Tripartite associations between Afrotropical bats, eukaryotic parasites,
and microbial symbionts
Lutz HL1,2,3, Gilbert JA1,2 , Dick
CW,3,4
1 Department of Pediatrics, University of California
San Diego, La Jolla, California, USA
2 Scripps Institution of Oceanography, University of
California San Diego, La Jolla, California, USA
3 Integrative Research Center, Field Museum of Natural
History, Chicago, Illinois, USA
4 Department of Biology, Western Kentucky University,
Bowling Green, Kentucky, USA
*Corresponding Author
Holly L. Lutz
Department of Pediatrics
University of California San Diego
9500 Gilman Drive
La Jolla, CA 92161
hllutz@health.ucsd.edu
ABSTRACT
Skin is the largest mammalian organ and the first defensive barrier
against the external environment. The skin and fur of mammals can host a
wide variety of ectoparasites, many of which are phylogenetically
diverse, specialized, and specifically adapted to their hosts. Among
hematophagous dipteran parasites, volatile organic compounds (VOCs) are
known to serve as important attractants, leading parasites to compatible
sources of blood meals. VOCs have been hypothesized to be mediated by
host-associated bacteria, which may thereby indirectly influence
parasitism. Host-associated bacteria may also influence parasitism
directly, as has been observed in interactions between animal gut
microbiota and malarial parasites. Hypotheses relating bacterial
symbionts and eukaryotic parasitism have rarely been tested among humans
and domestic animals, and have to our knowledge never been tested in
wild vertebrates. In this study, we use Afrotropical bats, hematophagous
ectoparasitic bat flies, and haemosporidian (malarial) parasites
vectored by bat flies as a model to test the hypothesis that the
vertebrate host microbiome is linked to parasitism in a wild system. We
identify significant correlations between bacterial community
composition of the skin and dipteran ectoparasite prevalence across four
major bat lineages, as well as striking differences in skin microbial
network characteristics between ectoparasitized and non-ectoparasitized
bats. We also identify links between the oral microbiome and presence of
malarial parasites among miniopterid bats. Our results support the
hypothesis that microbial symbionts may serve as indirect mediators of
parasitism among eukaryotic hosts and parasites.
Keywords: microbiome, bats, Chiroptera, Hippoboscoidea, malaria,
Haemosporidia, Afrotropics
1. INTRODUCTION
Animals are capable of hosting myriad biologically interdependent
symbionts including viruses, bacteria, archaea, and eukarya. Many
associations between eukaryotic parasites and hosts have ancient origins
(1, 2), and mounting evidence suggests that bacterial symbionts may be
responsible for mediating host-parasite interactions in ways that could
ultimately shape host evolution (3). For example, studies of human and
anthropophilic mosquito interactions have found that the human skin
microbiome can influence mosquito feeding preference, thereby affecting
transmission patterns of vector-borne pathogens (e.g. WNV, yellow
fever, dengue, malaria, etc.), and potentially imposing selective
pressures on human populations (4). Conversely, parasites may influence
the relative abundance of host-associated microbes in ways that
facilitate parasite transmission, as has been observed in rodent models
of Plasmodium transmission in which parasitism positively
correlates with abundance of skin-associated microbes that produce
volatile organic compound attractive to arthropod vectors ofPlasmodium (5-7). Despite the potential evolutionary significance
of interactions between animal hosts, microbial symbionts, and
eukaryotic parasites, such interactions have not been well studied in
wild vertebrates.
Bats (Mammalia: Chiroptera) are an ideal system for examining the
interactions between microbial symbionts, eukaryotic parasites, and
hosts. Bats are among the most speciose orders of mammals (second only
to the Rodentia), providing a diverse comparative phylogenetic framework
for hypothesis testing, and they harbor a great diversity of eukaryotic
parasites such as dipteran insects, haemosporidia, and helminths (8-11).
Bats are also associated with microbial pathogens of importance for
human health (e.g. Bartonella , Pasteurella , SARS
coronaviruses, filaviruses, rabies, Hendra and Nipah viruses (12-15)),
and serological surveys have supported the role of Afrotropical bats as
reservoirs for a number of viruses (16-18), making our understanding of
factors regulating parasite and pathogen transmission all the more
relevant. Inter- and intraspecific transmission of pathogens among bats
and other animals is an area of increasing concern in light of recent
zoonotic pandemics. Bat flies (Arthropoda: Hippoboscoidea), which are
obligate blood-feeding parasites of bats, are known to transmit several
pathogens of human relevance (19, 20) and also are the primary hosts for
bat-specific malarial parasites (Apicomplexa: Haemosporida). Bat flies
are typically host-specific (21, 22) and prevalence can vary widely
within a single species across different geographic locations (23).
Malarial parasites vectored by bat flies are also typically
host-specific but are observed less frequently in many bats relative to
ectoparasitic bat flies.
Bat flies are nutritionally dependent on their hosts and maintain
contact throughout most of their lives, living in fur and on skin
membranes and leaving their hosts only for brief reproductive periods
(24). How these parasites maintain host-specificity with bats over
evolutionary time is unknown, but blood protein and immunocompatibility
between host and parasite are thought to play a role (21, 25). The
proximal mechanism by which parasites locate “preferred” hosts is also
unknown, but as has been observed in other hematophagous parasites (e.g.
mosquitoes (26-28), tse-tse flies (29)), may involve host-associated
chemical cues. Chemical cues can be produced in a number of ways,
including via metabolic processes involving bacteria in the gut, oral
cavity, or on the skin (30, 31).
Here, we build on previous broad-scale studies of Afrotropical
bat-associated microbes (32, 33) to characterize parasitism by bat flies
and haemosporidia in four widespread lineages of bats. Using these data,
we test the hypothesis that the bat microbiome (skin, oral, and gut) is
linked to parasitism by two obligate, host-specific eukaryotic
parasites.
2. METHODS & MATERIALS
2.1 Host and parasite sampling
Sampling was conducted at sixteen field sites in Kenya and Uganda from
August to October 2016, using a combination of mist-netting and
hand-netting. Samples were taken from bats collected as voucher
specimens for biodiversity inventories, allowing for extensive
post-mortem sampling of skin and fur from multiple points on the body.
These included three biopsies from wing membrane, one from tail
membrane, one from the ear, one from the interscapular region of the
back, and one from the interclavicular region of the chest (Figure 1)
using 3mm sterile disposable biopsy punches (IntegraTMMiltex®). Biopsy samples from each individual were combined and stored
in sterile 95% ethanol. Whole tongues were collected (from apex to
root) and stored in 95% ethanol for oral microbiome analysis. We
collected ~2-4mL of blood from euthanized bats via
cardiac puncture. Blood was placed on Whatman FTA cards for nucleic acid
extraction, and 2-3 blood films per individual were prepared for
microscopic analyses. The remainder of each blood sample was stored in
cryovials placed in lN2. Following euthanasia, skin, and blood sampling,
bats were fumigated in ethyl acetate for 15 minutes and then examined
for ectoparasites. Presence or absence of dipteran parasites was noted,
and parasites were collected into 95% ethanol for taxonomic
identification. Bat flies were identified morphologically by examination
under magnification using a Leica MZ16 stereozoom microscope. Collected
specimens were compared to relevant taxonomic keys, to descriptions in
the alpha-literature, and to reference collections of the Field Museum
of Natural History, Chicago and the Bernice P. Bishop Museum, Honolulu.
Malarial parasite presence and taxonomic identity were determined as
described in Lutz et al. (2015). In brief, DNA was extracted from whole
blood using Qiagen DNeasy (Qiagen, Valencua, CA) and screened for the
presence of malarial parasites using triplicate PCR and Sanger
sequencing confirmation, followed by BLASTn to confirm parasite
taxonomy. All sampling was conducted in accordance with the Field Museum
of Natural History IACUC. Host and parasite vouchers are accessioned at
the Field Museum of Natural History (Chicago, IL, USA) (Table S1;S2).
2.2 Microbial DNA extraction, library preparation, and data generation
DNA was extracted using the MoBio PowerSoil 96 well soil DNA isolation
kit (catalog no. 12955-4; MoBio, Carlsbad, CA, USA) following the
standard Earth Microbiome Project protocol
(http://www.earthmicrobiome.org/). PCR amplification and sequencing were
performed as previously described in Lutz et al. (2019). Amplicon
sequence variants (ASVs) were identified using Deblur following standard
demultiplexing and quality filtering using the Quantitative Insights
into Microbial Ecology pipeline (QIIME2) (34). Skin, oral, and gut
libraries were rarefied to an even read depth of 5000 reads, 1000 reads,
and 1000 reads per library, respectively, based on rarefaction curve
estimates. 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; additional sequence library information is provided
in Table S2. Code for sequence processing and analyses can be viewed at
https://github.com/hollylutz/BatMP.
2.3 Statistical analyses
Alphadiversity and betadiversity analyses were performed using the
programming language R (35) and packages vegan2.4-2 (36), phyloseq (37),
dplyr (38), and ggplot2 (39). Differences in mean alphadiversity
measures (observed richness and Shannon index metrics) between
ectoparasitized and non-parasitized bats within families were assessed
using the Kruskal-Wallis test. PERMANOVA tests for differences in
betadiversity were performed using the adonis2 function (R package
vegan2.4-2 (36), with 1000 permutations.
We evaluated differences among skin associated microbial ASVs between
parasitized and non-parasitized bats grouped at the host family level by
ranking multinomial regression coefficients (hereafter referred to as
ranked differentials). This approach, implemented using the program
Songbird (40), relies on estimated centered log ratios of features
between sample groupings and thereby surpasses the need for absolute
measures of feature differentiation between groupings. Songbird
multinomial regressions were run for 100,000 epochs with a batch size of
3, minimum feature count of 5, a learning rate of 1e-5, and a
differential prior of 0.50. Ranked differentials were visualized using
the program Qurro (41).
To examine whether skin microbial communities differ in stability and
structure between parasitized and non-parasitized bats, we reconstructed
skin microbial networks using the R package Sparse Inverse Covariance
Estimation for Ecological Association Inference (SPIEC-EASI) (42). All
network datasets were filtered to contain only ASVs that appeared in at
least three individuals within each respective dataset and consisted of
skin microbial libraries grouped by host family and ectoparasite status.
Network results produced with SPIEC-EASI were summarized using the R
packages CAVnet (43) and igraph (44). Network stability was assessed by
sequentially removing network nodes (ordered by betweenness centrality
and degree) and observing natural connectivity (i.e. eigenvalue
of the graph adjacency matrix) as nodes are removed.
3. RESULTS
3.1 Microbiome, ectoparasite, and malarial parasite sampling and
detection.
We sampled 283 individuals representing eight species from four
chiropteran families (Hipposideridae, Miniopteridae, Rhinolophidae, and
Pteropodidae). Rarefaction and quality filtering of 16S rRNA libraries
resulted in the retention of 237 skin samples (29,270 ASVs, rarefied to
5000 reads), 202 oral samples (3,361 ASVs, rarefied to 1000 reads), and
230 gut samples (5,771 ASVs, rarefied to 1000 reads) for microbiota
profiling (Table 1). Hippoboscoid ectoparasites were recovered from all
host taxa, with an average prevalence of 51% (SD±13%). Malarial
parasitism was restricted almost entirely to the family Miniopteridae,
within which prevalence ranged from 47-65% (mean 53%±11%). All
malarial parasites observed in miniopterids belonged to the
haemosporidian genus Polychromophilus , and shared 99-100%
sequence similarity to Cytochrome b lineages previously
identified in Kenyan and Uganda miniopterid bats (11). We observed only
two non-miniopterid individuals of the species Rhinolophus
clivosus acrotis (Rhinolophidae) to be positive for malarial parasites,
which belonged to the genus Nycteria and exhibited 98% sequence
similarity to a Cytochrome b lineage previously identified in
Uganda. All other bats were negative for haemosporidia by molecular and
microscopic analyses (Table S1).
3.2 Associations between the bat microbiota and ectoparasitism.
No differences were observed between skin, oral, or gut microbial
alphadiversity and ectoparasitized or non-ectoparasitized bats at the
host family or species levels (p > 0.05,
Kruskal-Wallis) (Figure 2). However, subtle but significant differences
in skin-associated bacterial betadiversity were observed between
parasitized and non-parasitized bats for both weighted and unweighted
UniFrac metrics (p < 0.005, PERMANOVA). Differences
were only observed among the gut and oral microbiota using unweighted
but not weighted UniFrac metric (p < 0.002, PERMANOVA)
(Table 2), suggesting that differences were driven by the compositional
variance of rare taxa.
Multinomial regression analysis of the skin microbiome using Songbird
found models that included ectoparasite status of hosts significantly
outperformed null models (Miniopteridae pseudo Q2 =
0.12, Hipposideridae pseudo Q2 = 0.28, Rhinolophidae
pseudo Q2 = 0.17, Pteropodidae pseudo
Q2 = 0.26), allowing us to identify a number of ASVs
potentially associated with ectoparasitism (Figure 3). The bacterial
order Actinomycetales exhibited the greatest number of ASVs that were
differentially abundant between parasitized and non-parasitized bats.
Indeed, eight of fourteen ASVs found to be consistently associated with
the presence or absence of ectoparasites in all four host families
belonged to the order Actinomycetales, with the remaining six belonging
to the order Bacillales (phylum Firmicutes) and orders Burkholderiales,
Pseudomonadales, Rhizobiales, and Sphingomonadales (phylum
Proteobacteria) (Table 3).
Network analyses revealed striking differences in the topology and
stability of the skin microbiome in parasitized versus non-parasitized
bats, revealing a significant decrease in cluster size (p< 0.05, Mann-Whitney-Wilcoxon rank sum test) and median node
degree (p < 0.05, t test), as well as a
significant reduction in network connectivity (p <
0.05, t test) for parasitized bats from three of the four bat
families examined (pteropodids being the exception) (Figure 4).
3.3 Host microbiome and haemosporidian parasitism
Of the bat taxa sampled, only species belonging to the family
Miniopteridae exhibited malarial parasitism adequate for statistical
analysis. We observed no differences in alphadiversity of the skin,
oral, and gut microbiota of bats based on malarial infection status but
identified significant differences between unweighted UniFrac
betadiversity of the oral microbiota between malarial and non-malarial
bats (p < 0.002, PERMANOVA) (Table 4). Multinomial
regression analyses identified a number of bacterial ASVs associated
with malarial parasitism. Two ASVs exhibiting the greatest proportional
increase in malaria positive bats belonged to the species Pantoea
agglomerans and the genus Acinetobacter . ASVs most strongly
associated with absence of malarial parasites belonged to the family
Pasteurellaceae (Figure 5).