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).