MINI-REVIEW
Diversity and function of prevalent symbiotic marine bacteria
in the genus Endozoicomonas
Matthew J. Neave
1,2
& Amy Apprill
2
& Christine Ferrier-Pagès
3
& Christian R. Voolstra
1
Received: 25 June 2016 /Revised: 29 July 2016 /Accepted: 1 August 2016 / Published online: 24 August 2016
#
The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract Endozoicom onas bacteria are emerging as ex-
tremely diverse and flexible symbionts of numerous marine
hosts inhabiting oceans worldwide. Their hosts range from
simple invertebrate species, such as sponges and corals, to
complex vertebrates, such as fish. Although widely distribut-
ed, the functional role of Endozoicomonas within their host
microenvironment is not well understood. In this review, we
provide a summary of the currently recognized hosts of
Endozoicomonas and their global distribution. Next, the po-
tential functional roles of Endozoicomonas , particularly in
light of recent microscopic, genomic, and genetic analyses,
are discussed. These analyses suggest that Endozoicomonas
typically reside in aggregates within host tissues, have a free-
living stage due to their large genome sizes, show signs of host
and local adaptation, participate in host-associated protein and
carbohydrate transport and cycling, and harbour a high degree
of genomic plasticity due to the large proportion of transpos-
able elements residing in their genomes. This review will fin-
ish with a discussion on the methodological tools currently
employed to study Endozoicomonas and host interactions
and revie w future avenues fo r studying complex ho st-
microbial symbioses.
Keywords Endozoicomonas
.
Symbiosis
.
Marine
.
Coral reefs
Introduction
It is increasingly recognized that eukaryotic organisms rely on
bacterial associates to provide a diversity of functions, from
supplying nutrients and essential amino acids, to protection
from pathogenic microbes and degradation o f toxins
(McFall-Ngai et al. 2013). Despite only being recently de-
scribed (e.g. Kurahashi and Yokota 2007), the bacterial genus
Endozoicomonas (Gammaproteobacteria; Oceanospirillales)
has been reported to associate with a large diversity of marine
organisms, including cnidarians, poriferans, molluscs, anne-
lids, tunicates, and fish (Jensen et al. 2010; Morrow et al.
2012; Forget and Juniper 2013; Fiore et al. 2015;Katharios
et al. 2015). They are also globally distributed and have been
found living symbiotically with organisms in all major oceans
of the world (Neave et al. 2016). However, although they are
ubiquitously distributed, the functional role of
Endozoicomonas is unclear. Their suggested roles have
ranged from a beneficial symbiont required for healthy host
functioning to a pathogen that can rapidly cause host death
(Bourne et al. 2013; Katharios et al. 2015).
This mini-review provides an update on the ever-
expanding range of known Endozoicomonas hosts and global
distributions and addresses recent advancements in under-
standing the genetic potential and possible functions of bacte-
ria in the genus Endozoicomonas. We conclude with a discus-
sion on strategies for uncovering new insights into the lifestyle
of this cryptic and enigmatic genus, including emerging tools
for the study of microbial-animal symbioses, and provide rec-
ommendations for future work.
* Christian R. Voolstra
christian.voolstra@kaust.edu.sa
1
Red Sea Research Center, Division of Biological and Environmental
Science and Engineering, King Abdullah University of Science and
Technology (KAUST), Thuwal 23955-6900, Saudi Arabia
2
Woods Hole Oceanographic Institution, Woods Hole, MA, USA
3
Centre Scientifique de Monaco, 8 Quai Antoine 1er,
98000 Monaco, MC, Monaco
Appl Microbiol Biotechnol (2016) 100:8315–8324
DOI 10.1007/s00253-016-7777-0
History and prevalence of Endozoicomonas
in the scientific literature
The genus Endozoicomonas was described less than a decade
ago by Kurahashi and Yokota (2007)(Fig.1), after they iso-
lated an unknown gammaproteobacterial symbiont from the
sea slug Elysia ornata. Physiological and phylogenetic anal-
yses indicated that the creation of a new genus,
Endozoicomonas (i.e. monad living inside an animal), was
required (Kurahashi and Yokota 2007). After this initial de-
scription, references to Endozoicomonas remained relatively
scarce in the literature until 201 0, when Endozoicomonas
montiporae (Yang et al. 2010) was described, and coral sym-
bionts with similarity to Endozoicomonas were discovered
(Kvennefors et al. 2010). Kvennefors et al. (2010) also noted
that symbionts from earlier microbiome studies (before the
creation of the genus) were closely related to
Endozoicomonas. Examples include abundant
gammaproteobacterial symbionts termed B PA 1^ from the cor-
al Porites astreoides (Rohwer et al. 2002)and
Gammaproteobacteria from the coral Pocillopora damicornis
(Bourne and Munn 2005). These efforts to retroactively link
past studies to the Endozoicomonas genus, in addition to the
detection of Endozoicomonas in new hosts, resulted in a spike
in citations of Endozoicomonas within the scientific literature.
From 2013 onwards, more than 15 publications per year re-
ferred to the genus (Fig. 1). This relatively rapid rise in
Endozoicomonas publications, and the novelty of the genus,
has led to some taxonomic inconsistencies finding their way
into journal articles and databases. For example, the
Greengenes database (DeSantis et al. 2006) places the genus
Endozoicomonas in an apparently newly created family called
BEndozoicomonaceae^. However, despite not being explicitly
stated, the type description of Endozoicomonas contained nu-
cleotide information that placed the genus within the family
Hahellaceae (Kurahashi and Yokota 2007) in the order
Oceanospirillales and this taxonomy was maintained in
subsequent type descriptions (Yang et al. 2010; Nishijima
et al. 2013). The incorrect term Endozoicomonaceae has ap-
peared in a number of scientific publications (e.g. Dishaw
et al. 2014; Katharios et al. 2015; Lawler et al.
2016)p
aying
tribute to just how rapid this genus became scientifically pop-
ular. Confusion surrounding the initial naming of the genus
has also produced inconsistencies in the scientific literature.
For example, in March 2005 (prior to the Endozoicomonas
description), a sequence named BSpongiobacter
nickelotolerans^ was submitted to GenBank (#AB205011),
which is very similar (>97 % SSU rRNA identity) t o the
Endozoicomonas type strains. The associated taxonomic pa-
per describing the isolation of S. nickelotolerans from a ma-
rine sponge, however, was not published and the name ap-
pears to have been a bandoned (e.g. Pike et al. 2013;
McCauley et al. 2016). Nevertheless , se veral publicati ons
have referred to BSpongiobacter^ or to the BSpongiobacter/
Endozoicomonas^ group (e.g. Costa et al. 2012; La Rivière
et al. 2015).
The volume of genetic data available for Endozoicomonas
bacteria in NCBI’s GenBank (Benson et al. 2013) has also
rapidly increased (Fig. 1). Since the initial deposition of the
first 1437 bps SSU rRNA sequence of E. elysicola by
Kurahashi and Yokota (2007), Endozoicomonas nucleotide
information has steadily accumulated, reaching almost
100,000,000 bps by the start of 2016 (Fig. 1). Moreover, this
number only takes into account data retrieved from GenBank;
far more Endozoicomonas genetic information is available in
other databases, such as NCBI’s Sequence Read
Archive (SRA). Much of this rapid accumulation of genetic
information can be attributed to the move towards whole ge-
nome sequencing, rather than marker gene sequencing. The
first Endozoicomonas genome sequenced was E. elysicola in
2013 (Fig. 1) as part of the one thousand microbial genomes
project (Kyrpides et al. 2014). The following year, Neave et al.
(2014) released an updated version of the E. elysicola genome
plus two new genomes, E. montiporae (Yang et al. 2010
)and
Fig. 1 Prevalence of
Endozoicomonas in the scientific
literature as shown by the number
of publications referring to the
genus per year (red markers)and
the cumulative amount of
Endozoicomonas nucleotide
information uploaded to NCBI’s
GenBank (blue line)
8316 Appl Microbiol Biotechnol (2016) 100:8315–8324
E. numazuensis (Nishijima et al. 2013). Since then, the E.
montiporae genome has been re-sequenced (Ding et al.
2016), the genome of E. atrinae has been made available
(Hyun et al. 2014), the genome of an undescribed pathogenic
Endozoicomonas hasbeenanalysed(Kathariosetal.2015),
and several other Endozoicomonas genome projects are un-
derway (e.g. Appolinario et al. 2016). Most recently, Neave,
Michell, Apprill and Voolstra (Endozoicomonas genomes re-
veals functional adaptation and plasticity in bacterial strains
symbiotically associated with diverse marine hosts,
Submitted) applied single-cell genomics and metagenomic
binning to recruit four additional Endozoicomonas genomes
from native coral host assemblages.
Diversity and distribution of Endozoicomonas hosts
Endozoicomonas symbionts have a global distribution in nu-
merous marine hosts, from abyssal depths to warm photic
zones (Fig. 2). They are most frequently detected as coral sym-
bionts and occur throughout the global distribution of coral
reefs, from the Great Barrier Reef in Australia (Bourne et al.
2008;Lemaetal.2013), Papua New Guinea (Morrow et al.
2015), Indonesia and the Pacific (Yang et al. 2010;Neaveetal.
2016) to the Red Sea (Bayer et al. 2013b;Jessenetal.2013;
Neave et al. 2016; Ziegler et al. 2016), Indian Ocean (Neave
et al. 2016), an d t he Caribbean (Morrow et al. 2012;
Rodriguez-Lanetty et al. 2013)(Fig.2). Interestingly, different
coral species appear to harbour specific Endozoicomonas ge-
notypes (Neave et al. 2016). For example, the sympatric corals,
Stylophora pistillata and Pocillopora verrucosa, each contain
different Endozoicomonas genotypes, and these genotypes par-
tition differently across large geographic scales (Neave et al.
2016). Moreover , patterns of symbiont specificity seem to co-
align with differences in reproductive mode. For instance, the
spawning coral P. verrucosa globally associates with the same
Endozoicomonas symbionts, whereas the brooding coral S.
pistillata harbours different Endozoicomonas genotypes in dif-
ferent regions (Neave et al. 2016).
Although no quantitative methods have been applied to
Endozoicomonas, trends in cell abundance have been inferred
from SSU rRNA gene sequence abundances. These studies
have linked the abundance of Endozoicomonas to the abun-
dance of its coral host. For example, when the fungid coral
Ctenactis echinata grew in its preferred Red S ea habitat,
Endozoicomonas symbionts were more abundant than in habi-
tats of degraded quality (Roder et al. 2015). Moreover , reduced
abundances of the corals Acropora millepora and Porites
cylindrica near carbon dioxide seeps in Papua New Guinea
coincided with a 50 % reduction in Endozoicomonas symbionts
(Morrow et al. 2015). Anthropogenic pollution can similarly
decrease the abundance of Endozoicomonas bacteria. Near the
large Red Sea city of Jeddah, the corals P. verrucosa
and
Acr
opora hemprichii contained a lower proportion of
Endozoicomonas compared to corals further afield (Ziegler
et al. 2016). In addition, bleaching of the coral A. millepora
on the Great Barrier Reef induced a shift from
Endozoicomonas-like symbionts to a Vibrio-dominated com-
munity (Bourne et al. 2008). Lesioned P. astreoides colonies
also contained reduced Endozoicomonas sequence abundances,
compared to non-lesioned colonies (Meyer et al. 2014). These
studies suggest that Endozoicomonas bacteria are part of a
healthy coral microbiome and reductions in their abundance
may indicate unfavourable environmental conditions.
Gorgonians, commonly known as sea fans, are closely re-
lated to corals and also have a microbiome frequently domi-
nated by Endozoicomonas. In the Mediterranean, the main
gorgonian species (Eunicella cavolini, E. singularis,
E. verrucosa, Leptogorgia sarmentosa,andParamuricea
clavata) harbour a microbiome dominated by
Endozoicomonas symbionts (van de Water et al. In revision;
Bayer et al. 2013a; La Rivière et al. 2015). Interestingly,
Fig. 2 Global distribution and
diversity of Endozoicomonas host
organisms
Appl Microbiol Biotechnol (2016) 100:8315–8324 8317
patterns of host-specificity and possibly co-evolution between
the gorgonian species and their specific Endozoicomonas ge-
notype have also been observed (van de Water et al. In
revision; La Rivière et al. 2015). In addition, another octocoral
species, the red coral Corallium rubrum, also harbour
Endozoicomonas, although it is much less abundant compared
to the other gorgonians and is dominated by Spirochaetales
(van de Water et al. 2016). Outside of the Mediterr anean,
Endozoicomonas symbionts were found in the gorgonian E.
verrucosa living off the south-west coast of England
(Ransome et al. 2014), as well as in the Caribbean gorgonian
Pseudopterogorgia elisabethae (Correa et al. 2013). Although
corals and gorgonians dominate the Endozoicomonas
literature, these symbionts have also been found in a range
of other cnidarian species. Early work by Sch uett et al.
(2007) detected an Endozoicomonas strain with 98 % similar-
ity to E. elysicola and observed bacterial cell aggregates in the
tentacles of the sea anemone Metridium senile from
Helgoland in the North Sea. Although these aggregates were
not confirmed as Endozoicomonas,theyusedscanningelec-
tron microscopy to obtain detailed images of the bacterial cells
forming large aggregates within host tissues. More recently,
an Endozoicomonas strain with 99 % similarity to E. elysicola
was found in another sea anemone, Nematostella vectensis,
from a marsh of varying salinity conditions in
Massachusetts, USA (Har et al. 2015). Endozoicomonas sym-
bionts also dominated two jellyfish species, Mastigias cf.
papua and Tripedalia cf. cystophora, in several Indonesian
lakes (Cleary et al. 2016). These e xamples show that
Endozoicomonas bacteria are symbiotic with a large diversity
of cnidarian species and are often abundant and host species-
specific, suggesting an important and ancient evolutionary
relationship with lineage-specific evolution.
Endozoicomonas are also known to associate with a wide
range of other marine organisms (Fig. 2). A number of partic-
ularly interesting examples of the adaptability of
Endozoicomonas come from deep-sea hydrothermal vent
communities. Forget and Juniper (2013) collected the
tubeworm, Ridgeia piscesae, from the Juan de Fuca Ridge
in the north-east Pacific, which has high hydrogen sulphide
concentrations (~40 μmo
l/L), temperatures up to 41.9 °C, and
depths greater than 2000 m. Even in this extreme environ-
ment, R. piscesae contained abundant Endozoicomonas sym-
bionts (Forget and Juniper 2013). Moreover, Endozoicomonas
bacteria have been recovered from the gills of the hydrother-
mal vent snail Alviniconcha (Beinart et al. 2014), the gills of
the deep-water bivalve Acesta excavata (Jensen et al. 2010),
and from the tissues of deep-water corals (Meistertzheim et al.
2016). Endozoicomonas have also been associated with a
number of sponge species, which are one of the oldest groups
of metazoan invertebrates and often harbour a rich diversity of
microbial symbionts (Rua et al. 2014). In the Caribbean, the
giant barrel sponge (Xestospongia muta) not only contained
Endozoicomonas bacteria, but evidence of their transcription-
al activity was also detected (Fiore et al. 2015). Sponges in
Brazil (Rua et al. 2014), Japan (Nishijima et al. 2013), and
several other European seas also contain Endozoicomonas
symbionts (Esteves et al. 2013; Gardères et al. 2015). In ad-
dition, tunicates, which are basal chordates, have a
microbiome that consistently contains a substantial proportion
of Endozoicomonas, suggesting that these symbionts are core
members of the tunicate microbiome (Dishaw et al. 2014).
Although reports to date mostly associate Endozoicomonas
with marine invertebrates, several examples of associations
with fish have also emerged in the recent literature. In these
cases, fish were kept in artificial aquaculture environments
and Endozoicomonas bacteria are suspected to have caused
disease (Mendoza et al. 2013; Katharios et al. 2015). In both
examples, Endozoicomonas formed cyst-like aggregations on
the gills of the fish, resulting in epitheliocystis (Mendoza et al.
2013; Katharios et al. 2015).
The central picture that emerges is the remarkable ability of
Endozoicomonas bacteria to adapt to a wide range of hosts
and environments, from warm coral reefs to cold deep-sea
mussels, and their apparent ability to transition from beneficial
core microbiome members of corals and tunicates to disease-
causing pathogens in fish.
Function and genetic potential of Endozoicomonas
The proposed functions of Endozoicomonas can be summa-
rized into three categories: nutrient acquisition and provision,
structuring of the host microbiome, and roles in host health or
disease (Table 1). Nutrient acquisition spans from nitrogen and
carbon recycling (Nishijima et al. 2013; Forget and Juniper
2013;Correaetal.2013;Morrowetal.2015), or methane
and sulphur cycling (Bourne et al. 2013; Forget and Juniper
2013;Correaetal.2013;Dishawetal.2014;Morrowetal.
2015), to the synthesis of amino acids and other essential mol-
ecules (Neave, Michell, Apprill and Voolstra, Endozoicomonas
genomes reveals functional adaptation and plasticity in bacterial
strains symbiotically associated with diverse marine hosts,
Submitted). Bourne et al. (2013) found that the abundance of
Endozoicomonas-related sequences (referred to as
Oceanospirillales sp.1,3,5,and6)ininvertebratemicrobial
communities correlated with the presence of photosymbionts,
such as Symbiodinium algae in coral tissues. They suggested
that the photosymbionts provide carbon and sulphur to the bac-
teria from the large quant ities of dimethylsulfopropionate
(DMSP) produced (Bourne et al. 2013;Correaetal.2013).
On the other hand, Endozoicomonas bacteria are also found
in hosts without photosymbionts (Bourne et al. 2013). In addi-
tion to nutrient cycling, Endozoicomonas-related members may
also play a role in regulating bacterial colonization of the animal
host via the production of bioactive secondary metabolites or
8318 Appl Microbiol Biotechnol (2016) 100:8315–8324
probiotic mechanisms, such as competitive exclusion of patho-
genic bacteria (Bayer et al. 2013b;Jessenetal.2013;Ruaetal.
2014;Morrowetal.2015). Moreover, the loss of
Endozoicomonas is often characteristic of corals with lesions,
signs of disease, or if they are living in eutrophicated, warm, or
acidic environments. Therefore, the abundance of
Endozoicomonas seems to be linked with healthy colonies of
diverse coral species (Morrow et al. 2012; Bayer et al. 2013b;
Roder et al. 2015; Morrow et al. 2015; Ziegler et al. 2016).
The spatial location of Endozoicomona s bacteria within host
tissues may also have functional implications. For example,
Endozoicomonas frequently form aggregations in various host
habitats (Bayer et al. 2013b; Mendoza et al. 2013;Katharios
et al. 2015; Schreiber et al. 2016). In corals, fluorescent oligo-
nucleotides have been designed for Endozoicomonas and used
to hybridize to Endozoicomonas cells, confirming their resi-
dence within host tissues (Bayer et al. 2013b), where they
formed similar structures to cell-associated microbial aggre-
gates or CAMAs as previously described using histological
staining in corals (Work and Aeby 2014). More recently, using
catalyzed reporter deposition fluorescence in situ hybridization
(CARD-FISH) with this same probe, bacterial cells have been
better resolved within the autofluorescent coral tissues and it
was found that Endozoicom onas cells form dense aggregations
and can reside within the tentacles of corals (Fig. 3; Neave et al.
2016). Aggregations filled with thousands of bacterial cells
have also been found in anemone tentacles, and although
SSU rRNA gene sequence data demonstrates that
Endozoicomonas are in residence with these anemones, the
tentacle-associated cells have not yet been confirmed a s
Endozoicomonas (Schuett et al. 2007). Another probe devel-
oped for Endozoicomonas also identified cells residing in ex-
tracellular aggregations in sea squirts (Schreiber et al. 2016). In
fish, Endozoicomonas also form extremely dense aggregations
containing thousands of individuals surrounded by a thin tightly
enveloping membrane (Mendoza et al. 2013; Katharios et al.
2015). These formations, particularly the membrane barrier,
may provide protection from host immune cells or other host
responses to bacterial infection. Functionally, the aggregations
may act as centres of protein transformation and production that
could be beneficial for the host. Moreover, several genotypes of
Endozoicomonas
are known to inhabit individual hosts
Table 1 Suggested functions of
Endozoicomonas bacteria
Host organism Suggested function Reference
Fish Fish disease (Mendoza et al. 2013;
Katharios et al. 2015)
Sponge Sponge health (Gardères et al. 2015)
Bromopyrrole production for
feeding deterence
(Haber and Ilan 2014)
Carbohydrate fermentation/
nitrate reduction
(Nishijima et al. 2013)
Antibiotic production (Rua et al. 2014)
Tunicate Sulphur cycling/
nutrient metabolism
(Dishaw et al. 2014)
Hydrothermal vent snail Host nutrition/sulphur cycling
or breakdown of organic
compounds
(Beinart et al. 2014)
Hydrothermal
polychaete
Methane cycling/food degradation (Forget and Juniper 2013)
Scleractinian corals Quorum-sensing molecules (Bayer et al. 2013b)
Microbiome structuring (Jessen et al. 2013)
Antimicrobial activity/
N-acyl homoserine lactones
(Morrow et al. 2015)
Coral health (Meyer et al. 2014;
Roder et al. 2015;
Webster et al. 2016)
Coral health and/or disease (Ziegler et al. 2016)
Protection from bleaching (Pantos et al. 2015)
Dimethylsulfoniopropionate (DMSP)
metabolism/sulphur cycling
(Raina et al. 2009;
Bourne et al. 2013;
Correa et al. 2013)
Carbohydrate metabolism/
nutrient acquisition
(Correa et al. 2013;
Morrow et al. 2015)
Octocoral (gorgonians) Host health (Vezzulli et al. 2013;
Ransome et al. 2014)
Appl Microbiol Biotechnol (2016) 100:8315–8324 8319