites. Comparison of sample dates across all field sites revealed no obvious association between faecal parasite positivity and the month of specimen collection (Table 2). For example, samples collected in November and December at the TL2 site included a large fraction of malaria-positive specimens, but this was not the case for samples collected during these same months at the IK, KR and LA field sites. To examine the impact of climatic variation on bonobo parasite detection more directly, we used a statistical model shown to be strongly predictive of spatiotemporal variation in Laverania infection among wild-living chimpanzees (Erik Scully, unpublished results). This model, which was parameterised using PCR screening data from 2436 chimpanzee faecal samples collected at 55 locations across equatorial Africa7, showed that ambient temperature, daily temperature fluctuations and forest cover, but not rainfall, each influenced the probability of Laverania detection.

Table 2 Seasonal variation in parasite prevalence does not explain the geographic restriction of bonobo malaria Full size table

Using only specimens with known sampling dates and GPS coordinates for which land surface temperature and forest cover data were also available (Supplementary Table 5), we estimated the probability of Laverania infection for each of the 11 bonobo field sites. Assuming similar climatic influences on chimpanzee and bonobo parasite development and transmission, this analysis showed that at seven sites for which a sufficiently large number of samples were available, bonobos were significantly less frequently Laverania infected than predicted by the climate model. For the BN, IK, LA, LK and MZ sites, the model predicted a less than one in a million probability that a positive sample would not be detected if bonobos at these sites exhibited similar infection patterns as chimpanzees. Moreover, for the KR site, where only one sample was Laverania positive, seasonal variation could not explain this very low detection rate (Table 2). The rate of parasite detection at the TL2 site, where 27 of 113 samples with climate data were positive, was lower than, but not significantly different from, that predicted for a chimpanzee study site with similar ecological conditions. The very small sample sizes at BJ and BX sites lacked statistical power to detect differences, and the low predicted probability of infection at the ML site indicated that more sampling during months of higher infection probabilities would be necessary to confidently reject the climate model. Nevertheless, our sampling density at most sites was sufficient to conclude that the scarcity of infection in bonobos was not caused by biased sampling during seasonal troughs (Table 2). Thus, it appears that seasonal or climatic variation in parasite prevalence can be excluded as an explanation for the observed geographic restriction of bonobo Plasmodium infections.

Bonobo diet is not associated with faecal parasite detection

Wild apes consume a variety of plants, fruits, barks and piths, some of which have been reported to have antimalarial activity27,28,29. We thus asked whether our inability to detect Plasmodium infections at most bonobo field sites was due to the presence of certain plants, which upon ingestion would reduce parasite titres below the limits of faecal detection. To examine this possibility, we selected a subset of Laverania-positive (n = 18) and -negative (n = 51) bonobo faecal samples from endemic (TL2) and non-endemic (KR, IK, LG, LK) field sites, and characterised their plant content by targeting two regions of the chloroplast genome for high-throughput sequencing (Supplementary Table 6). These comprised a 500 bp fragment of the rbcL gene and a 750 bp fragment of the matK gene, both of which have been used as barcodes to identify land plants30,31,32, including in stool samples from endangered species33. Laverania-positive (n = 14) and -negative (n = 15) chimpanzee faecal samples were analysed for control (Supplementary Table 6).

Samples were sequenced to a mean depth of 16,054 matK and 21,995 rbcL paired-end reads, which were clustered into operational taxonomic units (OTUs) and assigned to taxonomic groups using a custom matK and rbcL reference database (Supplementary Fig. 4). Using a permutational multivariate analysis of variance (PERMANOVA) to compare unweighted UniFrac distances34 as a measure of large-scale differences in plant composition, we found small differences between faecal samples from bonobos and chimpanzees (matK: 2.0% of variance, p = 0.003; rbcL: 2.8% of variance, p < 10−6), but substantial differences between faecal samples from different study sites (matK: 19.8% of variance, p < 10−6; rbcL: 18.6% of variance, p < 10−6). However, no significant differences were observed between Laverania-positive and -negative faecal samples (matK: 1.2% of variance, p = 0.18; rbcL: 0.8% of variance, p = 0.71), suggesting that the lack of parasite detection was not associated with the abundance of certain plant phyla in the diet (Fig. 6a, b and Supplementary Fig. 5).

Fig. 6 Laverania infection of bonobos is not associated with particular faecal plant or microbiome constituents. A principal component analysis of unweighted UniFrac distances was used to visualise compositional differences of a, b plant (matK and rbcL) and c bacterial (16S rRNA) constituents in Laverania-positive (dark border) and -negative (light border) faecal samples from bonobos (blue) and chimpanzees (pink). The sample positions (shown for the first two components) do not indicate separate clustering of Laverania-positive and -negative samples Full size image

We also compiled a list of 466 African plant species (Supplementary Table 7), which have been reported to have potential antimalarial activity29,35,36, and looked for related matK and rbcL sequences in bonobo faecal samples from endemic and nonendemic field sites. Although a BLAST search identified 65 matK and 490 rbcL OTUs that shared 95% sequence identity with 3 and 17 of these putative antimalarial species, respectively, none was significantly more abundant at field sites where Laverania infections were absent (Supplementary Fig. 6). Similar results were obtained when the remaining plant OTUs were compared between endemic and nonendemic bonobo field sites. Finally, no compositional differences were observed in the plant content of Laverania-positive and -negative chimpanzee faecal samples (Fig. 6a, b). Although these analyses provide only a snapshot of bonobo and chimpanzee plant diet, they failed to identify an association between particular plant constituents and parasite detection in faecal samples.

The faecal microbiome does not predict Laverania infection

Plasmodium infections have been reported to influence the bacterial communities in the gut, with certain parasites causing intestinal dysbiosis37 and certain gut microbiota enhancing the host’s anti-parasite immune responses38. To examine potential interactions between the faecal microbiome and Laverania infection in bonobos, we used the same samples selected for plant analyses (Supplementary Table 6) for bacterial 16S rRNA sequencing (Laverania-positive and -negative chimpanzee samples again served as a control). Samples were sequenced to a mean depth of 65,132 reads, which were clustered into OTUs and assigned to taxonomic groups (Supplementary Fig. 7a). Examining Shannon diversity as a marker of dysbiotic outgrowth or loss of bacterial taxa, we failed to find significant differences in within-sample (alpha) diversity between specimens from Laverania-positive and -negative bonobos (or chimpanzees), or between specimens from endemic (TL2) and nonendemic (KR, IK, LG, LK) field sites (Supplementary Fig. 8a). Using unweighted UniFrac distance to compare between-sample (beta) diversity34, we found that as previously reported39 bonobo and chimpanzee faecal microbiomes differed in their bacterial composition (Fig. 6c and Supplementary Figs. 7b and 8b). Samples from the same field site were also often compositionally more similar to each other than to samples from other field sites (Supplementary Fig. 8b and c). Using PERMANOVA to examine the sources of this variation, we found that ape species accounted for 7.4% (p < 10−6), study site for 19.3% (p < 10−6) and Laverania positivity for 1.2% of the variance (p = 0.043), respectively. Considering only chimpanzee samples, study site accounted for 17.8% (p = 0.000018) and Laverania positivity for 4.0% of variance (p = 0.25). Comparing only samples from TL2 bonobos, differences among three sample locations (Fig. 5b) accounted for 14.6% (p < 10−6) and Laverania infection for 4.5% of variance (p = 0.0023). Thus, there was a small but significant compositional difference between the faecal microbiome of Laverania- positive and -negative bonobos at TL2 (the lack of significance in chimpanzees may be due to a smaller sample size).

Using Wilcoxon rank sum tests to look for OTUs that were driving these differences, we found one assigned to the family Ruminococcaceae that was significantly depleted, and two others assigned to family Lachnospiraceae and Prevotella copri that were significantly enriched in Laverania-positive TL2 bonobo samples (Supplementary Fig. 9). However, comparing samples from TL2 to nonendemic sites did not yield significantly higher UniFrac distance values than comparing samples between these nonendemic sites (Supplementary Fig. 8b). Thus, while the abundance of some bacterial taxa differed slightly between Laverania-positive and -negative bonobos at TL2, compositional differences between samples from TL2 and nonendemic sites were no greater than expected between any two random sites, thus failing to provide a microbial signature of Laverania infection for that site.