Integrative and Comparative Biology
Integrative and Comparative Biology, volume 55, number 6, pp. 972–985
doi:10.1093/icb/icv006
Society for Integrative and Comparative Biology
SYMPOSIUM
The Good, the Bad, and the Unknown: Microbial Symbioses of the
American Alligator
Sarah W. Keenan1,*,† and Ruth M. Elsey‡
*Department of Biology, Saint Louis University, Macelwane Hall, 3507 Laclede Avenue, St Louis, MO 63103, USA;
†
Department of Earth & Planetary Sciences, University of Tennessee, 1412 Circle Drive, Knoxville, TN 37996, USA;
‡
Louisiana Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, Grand Chenier, LA 70643, USA
From the symposium ‘‘Integrated Biology of the Crocodilia’’ presented at the annual meeting of the Society for
Integrative and Comparative Biology, January 3–7, 2015 at West Palm Beach, Florida.
1
E-mail: [email protected]
Synopsis Vertebrates coexist with microorganisms in diverse symbiotic associations that range from beneficial to detrimental to the host. Most research has aimed at deciphering the nature of the composite microbial assemblage’s genome, or
microbiome, from the gastrointestinal (GI) tract and skin of mammals (i.e., humans). In mammals, the GI tract’s microbiome aids digestion, enhances uptake of nutrients, and prevents the establishment of pathogenic microorganisms. However,
because the GI tract microbiome of the American alligator (Alligator mississippiensis) is distinct from that of all other
vertebrates studied to date, being comprised of Fusobacteria in the lower GI tract with lesser abundances of Firmicutes,
Proteobacteria, and Bacteroidetes, the function of these assemblages is largely unknown. This review provides a synthesis of
our current understanding of the composition of alligators’ microbiomes, highlights the potential role of microbiome
members in alligators’ health (the good), and presents a brief summary of microorganisms detrimental to alligators’
health (the bad) including Salmonella spp. and others. Microbial assemblages of the GI tract have co-evolved with their
vertebrate host over geologic time, which means that evolutionary hypotheses can be tested using information about the
microbiome. For reptiles and amphibians, the number of taxa studied at present is limited, thereby restricting evolutionary
insights. Nevertheless, we present a compilation of our current understanding of reptiles’ and amphibians’ microbiomes, and
highlight future avenues of research (the unknown). As in humans, composition of microbiome assemblages provides a
promising tool for assessing hosts’ health or disease. By further exploring present-day associations between symbiotic microorganisms in the microbiomes of reptiles and amphibians, we can better identify good (beneficial) and bad (detrimental)
microorganisms, and unravel the evolutionary history of the acquisition of microbiomes by these poorly-studied vertebrates.
Introduction
Defining symbioses in vertebrates
In the broadest sense, symbioses—positive, negative,
or neutral interactions between two organisms—
drive life. From nitrogen-fixing bacteria that live in
association with plant roots (e.g., Mylona et al. 1995)
to wasps that lay eggs in caterpillars (e.g., Whitfield
2002), life is a complex series of symbiotic associations. Vertebrates, including the American alligator
(Alligator mississippiensis), are colonized by diverse
assemblages of microorganisms in a variety of symbiotic interactions. However, distinguishing what
represents a ‘‘good’’ rather than a ‘‘bad’’ association
is not straightforward and has recently undergone a
transformation. In particular, the revelation that a
vertebrate contains an order of magnitude more microbial than eukaryotic cells (e.g., Karasov et al.
2011) has shifted our understanding of the role of
microorganisms in hosts’ health or disease (Hooper
and Gordon 2001).
Symbiotic associations between a vertebrate and
microorganisms occur on every surface from the
skin to the gastrointestinal (GI) tract lumen (Grice
et al. 2008; Human Microbiome Project Consortium
2012). Often, these interactions are beneficial for the
host or have a neutral impact on the host’s health.
Relative stability is disrupted through the establishment of pathogenic microbes or alteration to mutualistic communities (dysbiosis), negatively impacting
Advanced Access publication April 17, 2015
ß The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved.
For permissions please email: [email protected].
Microbial symbioses of the American alligator
health (e.g., Hooper and Gordon 2001). In alligators,
microbially caused diseases (see discussion below)
often are accompanied by physical manifestations,
such as inflammation or discoloration. However,
identifying ‘‘good’’ versus ‘‘bad’’ microbial colonization is not always straightforward (Cho and Blaser
2012), particularly in the GI tract. This review will
include a discussion of our current understanding of
‘‘good’’ (beneficial) and ‘‘bad’’ (detrimental) microbial associations in the American alligator. We also
highlight future directions for exploring the large
unknown or unexplored interactions between a vertebrate and its microorganisms in the context of reptiles and amphibians. Elucidating the nature of these
vertebrate–microbe interactions holds promise for
identifying symbiotic associations maintained over
geologic time as well as assessing the health or disease of the host.
Microenvironments of the GI tract
The GI tract of vertebrates consists of distinct organs
and tissues extending from the mouth to the colon.
Physiologically, each region of the GI tract plays a
specific role in the breakdown and uptake of nutrients (Karasov et al. 2011). Each of these regions presents distinct physiochemical conditions, availability
of nutrients, rates of turn-over in the lumen, surface
areas for attachment, and thus would be expected
to be inhabited by environment-specific and nichespecific microorganisms (Walter and Ley 2011).
For example, the pH of the stomach is highly
acidic (in alligators, pH ranges from 2.1 to 3.0
during feeding) (Keenan et al. 2013), while other
areas like the large intestine have a circum-neutral
pH (personal observation). For microorganisms, the
diversity of available nutrients as well as surfaces for
attachment present constraints on metabolism.
The skin as a microenvironment
The GI tract provides microbial assemblages with
relatively stable physiochemical conditions in each
distinct region along the digestive system. In some
vertebrates, including snakes and alligators, changes
in diet or feeding status result in seasonally-variable
physiochemical conditions (Costello et al. 2010;
Keenan et al. 2013). In contrast, microbial assemblages colonizing the skin of a vertebrate are faced
with a distinct suite of physical and chemical conditions. For semi-aquatic vertebrates like crocodilians,
the transition from land to water regularly throughout the day exposes microorganisms to widely variable temperature, salinity, UV light, and availability
of water and oxygen. The cutaneous microbiome of
973
alligators, and crocodilians in general, is currently
unknown. However, given the variability in composition of the microbiome observed in humans (e.g.,
Grice et al. 2008, 2009), a similar pattern would be
expected in alligators. A recent evaluation of the microbial diversity on human skin suggested that biogeographic and niche partitioning, driven both by
microenvironment and by the host’s individuality,
exerted controls on the resulting bacterial, archaeal,
and viral diversity (Oh et al. 2014).
Guided by these insights from humans, skin along
the dorsal and ventral surfaces would be expected to
contain dissimilar microbial assemblages due to the
distinct microenvironments present on these surfaces. Crocodilian skin is thick (epidermal and
dermal thickness of 30–150 mm and 4250 mm, respectively) (Alibardi 2011) and composed of -keratin
and collagen (Alibardi and Toni 2007; Dalla Valle
et al. 2009). Morphologically, dorsal and ventral surfaces are distinct. The dorsal surface is comprised of
ridges and troughs, due to the presence of ossified
bones or scutes, that form a complex surface. In
contrast, the ventral surface is more uniform with
thick, flat keratinized scutes. The surface morphology
of the ventral and particularly the dorsal surfaces
provides a large surface area for the attachment
and growth of microbial biofilms.
In addition to being morphologically distinct, the
dorsal and ventral surfaces are exposed to variable
physiochemical conditions on daily, as well as seasonal, intervals. Dorsal surfaces are regularly exposed
to variable UV, water, pH, salinity, and temperature
as alligators move from water to land. Ventral surfaces are likely to experience variable availability of
water and oxygen, but perhaps not as much exposure
to UV. These distinct physical and chemical conditions will exert large controls on composition of the
microbiome. Based on what has been observed on
the skin of humans, mice (e.g., Grice et al. 2008),
and amphibians (Kueneman et al. 2014), the greatest
diversity of microorganisms will likely be members
of Proteobacteria with lesser representation from
Actinobacteria and Firmicutes.
The good: microorganisms beneficial to
alligator health
Microorganisms are widely viewed as harmful or detrimental to an animal, often being associated with
disease or illness. However, as novel approaches toward characterizing the microbial world around us
have changed, particularly a departure from strictly
culture-based approaches (Hugenholtz et al. 1998),
microorganisms are now viewed in a different light.
974
S. W. Keenan and R. M. Elsey
The ability to obtain the genetics of microorganisms
within an environmental sample, such as an oral
swab, through ‘‘next generation sequencing’’, has
revolutionized our understanding of microbial ecology and diversity. Through these novel techniques,
characterizing the microbiome residing within and
on an organism is now feasible. In an animal, microbes are not only beneficial for the host’s health by
aiding the absorption of nutrients (Karasov et al.
2011), but may also be required for preventing the
proliferation of harmful microorganisms (PeraltaSanchez et al. 2014) and for the development of
the host’s immune system (Kau et al. 2011), including allergen sensitization (Stefka et al. 2014).
Establishment of a microbiome
The composition of a microbiome reflects the influence of a variety of interactions between a vertebrate
host and its environment, diet (prey items), genetics,
and interactions within the microbiome itself
(Fig. 1). The relative contribution of each component to the composition is unknown. Some studies
demonstrated the influence of geographic location or
environment on the composition (e.g., Yatsunenko
et al. 2012), suggesting that perhaps these sets of
variables exert the greatest control. Other studies,
including one of the Burmese python (Costello
et al. 2010) highlight the role of diet or feeding
status on modulating the composition of microbial
assemblages. Additionally, a similar conclusion has
been reached by several studies focusing on the microbial ecology of the GI tracts of humans and other
mammals (e.g., Nelson et al. 2013). The role of the
host’s genetics has also been proposed as a significant
modulator of the GI tract. Supporters of the idea
that a host’s genetics controls the composition of
microbiomes demonstrated the ability to reconstruct
hosts’ phylogenetics on the basis of the composition
and diversity of their resident microbial consortia
(e.g., Ochman et al. 2010). Interactions, including
competition among microbes within the gut (e.g.,
Roggenbuck et al. 2014) and between microbes and
the host itself also control membership of the assemblage (e.g., Artis 2008) (Fig. 1).
There are likely to be mixed contributions from
these four components—genetics, environment, diet
of the host, and interactions among members of the
microbiome—that shape a vertebrate’s microbiome
(Fig. 1). It is likely that throughout the course of a
vertebrate’s life, there are periods when the composition of the microbiome is altered due to aging (e.g.,
in humans; Heintz and Mair 2014), illness (e.g., Cho
and Blaser 2012), or changes to diet (e.g., Turnbaugh
Fig. 1 Schematic diagram of factors influencing the composition
of the microbiome of a vertebrate host.
et al. 2008). On longer, geologic timescales, the
emerging view of the GI tract’s microbiome as coevolved with the host suggests that membership of
the microbial assemblage has undergone a variety of
selective pressures resulting in the microbiome observed today (Ley et al. 2008b). Co-evolution between the vertebrate host and its resident microbial
assemblages creates strong selective pressures to keep
the beneficial microorganisms, and defend against
detrimental invaders. While determining if a certain
bacterial ‘‘species’’ is beneficial or harmful for the
host is still no small feat, a picture of the ‘‘core’’
microbiome for various vertebrates is starting to
emerge (Turnbaugh et al. 2007; Caporaso et al.
2011; Keenan et al. 2013).
The microbiome of alligators: composition and role in
health
Alligators are semi-aquatic, consuming prey derived
both from terrestrial and aquatic habitats (Platt et al.
1990; Shoop and Ruckdeschel 1990; Elsey et al.
1992). Alligators’ diets vary across their range due
to food-availability, sex, and ontogenetic stage of
the alligator (Elsey et al. 1992; Subalusky et al.
2009). Prey items include invertebrates and vertebrates (Platt et al. 1990; Shoop and Ruckdeschel
1990; Saalfeld et al. 2011). Soft tissues are broken
down within 1–2 days of consumption; however,
more resistant items, such as bones, teeth, and
nails, can remain in the GI tract for hundreds of
days (Garnett 1985; Barr 1997; Nifong et al. 2012).
Starting with initial consumption, oral processing of
prey varies, depending on prey-size. In some circumstances, prey is crushed with several strong bites
(Erickson et al. 2003). If small enough, prey may
be swallowed without biting or crushing. Within
975
Microbial symbioses of the American alligator
the oral cavity, the composition of the microbial assemblage not only reflects the endogenous, resident
microorganisms but also those acquired through contact with the environment (e.g., sediment, water, and
vegetation). In alligators and other crocodilians that
employ basking as a method of thermoregulation
(Lang 1987; Seebacher et al. 2003), visible green to
brown biofilms along the tongue indicate the establishment of environmentally-derived microorganisms
(e.g., Proteobacteria and Actinobacteria). At the level
of the bacterial class, oral communities in actively feeding alligators are dominated by Clostridia, Fusobacteria,
and members of Proteobacteria (Fig. 2A). Members of
the same classes are also present in winter, fasting alligators (Keenan et al. 2013).
The stomach presents a microenvironment distinct
from that of the rest of the GI tract. Highly acidic pH, as
well as an influx of transient, food-derived microorganisms, results in a dynamic system. Assemblages of
free-living and attached bacteria were similar, and consisted overwhelmingly of Clostridia (80% of the assemblage) (Fig. 2B; Keenan et al. 2013). Similarly, in
the duodenum, Clostridia are numerically dominant,
with lesser contributions from Epsilonproteobacteria
and unclassified Bacteria (Fig. 2C).
The intestines have highly folded mucosa, leading
to high surface-area:volume ratios that facilitate the
uptake of nutrients. These surfaces also provide areas
for microbial colonization and the development of
biofilms. In the duodenum and ileum, Clostridia
and Bacilli dominated the bacterial assemblage.
Notably, Fusobacteria, which were prominent in the
mouth, as well as in the lower GI tract, formed a
relatively minor fraction of the assemblage, representing approximately 2–6%. In the colon as well
as in the feces, Fusobacteria become the dominant
phylum. In the colon and feces, Clostridia are also
significant members of the assemblage. The diversity
of tissues and functions along the length of the GI
tract, as well as the distinct physiochemical environments, would be expected to result in diverse
microbial assemblages, and this is observed along
the length of the GI tract, particularly in the less
well-represented classes (Dethlefsen et al. 2007;
Keenan et al. 2013; Keenan 2014a, 2014b).
Using the insights gained for different bacterial
groups in modulating the health or disease of
humans, we can begin to piece together likely functional roles for different bacteria in modulating alligators’ health (or disease). Changes in the ratio of
Firmicutes and Bacteroidetes, dominant both in
humans and in alligators, have been linked to
changes in overall health of the host, particularly
obesity (Ley et al. 2005; Turnbaugh et al. 2006,
2008, 2009; Keenan et al. 2013). In farm-raised alligators, which contained greater mesenteric fat deposits than their wild counterparts, we noted
similar shifts in composition of the microbiomes as
observed in humans (Keenan et al. 2013).
Specifically, an increase in Firmicutes relative to
Bacteroidetes in humans, mice, and alligators, supports a direct link between these two phyla and overall health of the host; however, specific links directly
identifying obesity have recently been questioned
(e.g., Fincane et al. 2014). The recent discovery of
similar Fusobacteria-dominated microbiomes in vultures by Roggenbuck et al. (2014) provided new insights into the role of Fusobacteria in modulating the
health of alligators. Roggenbuck et al. (2014) demonstrated that members of Fusobacteria as well as
Clostridia in the guts of vultures provided a level
of defense to the host, allowing consumption of partially decomposed carrion containing bacterially-produced toxins. The authors suggested that
Fusobacteria and Clostridia, dominant members of
the microbiome, may be integral to the host’s health.
The microbiome plays a critical role in establishing the host’s immune system, immune responses,
and resistance to disease (Turnbaugh et al. 2006).
Studies have shown that alligators exhibit a febrile response to infection (Merchant et al. 2007) and have a
strong capacity to resist infection. Additionally, their
blood has antibacterial (Merchant et al. 2003),
amoebacidal (Merchant et al. 2004), and antiviral
properties (Merchant et al. 2005). It may be that
wild alligators do succumb to microbially-caused
malignancies but, after death in the wild, carcasses
may rapidly deteriorate in ambient heat and humidity, and are not recovered by researchers. Part of
alligators’ exceptional resistance to disease may be
due to a tight co-evolution with their gut microbiome, thereby helping to hone their immune response (Merchant et al. 2003, 2006; Keenan et al.
2013). Similar suggestions for a link between development of the immune system and the composition
of the microbiome in humans have transformed our
understanding of the role of microorganisms in
hosts’ health and disease (e.g., O’Hara and
Shanahan 2006; Kau et al. 2011).
The bad: detrimental microorganisms
associated with alligators
Microbial pathogens affecting alligators
Despite a robust immune system, alligators are susceptible to microbial infection from a variety of
pathogens. Gram-negative septicemia has been
noted in captive alligators. In one study, six bacterial
976
S. W. Keenan and R. M. Elsey
Fig. 2 Average abundance of different classes of bacteria from wild and farm-raised alligators, post-feeding. Samples were obtained
along the length of the GI tract, as indicated by letters A–F: (A) mouth/esophagus, (B) stomach, (C) duodenum, (D) ileum, (E) colon,
(F) feces (data available from Keenan et al. [2013] and the Supplementary Information file described in that paper).
977
Microbial symbioses of the American alligator
species were implicated in septicemia (family
Enterobacteriaceae) and were not previously associated with disease in alligators (Novak and Seigel
1986). There are numerous other bacterial as well
as fungal species that have been isolated from infected alligators (Table 1). While far from complete,
the list of known pathogens (Table 1) spans many of
the phyla that also contain normal members of the
microbiome of alligators, including Actinobacteria,
Firmicutes, Bacteroidetes, Proteobacteria, and
Fusobacteria. Identifying microbially-caused diseases
in wild populations is difficult, if not impossible,
without subsequent analyses of the sites of infection.
Research assessing wild crocodiles’ abnormalities and
injuries, which have the potential to become infected,
are relatively limited. A thorough study of 1345 wildcaught saltwater crocodiles (Crocodylus porosus) only
identified three (0.2%) with ‘‘growths’’ (Webb and
Messel 1977), possibly former sites of microbial infection. A similar survey of 797 wild-caught freshwater crocodiles (Crocodylus johnstoni) noted only six
‘‘growths’’, three of which appeared to be sites of
microbial infection (Webb and Manolis 1983). A
smaller study of diseases of 144 wild-caught Nile
crocodiles (Crocodylus niloticus) did not reveal any
growths or tumors due to prior microbial infection,
and the only external lesions were an old bite and a
recent puncture from a bite (Leslie et al. 2011). A
review by Huchzermeyer (2003) of transmissible
crocodilian diseases (viral, bacterial, fungal, and parasitic) highlights the physical manifestations of microbial infections in crocodilians.
Microbes associated with alligator bites on humans
Although rare, alligators can attack or incidentally bite
humans, particularly when humans participate in
Table 1 Isolated microbial pathogens affecting the health of alligators
Microbial pathogen
Citation (not necessarily first cited report)
Ascomycota
Aspergillus ustus
Aspergillus fumigatus
Penicillium sp.
Scopulariopsis sp.
Jasmin et al. (1968)
Jasmin et al. (1968)
Williamson et al. (1963)
Scott (1995)
Gammaproteobacteria
Acinetobacter junii/johnsonii
Acinetobacter spp.
Aeromonas hydrophila
Chryseomonas luteola
Citrobacter braakii
Citrobacter freundii
Edwardsiella tarda
Enterobacter agglomerans
Enterobacter cloacae
Escherichia coli
Klebsiella oxytoca
Morganella morganii
Proteus mirabilis
Proteus sp.
Proteus vulgaris
Pseudomonas spp.
Salmonella arizonae
Serratia marcescens
Vibrio parahaemolyticus
Nevarez et al. (unpublished data)
Nevarez et al. (unpublished data)
Novak and Seigel (1986); Sartain and Steele (2009)
Nevarez et al. (unpublished data)
Nevarez et al. (unpublished data)
Novak and Seigel (1986); Sartain and Steele (2009)
Wallace et al. (1966)
Novak and Seigel (1986)
Nevarez et al. (unpublished data)
Nevarez et al. (unpublished data)
Novak and Seigel (1986)
Novak and Seigel (1986)
Nevarez et al. (unpublished data)
Novak and Seigel (1986)
Wallace et al. (1966); Sartain and Steele (2009)
Sartain and Steele (2009)
Nevarez et al. (unpublished data)
Novak and Seigel (1986)
Nevarez et al. (unpublished data)
Actinobacteria
Cellumonas/Microbacterium
Corynebacterium argentoratense
Corynebacterium glucuronolyticum
Corynebacterium jeikeium
Corynebacterium propinquum
Micrococcus spp.
Rhodococcus spp.
Nevarez
Nevarez
Nevarez
Nevarez
Nevarez
Nevarez
Nevarez
Bacteroidetes
Bacteroidetes spp.
Chryseobacterium indologenes
Sartain and Steele (2009)
Nevarez et al. (unpublished data)
Firmicutes
Clostridium spp.
Enterococcus spp.
Mycoplasma alligatoris
Staphylococcus spp.
Sartain and Steele (2009)
Sartain and Steele (2009)
Clippinger et al. (2000)
Nevarez et al. (unpublished data)
Fusobacteria
Fusobacterium varium
Sartain and Steele (2009)
et
et
et
et
et
et
et
al.
al.
al.
al.
al.
al.
al.
(unpublished
(unpublished
(unpublished
(unpublished
(unpublished
(unpublished
(unpublished
data)
data)
data)
data)
data)
data)
data)
978
recreational activities in the water (Conover and
Dubow 1997). In addition to the traumatic injuries
from alligator bites, victims may suffer infections
from various microorganisms, particularly Gram-negative bacteria (Langley 2005). Numerous pathogenic
species (aerobic and anaerobic bacteria; fungi) have
been cultured from the mouths of crocodilians
(Langley 2005). The oral flora of the American alligator
has been described, and recommended initial antibiotic
therapy for alligator bites has been recommended
(Flandry et al. 1989). A wide variety of microorganisms
were cultured from alligators and water samples in the
study by Flandry et al. (1989) including 20 aerobic and
18 anaerobic bacterial species, and 20 fungal species.
The most frequently isolated microorganisms included
members of Proteobacteria (Aeromonas hydrophila,
Proteus vulgaris) in addition to Pseudomonas spp.,
Clostridium spp., and Bacteroides spp. Our work characterizing the oral microbiome of alligators (Fig. 2;
Keenan et al. 2013), the work of Flandry et al. (1989)
and others (Table 1) captured many of the dominant
members of the assemblage in their culture-based
work, but missed some of the other dominant taxa,
namely Fusobacteria. Sartain and Steele (2009) reported a traumatic amputation of an upper extremity
in a youth following an alligator bite, and summarized
the literature on microorganisms associated with alligator bites (Table 1).
The unknown: current and future
research on microbiomes
The ability to employ culture-independent methods
to evaluate microorganisms in environmental samples, including tissues of the GI tract, has revolutionized our view of the GI system and our
understanding of health and disease. The application
of next-generation sequencing has revealed some
striking patterns in the GI tract’s microbial assemblages. Notably, all vertebrate GI tracts studied to
date contain a similar and conservative assemblage
of a few bacterial phyla, dominated by Firmicutes,
Bacteroidetes, and Proteobacteria, with lesser representation by Fusobacteria, Actinobacteria, and several
other phyla (Ley et al. 2008a, 2008b). In some taxa,
including mammals (e.g., humans and mice) and
reptiles (e.g., alligators), the ratio of Firmicutes to
Bacteroidetes has been used as an indicator of obesity or health of the host (Ley et al. 2005; Turnbaugh
et al. 2006, 2008, 2009; Keenan et al. 2013).
Elucidating the role of Fusobacteria in the GI tract
At the phylum-level, Fusobacteria are poorly described in terms of environmental distribution,
S. W. Keenan and R. M. Elsey
metabolic activities, and even their phylogenetic position on the tree of life (Mira et al. 2004; Battistuzzi
and Hedges 2008). One estimate places their origin
among the earliest branches on the tree of life
around 3.5 billion years ago (Battistuzzi and
Hedges 2008), while another suggests a more recent
origin around 400 million years ago (Mira et al.
2004), coincident with the evolution of vertebrates
and the GI tract.
Part of this uncertainty with respect to phylogenetic
placement is due to limited studies of non-pathogenic
Fusobacteria, essentially leaving our understanding of
genetics, metabolic functioning, and distribution of
this phylum incomplete. Fusobacteria have been recovered from a narrow diversity of environmental samples,
including the oral cavity of humans (e.g., Bradshaw
et al. 1998), liver abscesses, foot rot, oral necrotic lesions, farm soil (Langworth 1977), and several other
environmental locations (e.g., 100 cm-deep snow,
Moller et al. 2013). Members of this phylum have largely been studied in the context of pathogens of
humans and other animals (e.g., Fusobacterium necrophorum) (Langworth 1977; Nagaraja et al. 2005). They
are Gram-negative, anaerobic, non-spore-forming, and
when isolated from an animal, are considered to be an
indication of disease (Langworth 1977; Tan et al. 1996).
They often are viewed as opportunistic pathogens,
flourishing when the host is physically or immunecompromised by injury or illness (Tan et al. 1996).
The overwhelming majority of research on
Fusobacteria relates directly to human oral health.
The presence of Fusobacteria is widely associated
with the formation of biofilm in the mouth, leading
to dental carries and the buildup of plaque (Munson
et al. 2004). However, they form a minor component
of the gut microbiome of humans, suggesting limited
to no colonization of the digestive system other than
the oral cavity. In contrast, there is a dominance of
Fusobacteria in the lower GI tract of alligators
(Keenan et al. 2013; Keenan 2014a, 2014b). More
recently, this phylum was found to dominate vulture’s microbiome (Roggenbuck et al. 2014). From
the few studies describing Fusobacteria isolated from
environmental samples, they are characterized as
being involved in anaerobic fermentation and in degradation of cellulose (van Gylswyk 1980). Alligators
frequently consume plant-derived organics during
feeding. While not yet demonstrated, the initial
source of Fusobacteria inoculating alligators may be
the vegetation used in nest construction, with the
first inoculants being hatchlings. Alligators’ nests
are constructed of local vegetation, including
Spartina patens (Joanen 1969). The high cellulose
content of this nest material would suggest the
979
Microbial symbioses of the American alligator
presence of cellulose-degrading microorganisms,
which may contain members of Fusobacteria.
Unraveling the initial source of members of
Fusobacteria in alligators’ microbiome is an unexplored and promising avenue for future discoveries
related to the acquisition of a microbiome, as well as
to the development of the immune system.
The ability of Fusobacteria to form biofilms in the
human mouth (e.g., Munson et al. 2004) suggests
that they may be functioning in a similar manner
in the gut of alligators. Additionally, Roggenbuck
et al. (2014) recently suggested that the presence of
this phylum in the two vertebrate taxa, alligators and
vultures, both of which may scavenge partially
decomposed prey (e.g., Platt et al. 2014), likely reflects a competitive advantage of Fusobacteria in the
lower GI tract, and suggests tolerance of toxins produced by pathogens like Clostridia in decomposing
food. Fusobacteria are often pathogenic in humans
(e.g., Langworth 1977), but for vultures, Fusobacteria
are suggested to provide critical functions for the
host’s health, immunity, and potentially for the
uptake of nutrients through the breakdown of carrion (Roggenbuck et al. 2014). We suggest that
Fusobacteria may benefit alligators in a similar way,
given both the similarity in diet as well as the numerical abundance of Fusobacteria in their GI tracts
compared with vultures. However, until this phylum
is studied more fully for the range of vertebrate
hosts, its functional role(s) will remain uncertain.
Microbiomes of non-mammalian vertebrates
The greatest amount of research into vertebrates’
microbiomes has focused almost exclusively on
mammals, more specifically humans, despite representing just a single vertebrate species. This work
continues to provide novel insights into health
(Cho and Blaser 2012) and disease (Ley et al. 2005;
Turnbaugh et al. 2009; Subramanian et al. 2014), and
the intimate association between a vertebrate and its
microbial symbionts. Unsurprisingly, the majority of
studies of the microbiomes of non-mammal vertebrates relate directly to agriculture, the diet of
humans, animal husbandry (particularly of broiler
chickens) (e.g., Bjerrum et al. 2006), and aquaculture
(e.g., Llewellyn et al. 2014).
In addition to research on the microbiome of the
American alligator (Keenan et al. 2013; Keenan
2014a, 2014b) (Fig. 3) there are a limited, but growing,
number of studies focusing on other reptiles, e.g.,
Burmese pythons (Costello et al. 2010), marine and
terrestrial iguanas, giant tortoise (Hong et al. 2011;
Lankau et al. 2012), and on amphibians, e.g., leopard
frogs (Kohl et al. 2013) and various species from
Northern California (Kueneman et al. 2014) (Table
2). The insights gained from reptilian, amphibian,
Fig. 3 Number of publications per year from 1962 to January 2015 describing microbiomes in selected groups of vertebrates. Data
were compiled through searches in PubMed for key words (e.g., reptile microbiome), and screening the resulting publications for
relevant articles, and binning them according to year of publication.
Alligator, Alligator mississippiensis (feces)
Burmese python (fasted,
pooled GI tract)
Land iguana (feces)
Marine iguana (feces)
Giant tortoise (feces)
Green iguana (feces)
Vultures
Turkey vulture (skin)
Turkey vulture (gut)
Black vulture (skin)
Black vulture (gut)
Amphibians Northern leopard frog
tadpole, Lithobates pipiens
(intestine)
Northern leopard frog adult,
Lithobates pipiens
(intestine)
Western toad, Anaxyrus
boreas (skin)
California newt, Taricha
torosa (skin)
Pacific tree frog, Pseudacris
regilla (skin)
American bullfrog, Lithobates
catesbeianus (skin)
Reptiles
Vertebrate host
(sample location)
30
11
et al.
et al.
et al.
et al.
et al.
24
23
26
26
16
et al.
et al.
47
et al.
Roggenbuck
(2014)
Roggenbuck
(2014)
Roggenbuck
(2014)
Roggenbuck
(2014)
Kueneman
(2014)
Kueneman
(2014)
Kueneman
(2014)
Kueneman
(2014)
8
16
30
4
2
Kohl et al. (2013)
(2011)
(2011)
(2011)
(2011)
7
al.
al.
al.
al.
Kohl et al. (2013)
et
et
et
et
3
Costello et al. (2010)
Hong
Hong
Hong
Hong
9
Source of data
Keenan (2014a)
3.0
31.1
6.1
21.2
11.8
0.8
0.8
0.7
0.32
0.01
0.6
0
0
0
0.5
69.6
39.3
60.8
34
67.1
8.9
11.8
3.0
3.7
66.1
36.6
63.9
75.1
81.1
74
61.8
16.7
4.6
0
12.1
0
3.7
5.9
3.7
8.9
22.8
2.43
4.2
8.2
4.4
10.1
20.6
5
31.3
5.4
22.6
11.2
51.3
53.7
74.7
30.3
10.4
54.9
1.4
0.6
2
3.1
10.1
2.7
0
0
0
0
0
0
0
0
0
0.09
0
0
0.1
0
0
0
10.6
1.2
15.5
0.2
1.9
6.3
2.2
26.6
0.08
1.13
1.3
0.6
0.8
0.1
0.6
0.08
3.7
0
0.8
0
0.5
2.0
0.7
0
0
0
0
0
0.1
0
0
0.1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3.9
0
0
0
0
0
0.2
0.8
3.0
0.8
0.03
2.66
0.2
1
0.1
1
0.6
4.3
0
0
0
0
0
0
0
0
0
0
26.6
14.5
10.4
10.1
0
1.4
7.5
1.5
8.9
0.3
21.70
18.70
11.90
29.00
0.27
2.21
1.80
0.00
1.00
1.60
1.90
0.12
Number of
Animals (n) Fusobacteria Firmicutes Bacteroidetes Proteobacteria Acidobacteria Actinobacteria Cyanobacteria Deferribacteres Verrucomicrobia Unclassified Other
Microbiome phylum-level composition
Table 2 Percent representation of bacterial phyla in microbiomes from reptiles, amphibians, and vultures
980
S. W. Keenan and R. M. Elsey
Microbial symbioses of the American alligator
and avian species have the potential to transform our
view of microbiomes. For example, in alligators and
more recently in vultures, Fusobacteria dominated
the biota of the microbiome (Keenan et al. 2013;
Roggenbuck et al. 2014), hinting that diet might be
exerting a large control on the biome’s composition.
Both the co-evolved microbial assemblages and the
vertebrate host preserve two evolutionary records.
One of the goals of research on microbiomes is to
utilize the evolutionary record of acquisition of microbial assemblages to decipher the evolutionary relationships among vertebrate hosts (e.g., Ochman
et al. 2010). However, the question then stands as
to what extent can the composition of microbiomes
be used to reconstruct vertebrate hosts’ phylogenetic
affiliations? The discovery that microbiome composition of hominids directly correlated with hominid
phylogenetic relationships opened the door to using
the microbiome as a tool to evaluate the evolution of
vertebrates (Ochman et al. 2010). Using the current
body of knowledge on composition of the microbiomes of reptiles and amphibians, it is possible to
begin to address questions related to membership in
microbial assemblages and to hosts’ evolutionary relationships. For example, with the available data it is
possible to ask basic questions relating to the
981
similarity or differences in microbiome membership
as a function of hosts’ phylogenetic relationships,
diet, or environment. These comparisons can help
elucidate the presence of shared, ancestral microbial
assemblages, as well as identify the acquisition of
more recent symbiotic microorganisms.
Data obtained from the American alligator
(Keenan et al. 2013) were compared with those available in the published literature for other reptilian
and amphibian taxa through May 2014 (Table 2).
The resulting dataset incorporated six reptilian and
six amphibian taxa. In addition, data recently available from the microbiome of vultures were also included as similarities were noted in the abundance of
Fusobacteria (Roggenbuck et al. 2014). Datasets were
deemed adequate for comparative purposes if one of
the goals of the prior study was to evaluate composition of the microbiome, if bacterial phyla were displayed visually or in tables, and if details of type of
sample (e.g., gut or skin) were provided.
Despite the limited number of reptilian and amphibian taxa studied to date, interesting patterns are
beginning to emerge. As observed in all other vertebrates, there is a ‘‘core’’ assemblage dominated by
representatives of Bacteroidetes, Firmicutes, and
Proteobacteria (Fig. 4). Compared with other reptiles
Fig. 4 Unconstrained cluster analysis of bacterial phyla composing microbiomes of reptiles and amphibians, based on unpaired groups
and Bray–Curtis dissimilarity. Normalized bacterial phyla are presented above the dendogram, highlighting the species-specific variability
in the microbiomes’ composition (see Table 2 for references; figure modified from Keenan [2014b]).
982
and to vultures, the American alligator’s microbiome
is distinct due to an overwhelming dominance of
Fusobacteria (Fig. 4). Samples obtained from
pythons, iguanas, tortoises, and leopard frogs contained a dominance of Firmicutes, with lesser representation of Bacteroidetes. Microbiomes representative
of the bullfrog, treefrog, newt, and western toad have
a larger dominance of Proteobacteria. If these results
were examined out of context of the source of the
sample, it could be suggested that perhaps reptiles
and amphibians could be distinguished by the relative dominance of Firmicutes or Proteobacteria
(Fig. 5A). However, when the source of the sample
is included in the analysis, a very different picture
emerges in which the influence of the type of sample
(e.g., skin swab or the contents of the GI tract) exerts
a large control on how samples begin to cluster
(Fig. 5B). This represents a significant obstacle for
evaluating the composition of microbiomes in
broader contexts, including the relative influence of
the host’s diet, environment, and genetics on members of the microbial assemblage. Additionally, due
to these differences in types or locations of samples,
attempts to use microbiome composition as a
window into symbioses maintained over time is extremely challenging. Further sampling of the microbiomes of the skin and/or GI tract from additional
reptilian and amphibian taxa may make phylogenetic
reconstructions an achievable goal.
Functional characterization of microbes and
microbial assemblages
The potential for identifying microorganisms involved in health or disease relies on an understanding of their metabolic functioning. Technological
advances have made characterizing the ‘‘who’s
there’’ relatively straight-forward—it’s the ‘‘what
are they doing’’ that remains more difficult to
answer. For the GI tract’s microbiome, understanding microbial function is not a trivial issue. New
analytical approaches, including metagenomics (e.g.,
Illumina-based analyses; Qin et al. 2010) and transcriptomics (e.g., RNA-Seq; David et al. 2014), are
transforming our view of how microbiomes function
in humans. In addition to serving a direct role
through their metabolic activity, microorganisms
have the potential of directly influencing the host
through other interactions. The potential for bi-directional communication between microorganisms
and their vertebrate host via the ‘‘brain–gut axis’’
suggests that the microbiome may be significantly
influencing the host’s metabolic, physiological,
and immune functions (Bercik et al. 2012).
S. W. Keenan and R. M. Elsey
Fig. 5 Non-metric multidimensional scaling (NMDS) plots of
bacterial phyla in microbiomes of reptiles, amphibians, and vultures, based on Euclidean distances (data in Table 2). (A)
Grouping vertebrate taxa by Class revealed two distinct clusters—reptiles and amphibians—suggestive of host-specific differences in composition of the microbiome. Note the overlap with
vultures highlighted in their own group. (B) Grouping taxa of
hosts by the type of sample examined also reveals distinct groups
(e.g., GI versus cutaneous), which suggests potential tissue-specific or organ-specific microbiomes. These findings highlight limitations with respect to using samples derived from distinct types
of tissue when trying to decipher an evolutionary or ecological
control on the composition of microbiomes (figure modified from
Keenan [2014b]).
Additionally, as part of the forces involved in establishment of the microbiome and its co-evolution
with the host, the host has the ability to sense beneficial or harmful microbes through epithelial cells
(Artis 2008). Through the incorporation of these
novel analytical techniques in studies of alligator
microbiomes, elucidating the funcational and metabolic role(s) of specific bacteria, particularly members of Fusobacteria, may be possible.
Conclusions
The vertebrate microbiome, both of the skin and the
GI tract, is vastly underexplored, particularly for
Microbial symbioses of the American alligator
reptiles and amphibians (Fig. 3). Given the integral
role of the microbiome for maintaining hosts’ health,
characterizing the composition and function of assemblages may aid with diagnosing health or disease.
Additionally, the GI tract can be considered as an
ecosystem in itself, worthy of exploring for the discovery of potentially new microbial taxa. Based on
the current work on alligators’ microbiome, it is
highly likely that a similarly distinct microbial consortium will be observed in other crocodilian species,
distinct from other vertebrate taxa.
In alligators and other crocodilian taxa, a highly
adapted and effective immune system may be due to
a tight co-evolution with their resident microbiome.
Likely, it is through intimate interactions with their
gut microbiome that alligators have thrived in pathogen-rich environments. Future work evaluating the
connection between microbial assemblages in the GI
tract, acquisition of the assemblage after hatching,
and development of the immune system are likely
to provide new insights into this enigmatic group
of reptiles.
Acknowledgments
The authors would like to acknowledge Val Lance for
his persistence in organizing the symposium,
Integrative Biology of the Crocodilia, in which ideas
developed in this paper were presented. They would
also like to thank the Louisiana Department of
Wildlife and Fisheries for access to salvaged alligators, and Annette Engel (University of Tennessee) for
collaborating on the project, and reading earlier versions of this manuscript.
Funding
This work was partly supported by the Department
of Earth & Planetary Sciences at the University of
Tennessee, Knoxville [to S.W.K.].
References
Alibardi L. 2011. Histology, ultrastructure, and pigmentation
in the horny scales of growing crocodilians. Acta Zool
92:187–200.
Alibardi L, Toni M. 2007. Characterization of keratins and
associated proteins involved in the corneification of crocodilian epidermis. Tissue Cell 39:311–23.
Artis D. 2008. Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut.
Nat Rev Immunol 8:411–20.
Barr B. 1997. Food habits of the American alligator, Alligator
mississippiensis, in the southern Everglades. Coral Gables
(FL): University of Miami.
983
Battistuzzi FU, Hedges SB. 2008. A major clade of prokaryotes
with ancient adaptations to life on land. Mol Biol Evol
26:335–43.
Bercik P, Collins SM, Verdu EF. 2012. Microbes and the gutbrain axis. Neurogastroent Motil 24:405–13.
Bjerrum L, Engberg RM, Leser TD, Jensen BB, Finster K,
Pedersen K. 2006. Microbial community composition of
the ileum and cecum of broiler chickens as revealed by molecular and culture-based techniques. Poult Sci 85:1151–64.
Bradshaw DJ, Marsh PD, Watson GK, Allison C. 1998. Role
of Fusobacterium nucleatum and coaggregation in anaerobe
survival in planktonic and biofilm oral microbial communities during aeration. Infect Immun 66:4729–32.
Caporaso JG, Lauber CL, Costello EK, Berg-Lyons D,
Gonzalez A, Stombaugh J, Knights D, Gajer P, Ravel J,
Fierer N, et al. 2011. Moving pictures of the human microbiome. Genome Biol 12:R50.
Cho I, Blaser MJ. 2012. The human microbiome: at the interface of health and disease. Nat Rev Genet 13:260–70.
Clippinger TL, Bennett RA, Johnson CM, Vliet KA, Deem SL,
Oros J, Jacobson, Schumacher IM, Brown DR, Brown MB.
2000. Morbidity and mortality associated with a new mycoplasma species from captive American alligators (Alligator
mississippiensis). J Zoo Wildl Med 31:303–14.
Conover MR, Dubow TJ. 1997. Alligator attacks on humans
in the United States. Herpetol Rev 28:120–4.
Costello EK, Gordon JI, Secor SM, Knight R. 2010.
Postprandial remodeling of the gut microbiota in
Burmese pythons. ISME J 4:1375–85.
Dalla Valle L, Nardi A, Gelmi C, Toni M, Emera D,
Alibardi L. 2009. Beta-keratins of the crocodilian epidermis:
composition, structure, and phylogenetic relationships.
J Exp Zool B Mol Dev Evol 312:42–57.
David LA, Maurice CF, Carmody RN, Gootenberg DB,
Button JE, Wolfe BE, Ling AV, Devlin AS, Varma Y,
Fischbach MA, et al. 2014. Diet rapidly and reproducibly
alters the human gut microbiome. Nature 505:559–63.
Dethlefsen L, McFall-Ngai MJ, Relman DA. 2007. An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature 449:811–8.
Elsey RM, McNease L, Joanen T, Kinler N. 1992. Food habits of
native wild and farm-released juvenile alligators. Proceedings
of the Southeastern Association of Fish and Wildlife Agencies
Annual Conference, Corpus Christi, Texas Oct 25–28. Vol. 46,
p. 57–66.
Erickson GM, Lappin AK, Vliet KA. 2003. The ontogeny of
bite-force performance in the American alligator (Alligator
mississippiensis). J Zool 260:317–27.
Fincane MM, Sharpton TJ, Laurent TJ, Pollard KS. 2014. A
taxonomic signature of obesity in the microbiome? Getting
to the guts of the matter. PLoS One 9:e84689.
Flandry F, Lisecki MEJ, Domingue GJ, Nichols RL, Greer DL,
Haddad RJ. 1989. Initial antibiotic therapy for alligator
bites: characterization of the oral flora of Alligator mississippiensis. South Med J 82:262–6.
Garnett S. 1985. The consequences of slow chitin digestion on
crocodilian diet analyses. J Herpetol 19:303–4.
Grice EA, Kong HH, Conlan S, Deming CB, Davis J,
Young AC, Bouffard GG, Blakesley RW, Murray PR,
Green ED, et al. 2009. Topographical and temporal diversity of the human skin microbiome. Science 324:1190–2.
984
Grice EA, Kong HH, Renaud G, Young AC, Bouffard GG,
Blakesley RW, Wolfsberg TG, Turner ML, Segre JA. 2008.
A diversity profile of the human skin microbiota. Genome
Res 18:1043–50.
Heintz C, Mair W. 2014. You are what you host: microbiome
modulation of the aging process. Cell 156:408–11.
Hong PY, Wheeler E, Cann IKO, Mackie RI. 2011. Phylogenetic
analysis of the fecal microbial community in herbivorous land
and marine iguanas of the Galápagos Islands using 16S rRNAbased pyrosequencing. ISME J 5:1461–70.
Hooper LV, Gordon JI. 2001. Commensal host–bacterial relationships in the gut. Science 292:1115–8.
Huchzermeyer FW. 2003. Crocodiles: biology, husbandry, and
diseases. Wallingford, UK: CABI Publishing. p. 337.
Hugenholtz P, Goebel BM, Pace NR. 1998. Impact of cultureindependent studies on the emerging phylogenetic view of
bacterial diversity. J Bacteriol 180:4765–74.
Human Microbiome Project Consortium. 2012. Structure,
function and diversity of the healthy human microbiome.
Nature 486:207–14.
Jasmin AM, Carrol JM, Baucom JN. 1968. Pulmonary aspergillosis of the American alligator (Alligator mississippiensis).
Am J Vet Clin Pathol 2:93–5.
Joanen T. 1969. Nesting ecology of alligators in Louisiana.
Proceedings of the Southeastern Association Game Fish
Commissioners Conference, Mobile, Alabama, Oct 19–22.
Vol. 23, p. 141–51.
Karasov WH, Martinez del Rio C, Caviedes-Vidal E. 2011.
Ecological physiology of diet and digestive systems. Ann
Rev Physiol 73:69–93.
Kau AL, Ahern PP, Griffin NW, Goodman AL, Gordon JI.
2011. Human nutrition, the gut microbiome and the
immune system. Nature 474:327–36.
Keenan SW. 2014a. Freshwater vertebrate animal metagenomics, alligatorinae. In: Nelson K, editor. Encyclopedia
of metagenomics. Berlin: Springer-Verlag. p. 1–12.
Keenan SW. 2014b. Gastrointestinal microbial diversity and
diagenetic alteration of bone from the American alligator
(Alligator mississippiensis) [dissertation]. [Knoxville (TN)]:
The University of Tennessee. p. 241.
Keenan SW, Engel AS, Elsey RM. 2013. The alligator gut
microbiome and implications for archosaur symbioses. Sci
Rep 3:2877.
Kohl KD, Cary TL, Karasov WH, Dearing MD. 2013.
Restructuring of the amphibian gut microbiota through
metamorphosis. Environ Microbiol Rep 5:899–903.
Kueneman JG, Parfrey LW, Woodhams DC, Archer HM,
Knight R, McKenzie VJ. 2014. The amphibian skin-associated microbiome across species, space and life history
stages. Mol Ecol 23:1238–50.
Lang JW. 1987. Crocodilian thermal selection. In: Webb GJW,
Manolis SC, Whitehead PJ, editors. Wildlife management:
Crocodiles and alligators. Sydney: Surrey Beatty & Sons.
p. 301–17.
Langley RL. 2005. Alligator attacks on humans in the United
States. Wilderness Environ Med 16:119–24.
Langworth BF. 1977. Fusobacterium necrophorum: its characteristics and role as an animal pathogen. Bacteriol Rev
41:373–90.
Lankau EW, Hong PY, Mackie RI. 2012. Ecological drift
and local exposures drive enteric bacterial community
S. W. Keenan and R. M. Elsey
differences within species of Galápagos iguanas. Mol Ecol
21:1779–88.
Leslie AJ, Lovely CJ, Pittman JM. 2011. A preliminary disease
survey in the wild Nile crocodile (Crocodylus niloticus) population in the Okavango Delta, Botswana. J S Afr Vet Assoc
82:155–9.
Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD,
Gordon JI. 2005. Obesity alters gut microbial ecology. Proc
Natl Acad Sci USA 102:11070–5.
Ley RE, Hamady M, Lozupone C, Turnbaugh PJ, Ramey RR,
Bircher JS, Schlegel ML, Tucker TA, Schrenzel MD,
Knight R, et al. 2008a. Evolution of mammals and their
gut microbes. Science 320:1647–51.
Ley RE, Lozupone CA, Hamady M, Knight R, Gordon JI.
2008b. Worlds within worlds: evolution of the vertebrate
gut microbiota. Nat Rev Microbiol 6:776–88.
Llewellyn MS, Boutin S, Hoseinifar SH, Derome N. 2014.
Teleost microbiomes: the state of the art in their characterization, manipulation and importance in aquaculture
and fisheries. Front Microbiol 5:207.
Merchant M, Thibodeaux D, Loubser K, Elsey RM. 2004.
Amoebacidal effects of serum from the American alligator
(Alligator mississippiensis). J Parasitol 90:1480–3.
Merchant M, Williams S, Trosclair PL 3rd, Elsey RM, Mills K.
2007. Febrile response to infection in the American alligator
(Alligator mississippiensis). Comp Biochem Physiol A Mol
Integr Physiol 148:921–5.
Merchant ME, Mills K, Leger N, Jenkins E, Vliet KA,
McDaniel N. 2006. Comparisons of innate immune activity
of all known living crocodylian species. Comp Biochem
Physiol B Biochem Mol Biol 143:133–7.
Merchant ME, Pallansch M, Paulman RL, Wells JB, Nalca A,
Ptak R. 2005. Antiviral activity of serum from the
American alligator (Alligator mississippiensis). Antiviral Res
66:35–8.
Merchant ME, Roche C, Elsey RM, Prudhomme J. 2003.
Antibacterial properties of serum from the American alligator (Alligator mississippiensis). Comp Biochem Phys B
136:505–13.
Mira A, Pushker R, Legault BA, Moreira D, Rodriques-Valera F.
2004. Evolutionary relationships of Fusobacterium nucleatum
based on phylogenetic analysis and comparative genomics.
BMC Evol Biol 4:50.
Moller AK, Soborg DA, Al-Soud WA, Sorensen SJ, Kroer N.
2013. Bacterial community structure in High-Arctic snow
and freshwater as revealed by pyrosequencing of 16S rRNA
genes and cultivation. Polar Res 32:17390.
Munson MA, Banerjee A, Watson TF, Wade WG. 2004.
Molecular analysis of the microflora associated with
dental caries. J Clin Microbiol 42:3023–9.
Mylona P, Pawlowski K, Bisseling T. 1995. Symbiotic nitrogen-fixation. Plant Cell 7:869–85.
Nagaraja TG, Narayanan SK, Stewart GC, Chengappa MM. 2005.
Fusobacterium necrophorum infections in animals: pathogenesis
and pathogenic mechanisms. Anaerobe 11:239–46.
Nelson TM, Rogers TL, Brown MV. 2013. The gut bacterial
community of mammals from marine and terrestrial habitats. PLoS One 8:e83655.
Nifong JC, Rosenblatt AE, Johnson NA, Barichivich W,
Silliman BR, Heithaus MR. 2012. American alligator
Microbial symbioses of the American alligator
digestion rate of blue crabs and its implications for stomach content analysis. Copeia 3:419–23.
Novak SS, Seigel RA. 1986. Gram-negative septicemia in American
alligators (Alligator mississippiensis). J Wildl Dis 22:484–7.
Ochman H, Worobey M, Kuo CH, Ndjango JB, Peeters M,
Hahn BH, Hugenholtz P. 2010. Evolutionary relationships
of wild hominids recapitulated by gut microbial communities. PLoS Biol 8:e1000546.
Oh J, Byrd AL, Deming C, Conlan S, Comparative
Sequencing Program NISC, Kong HH, Segre JA. 2014.
Biogeography and individuality shape function in the
human skin metagenome. Nature 514:59–64.
O’Hara AM, Shanahan F. 2006. The gut flora as a forgotten
organ. EMBO Rep 7:688–93.
Peralta-Sanchez JM, Soler JJ, Martin-Platero AM, Knight R,
Martinez-Bueno M, Moller AP. 2014. Eggshell bacterial
load is related to antimicrobial properties of feathers
lining barn swallow nests. Microb Ecol 67:480–7.
Platt SG, Brantley CG, Hastings RW. 1990. Food habits of
juvenile American alligators in the upper Lake
Pontchartrain estuary. Northeast Gulf Sci 11:123–30.
Platt SG, Elsey RM, Trosclair PL III, Linscombe JT. 2014.
Alligator mississippiensis (American alligator) cannibalism.
Herp Rev 43:488–9.
Qin J, Li R, Raes J, Arumugam M, Burgdorf KS,
Manichanh C, Nielsen T, Pons N, Levenez F, Yamada T,
et al. 2010. A human gut microbial gene catalogue
established by metagenomic sequencing. Nature 464:59–65.
Roggenbuck M, Schnell IB, Blom N, Bælum J, Bertelsen MF,
Pontén TS, Sørensen SJ, Gilbert MTP, Graves GR,
Hansen LH. 2014. The microbiome of New World vultures.
Nat Commun 5:5498.
Saalfeld DT, Conway WC, Calkins GE. 2011. Food habits of
American alligators (Alligator mississippiensis) in East Texas.
Southeast Nat 10:659–72.
Sartain SE, Steele RW. 2009. An alligator bite. Clin Pediatr
(Phila) 48:564–7.
Scott TP. 1995. Disease surveillance in the American alligator
(Alligator mississipiensis). Proceedings of the Wildlife
Production, Management and Utilization Conference,
College Station, Texas, Oct 20–21. p. 1–12.
Seebacher F, Elsey RM, Trosclair PL. 2003. Body temperature null distributions in reptiles with nonzero heat
capacity: seasonal thermoregulation in the American alligator
(Alligator mississippiensis). Physiol Bioch Zool 76:348–59.
Shoop CR, Ruckdeschel CA. 1990. Alligators as predators on
terrestrial mammals. Am Midl Nat 124:407–12.
Stefka AT, Feehley T, Tripathi P, Qiu J, McCoy K,
Mazmanian SK, Tjota MY, Seo GY, Cao S, Theriault BR,
et al. 2014. Commensal bacteria protect against food
allergen sensitization. Proc Natl Acad Sci USA 111:13145–50.
Subalusky AL, Fitzgerald LA, Smith LL. 2009. Ontogenetic
niche shifts in the American Alligator establish functional
985
connectivity between aquatic systems. Biol Conserv
142:1507–14.
Subramanian S, Huq S, Yatsunenko T, Haque R, Mahfuz M,
Alam MA, Benezra A, DeStefano J, Meier MF, Muegge BD,
et al. 2014. Persistent gut microbiota immaturity in malnourished Bangladeshi children. Nature 510:417–21.
Tan ZL, Nagaraja TG, Chengappa MM. 1996. Fusobacterium
necrophorum infections: virulence factors, pathogenic
mechanism and control measures. Vet Res Commun
20:113–40.
Turnbaugh PJ, Backhed F, Fulton L, Gordon JI. 2008. Dietinduced obesity is linked to marked but reversible
alterations in the mouse distal gut microbiome. Cell Host
Microbe 3:213–23.
Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL,
Duncan A, Ley RE, Sogin ML, Jones WJ, Roe BA,
Affourtit JP, et al. 2009. A core gut microbiome in obese
and lean twins. Nature 457:480–4.
Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM,
Knight R, Gordon JI. 2007. The human microbiome project. Nature 449:804–10.
Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V,
Mardis ER, Gordon JI. 2006. An obesity-associated gut
microbiome with increased capacity for energy harvest.
Nature 444:1027–31.
van Gylswyk NO. 1980. Fusobacterium polysaccharolyticum
sp.nov., a gram-negative rod from the rumen that produces
butyrate and ferments cellulose and starch. J Gen Microbiol
116:157–63.
Wallace LJ, White FH, Gore HL. 1966. Isolation of
Edwardsiella tarda from a sea lion and two alligators.
J Am Vet Med Assoc 149:881–3.
Walter J, Ley R. 2011. The human gut microbiome: ecology
and recent evolutionary changes. Ann Rev Microbiol
65:411–29.
Webb GJW, Manolis SC. 1983. Crocodylus johnstoni in the
McKinlay River Area N. T. V.: abnormalities and injuries.
Aust Wildl Res 10:407–20.
Webb GJW, Messel H. 1977. Abnormalities and injuries in the
Estuarine crocodile, Crocodylus porosus. Aust Wildl Res
4:311–9.
Whitfield JB. 2002. Estimating the age of the polydnavirus/
braconid wasp symbiosis. Proc Natl Acad Sci USA
99:7508–13.
Williamson WM, Tilder EB, Getty RE. 1963. Mycotic
infections occurring during an eight year period at the
Chicago Zoological Park, Brookfield, Illinois. Bijdr Dierkd
33:83–5.
Yatsunenko T, Rey FE, Manary MJ, Trehan I, DominguezBello MG, Contreras M, Magris M, Hidalgo G,
Baldassano RN, Anokhin AP, et al. 2012. Human gut
microbiome viewed across age and geography. Nature
486:222–7.