4. DISCUSSION
In this study we identify associations between obligate ectoparasitic bat flies and the skin microbiome of four Afrotropical bat families, and limited association between rare taxa in the gut and oral microbiota. Network analyses identified consistent, stable, and taxonomically rich clusters of bacteria on the skin of non-ectoparasitized bats, compared to relatively disconnected and apparently transient bacteria on the skin of bats harboring ectoparasites. In addition to these links between ectoparasitism and the bat microbiome, we found a significant association between the oral microbiota and infection by malarial parasites among bats belonging to the family Miniopteridae. These results are the first to examine links between the microbiota and eukaryotic parasitism in wild bats and support the hypothesis that parasitism may be in part mediated by host-associated bacteria.
We found a number of ASVs belonging to the genus Mycoplasma that were associated with the presence of ectoparasites across multiple bat families, as well as ASVs that were positively associated with ectoparasitism in all four bat families studied, suggesting possible convergence of bacterial associations with hippoboscoid ectoparasitism among these hosts. Bacteria positively correlating with ectoparasitism included Actinomycetales ASVs in the genera Corynebacterium ,Dermacoccus , Janibacter , and Kocuria . Some bacteria in these genera have been shown experimentally to produce VOCs that are attractive to other host-seeking hematophagus arthropods including anopheline mosquitoes (26, 45) and Rhodnius prolixus kissing bugs (the primary vector of Chagas disease) (46), and it is therefore interesting to find them consistently associating with blood-feeding hippoboscoid across divergent bat families. Although we did not quantify or characterize VOCs in this study, we hypothesize that the bacterial ASVs in these genera may be producing similar VOCs, such as sulfur-containing compounds identified in the head space ofCorynebacterium minutissimum (e.g. dimethylsulfide, dimethyltetrasulfide, octasulfur) associated with anopheline mosquito attraction to humans (45). Further validation is certainly needed.
Network analyses showed that presence of hippoboscoid parasites was significantly associated with a reduction in the size and stability of skin microbial clusters, with non-parasitized bats exhibiting fewer clusters that contained greater microbial diversity. The differences in these network statistics were shared by all four bat families in the study and were significant for all but pteropodid fruit bats. Similar patterns have been observed in human-mosquito interactions, in which individuals with lower bacterial diversity on the skin are significantly more attractive to blood-seeking mosquitoes than individuals with higher diversity (27). In humans, skin bacteria play a known role in attracting mosquitoes via their production of VOCs and studies have shown that variation in skin microbial community composition can increase or decrease human attractiveness to blood-seeking mosquitoes (7, 27, 28). Similar mechanisms may be at play in the bat-ectoparasite system, particularly given the shared evolutionary history of dipterans (47).
As suggested by studies of human-mosquito interactions (7, 27, 48), bacteria positively associated with increased rates of blood-feeding dipteran host selection may be producing VOCs on which the insects rely to identify their hosts. Bacteria that are negatively associated with such insects may be consuming the products of the former, or may be producing VOCs of their own that mask those of the former (suggested by Verhulst et al. (27)). To better understand the mechanisms underlying these correlations in wild populations, future experiments should consider including sampling and characterization of VOCs in vivothrough mass spectrometry and other metabolomics approaches.
Associations between the oral microbiome and malarial parasitism were supported by unweighted UniFrac diversity metric analysis, suggesting that ASVs contributing to observed differences are relatively rare among the oral microbiota. Upon further investigation of differential microbiota abundances, we found a bacterial ASV belonging to the speciesPantoea agglomerans to be most strongly associated with miniopterid bats infected with malaria. Interestingly, P. agglomerans has been the target of numerous paratransgenesis experiments aimed at controlling the transmission of malarial parasites (Plasmodium spp.) in anopheline and culicine mosquitoes (49). A common constituent of the dipteran midgut, P. agglomerans has been associated with the production of ‘Immunopotentiator from P. agglomerans 1’ (IP-PA1), a broad-spectrum antibiotic effective against bacterial, fungal, and viral pathogens (50). How and why this bacterium is associated with the oral microbiome of malarial bats requires more in-depth investigation. As no other bat groups experienced rates of malarial parasitism adequate for statistical analyses, we were unable to explore this relationship further. Future studies that incorporate greater sampling of malaria-positive species may reveal more robust microbial associations, as have been documented in numerous experiments with controlled rodent and human malaria infections (5-7, 48, 51, 52).
Although we cannot ascertain causality of differences in the microbial composition of skin in this study, our results support the hypothesis that these differences may provide a mechanism by which ectoparasites can locate or distinguish hosts. Alternatively, observed differences in microbial composition could result from microbial transfer from parasites to hosts – indeed Mycoplasma bacteria, which were commonly associated with ectoparasitism in our study, are a common constituent of the hippoboscoid bat fly microbiome (53). Bat flies spend the majority of their lives living on the skin and fur of their chiropteran hosts, providing ample opportunity for the exchange of microbes between bat and bat fly. Moreover, even some host-species specific bat flies readily transfer between intraspecific host individuals (54) effectively utilizing the host population as habitat. Our analysis of the skin microbiota identified significant differences in microbial betadiversity as well as differentially abundant bacteria between parasitized and non-parasitized bats at the host family level, but we were unable to ascertain the origin of these bacteria. Bacteria associated with parasitized bats may have originated in the bat flies themselves or may have been acquired from the environment. Given the known effect of locality and apparent absence of host phylogenetic signal in microbial community composition of skin (33), one possible explanation is that local environmental variables play a greater role in determining host-bacteria associations in bats. Indeed, in North America, multiple bat species have been found to share many bacterial genera with soil and plant material (55), and the bat skin microbiome has previously been documented to shift at the colony level over time (56). Thus, local conditions and bacterial composition of bat roosts are likely playing an important role in driving the composition of skin bacteria, and thereby potentially influencing which individuals become parasitized. Ecological and behavioral studies of bats have also observed that many species exhibit localized migration between caves, and it has been suggested that this behavior may be associated with the avoidance of parasites and pathogens (57). Longitudinal analyses of individuals will provide much-needed insight into the effect of local migration on skin microbial community composition and ectoparasite prevalence.
ACKNOWLEDGMENTS
We thank the Kenya Wildlife Service and the Uganda Wildlife Authority for permission to conduct research in national parks, and Simon Musila of the National Museums of Kenya for his logistical support. For assistance in the field, we thank Mike Bartonjo of the National Museums of Kenya, Phausia Kweyu of Karatina University, Dr. Robert Kityo, Sadic Babyesiza, Solomon Sebuliba, and Cissy Akoth of the Makerere University Zoological Museum, and Drs. Brian Amman, Jonathan Towner, and Rebecca Tiller of the Centers for Disease Control and Prevention for their logistical assistance. HLL was supported by the National Science Foundation Postdoctoral Research Fellowship in Biology (award number 1611948).
AUTHOR CONTRIBUTIONS
HLL conceived and designed study, and performed field work, laboratory and data analyses. JAG guided statistical analyses and provided laboratory support. CWD provided taxonomic identification of hippoboscoid parasites. All authors contributed to writing of the manuscript.
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FIGURE LEGENDS
Figure 1. Diagram of host-parasite associations and sampling. A) Bat host, with orange circles indicating locations from which skin and fur samples were collected; dashed circle indicates interscapular sampling region on dorsal side of bat. B) hippoboscoid bat fly, C) bat red blood cells infected by malarial haemosporidian parasites. Illustration by Madison Erin Mayfield @MEMIllustration.
Figure 2. Alphadiversity among the skin, oral, and gut microbiota of bats grouped by family and ectoparasite status.
Figure 3. Ranked differential features associated with skin of ectoparasitized bats grouped by host family; full range of ranked features (including negatively associated features) shown in gray inset. Highlighted features include those observed in all four bat families (red), those observed in three of the four bat families (orange), and features belonging to the genus Mycoplasma that were also shared by three of four bat families. Bars of highlighted features have been enlarged for clarity.
Figure 4. Network characteristics of the skin microbiome among ectoparasitized and non-parasitized bats grouped by host family, including A) cluster size density (* indicates p -value < 0.05, Mann-Whitney-Wilcoxon rank sum test), B) degree distribution (* indicates p -value < 0.05, t-test), and C) Fruchterman-Reingold network topology colored by individual network clusters.
Figure 5. Ranked differential features associated with oral of malarial miniopterid bats. Highlighted are the two highest-ranked features associated with presence of malarial parasites (P. agglomerans(red), Acinetobacter sp. (orange)), and the most abundant features associated with absence of malarial parasitism (ASVs in the Pasteurellaceae family (dark green)).
DATA AVAILABILITY STATEMENT
All 16S rRNA sequence data are publicly available via the QIITA platform (https://qiita.ucsd.edu) under the study identifier (ID) 11815 and the European Bioinformatics Institute (EBI) under accession number PRJEB32520. Code for sequence processing and analyses can be viewed at https://github.com/hollylutz/BatMP. Host and parasite vouchers are accessioned at the Field Museum of Natural History (Chicago, IL, USA).