Abdominal setae and midgut bacteria of the mudshrimp Pestarella tyrrhena

Abstract

We investigated the diversity of the bacterial 16S rRNA genes occurring on the abdominal setal tufts and in the emptied midgut
of the marine mudshrimp Pestarella tyrrhena (Decapoda: Thalassinidea). There were no dominant phylotypes on the setal tufts. The majority of the phylotypes belonged
to the phylum Bacteroidetes, frequently occurring in the water column. The rest of the phylotypes were related to anoxygenic
photosynthetic α-Proteobacteria and to Actinobacteria. This bacterial profile seems more of a marine assemblage rather than
a specific one suggesting that no specific microbial process can be inferred on the setal tufts. In the emptied midgut, 64
clones were attributed to 16 unique phylotypes with the majority (40.6%) belonging to the γ-Proteobacteria, specifically to
the genus Vibrio, a marine group with known symbionts of decapods. The next most abundant group was the ɛ-Proteobacteria (28.1%), with members
as likely symbionts related to the processes involving redox reactions occurring in the midgut. In addition, phylotypes related
to the Spirochaetes (10.9%) were also present, with relatives capable of symbiosis conducting a nitrite associated metabolism.
Entomoplasmatales, Bacteroidetes and Actinobacteria related phylotypes were also found. These results indicate a specific
bacterial community dominated by putative symbiotic Bacteria within the P. tyrrhena’s midgut.

KeywordsBacteria-Setal tuft-Midgut-16S rRNA diversity-Thalassinidea-Decapoda-Pestarella tyrrhena

Full text

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Central European Journal of Biology
Abdominal setae and midgut Bacteria
of the mudshrimp Pestarella tyrrhena
*E-mail: kkormas@uth.gr
Received 17 May 2009; Accepted 21 August 2009
Abstract: We investigated the diversity of the bacterial 16S rRNA genes occurring on the abdominal setal tufts and in the emptied midgut of the
marine mudshrimp Pestarella tyrrhena (Decapoda: Thalassinidea). There were no dominant phylotypes on the setal tufts. The majority
of the phylotypes belonged to the phylum Bacteroidetes, frequently occurring in the water column. The rest of the phylotypes were
related to anoxygenic photosynthetic α-Proteobacteria and to Actinobacteria. This bacterial profile seems more of a marine assemblage
rather than a specific one suggesting that no specific microbial process can be inferred on the setal tufts. In the emptied midgut, 64
clones were attributed to 16 unique phylotypes with the majority (40.6%) belonging to the γ-Proteobacteria, specifically to the genus
Vibrio, a marine group with known symbionts of decapods. The next most abundant group was the ε-Proteobacteria (28.1%), with
members as likely symbionts related to the processes involving redox reactions occurring in the midgut. In addition, phylotypes
related to the Spirochaetes (10.9%) were also present, with relatives capable of symbiosis conducting a nitrite associated metabolism.
Entomoplasmatales, Bacteroidetes and Actinobacteria related phylotypes were also found. These results indicate a specific bacterial
community dominated by putative symbiotic Bacteria within the P. tyrrhena’s midgut.
© Versita Warsaw and Springer-Verlag Berlin Heidelberg.
Keywords: Bacteria • Setal tuft • Midgut • 16S rRNA diversity • Thalassinidea • Decapoda • Pestarella tyrrhena
1Department of Zoology – Marine Biology, School of Biology, University of Athens,
157 84 Panepistimiopoli Zografou, Greece
2Department of Ichthyology & Aquatic Environment, School of Agricultural Sciences,
University of Thessaly,
384 46 Nea Ionia, Greece
#Present address:
Andalucian Institute of Marine Sciences, Campus Río San Pedro,
11510 Puerto Real, Cádiz, Spain
Alexandra Demiri1, Alexandra Meziti2, Sokratis Papaspyrou1#,
Maria Thessalou-Legaki1, Konstantinos Ar. Kormas2*
Research Article
1. Introduction
Symbiosis between prokaryotes and eukaryotes
are widespread throughout many ecosystems and
determines many aspects of the physiology, ecology
and evolution of the hosts [1]. Prokaryotes can dwell
in internal (e.g. digestive tract) or external (mouth
parts, gills or special organs) body parts of a variety of
invertebrates, and are involved in different metabolic
activities of their hosts either as symbionts or parasites
[2-4]. In particular, gut microbiota has been one of the
most ancient and widespread types of symbiosis and
has offered evolutionary opportunities to the hosts
based on nutritional alternatives [5].

through their relationships with the host, however
nowadays the efforts are made toward independent
examination, encompassing either culture-independent
or culturing approaches [1]. Since a culture based approach
has significant limitations, a 16S rRNA gene analysis is
the most widely used method to analyse the microbial
diversity of a heterogeneous sample [6]. The majority
of the studies on bacteria associated with crustaceans,
have been focused either on the hindgut where symbiotic
bacteria are most likely to be found since there they
can access food remnants without competing with their
host for nutrients absorption [4,7-9] or on epibionts
[e.g. 10,11].
Initially, symbiotic prokaryotes were examined
Cent. Eur. J. Biol. • 4(4) • 2009 • 558–566
DOI: 10.2478/s11535-009-0053-x
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Abdominal setae and midgut Bacteria
of the mudshrimp Pestarella tyrrhena

shrimp that lives in complex burrows in marine sediments
along the coast of the Mediterranean Sea and the Atlantic
Ocean [12]. The mudshrimp feeds primarily on detritus
encountered during its burrowing activities [13]. It has
also been observed that the mudshrimp accumulates
detritus, mainly seagrass detritus, in burrowing chambers
where decomposition occurs. As this detritus is often of
poor quality, it has been suggested that the mudshrimp
uses this detritus to “garden” bacteria, with a higher C:N
ratio. The mudshrimp later feeds on this detritus and any
bacteria that have grown on it [13-16]. The mudshrimp
bears tufts of setal hair on its dorsal side. Although the
exact functions of the setal tufts is unknown, they seem
to play a role in the sense of touch when P. tyrrhena
comes into contact with the burrow walls [14]. In order to
investigate whether this tuft harbours a specific bacterial
community of a possibly specialized ecophysiological
process we investigated its 16S rRNA gene diversity.
Recently, one of the phylotypes of the current study was
found to occur in the setae of the deep-sea hydrothermal
vent decapod Kiwa hirsuta [15] suggesting a universal
specific association in marine decapods. Since the
feeding behaviour of the genus Pestarella suggests that
mouth parts are only for mechanical process of food [14]
and thus it is not expected to have resident symbionts,
we did not investigate the epibionts on the mouthparts.
In the same individuals, we also investigated the
resident bacterial community in the midgut of P. tyrrhena
using the same technique in order to infer benefits to
the animals. We also aimed at investigating the bacterial
community of the hindgut but no amplifiable DNA was
retrieved. Some of our retrieved sequences were also
closely related to gut bacterial phylotypes of the deep-
sea hydrothermal vent shrimp Rimicaris exoculata (M-A
Cambon-Bonavita, personal communication). Such
co-occurrence patterns are the first step for defining
potentially interacting organisms and interaction
networks in highly complex systems, like the invertebrate
– bacteria associations.
Pestarella tyrrhena is a deposit feeding thalassinid
2. Experimental Procedures
Pestarella tyrrhena specimens were collected from
Vravrona Bay (eastern Attica, Greece) using a handheld
bait pump in September 2005 and the animals were
transported to the laboratory in less than 6 h. Immediately
upon return to the laboratory, whole setal tufts –with no
cuticle included– from five mudshrimps were detached
from the third, fourth and fifth abdominal body part using
a sterilized scalpel, and frozen immediately at -80°C
until further analysis.

of the resident midgut bacterial community. Based on
knowledge from previous studies [14, Thessalou-Legaki
& Papaspyrou, unpublished data], these animals were
left for 24 hours in individual aquaria kept in the dark (with
a screen mesh near the bottom to avoid coprophagy,
which has been observed in this species [14]) in order
to empty their guts and avoid bacteria associated with
faecal pellets. The emptied midgut from each of the five
individuals was detached aseptically after incision under
a microscope, midguts were pooled in order to have
adequate amounts of amplifiable prokaryotic DNA, and
stored at -80°C until DNA extraction. Although we did not
analyse individual specimens as is the common practice
in fingerprinting techniques (e.g. T-RFLP, DGGE), our
clone library average (see below) allowed us to reveal
as many as possible of the occurring phylotypes, since
our intention was not to investigate individual differences
in gut microbial communities.
In this paper we used clone libraries of the bacterial
16S rRNA genes. Our data are indicative of the
structure of the gut bacteria communities of the decapod
Pestarella tyrrhena and adds information to the existing
literature. DNA was extracted using the Ultra Clean Soil
DNA Isolation Kit (MO BIO Laboratories, Inc., USA)
according to the manufacturer’s instructions. From both
DNA samples c.a. 1500 bp of the 16S rRNA gene were
amplified by PCR using the bacterial primers BAC8f
(5’-AGAGTTTGATCCTGGCTCAG-3’) and BAC1492r
(5’-CGGCTACCTTGTTACGACTT-3’) on a thermal
cycler (MyCycler, Bio-Rad Inc., USA). After PCR
optimisation, the conditions were 1 min pre-PCR hold at
94°C, followed by 30 or 27 cycles for the setal tuft and
midgut samples, respectively, of denaturation at 94°C for
45 sec, annealing at 52.5°C for 45 sec and elongation
at 72°C for 2min, and a final 7 min post-PCR elongation
at 72°C. The PCR products were stained with ethidium
bromide and visualized on a 1.2% agarose gel under
UV light.
Bands from both samples were excised and purified
with the NucleoSpin®ExtractII kit (Macherey-Nagel,
Germany) according to the manufacturer’s instructions.
Purified products were A-tailed in order to improve
cloning efficiency. For the setal tuft sample A-tailing was
performed by mixing 25 µl of purified PCR product, 11.9 µl
purified PCR water, 3 µl of MgCl2 (25 mM), 5 µl of
deoxynucleoside triphosphate (2 mM), 5 µl of Buffer10x
(200 M m Tris, pH=8.55) and 0.1 µl Taq polymerase.
The midgut sample was A-tailed by mixing 7 µl of purified
PCR product, 0.8 µl purified PCR water, 0.4 µl of MgCl2
(25 mM), 1 µl of deoxynucleoside triphosphate (2 mM),
0.6 µl of Buffer10x (200 mM Tris, pH=8.55) and 0.2 µl
Taq polymerase. The samples were incubated for 10 min
The same animals were used for the investigation
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A. Demiri et al.
at 72°C in the thermal cycler. For the cloning procedure
material and protocols of the TOPO XL PCR Cloning Kit
(Invitrogen Co., USA) were used. The resulting clones
were checked for the right insert (c.a. 1500 bp) by PCR
using the primers M13f (5’-GTAAAACGACGGCCAG-3’)
and M13r (5’-CAGGAAACAGCTATGAC-3’),
consisted of a pre-PCR hold for 2 min at 94°C, then 30
cycles of a 1 min denaturation at 94°C, 1 min annealing
at 55°C, 1 min elongation at 72°C, and at last a 7 min
post-PCR hold at 72°C. Clones were grown in liquid
cultures and then the plasmids were purified using
NucleoSpin®Plasmid QuickPure
Germany) according to the manufacturer’s instructions.
Sequencing of the inserts in the purified plasmids
was performed by Macrogen Inc. (Korea) using capillary
electrophoresis and the BigDye Terminator kit (Applied
Biosystems Inc., USA) with the primers M13f or M13r.
For each clone, forward and reverse sequences were
assembled and afterwards checked for chimeras on
CHIMERA_CHECK function of the Ribosomal Database
Project II [17]. The assembled sequences were
compared to already known sequences in GenBank
for identification of closest relatives through the BLAST
function (http://www.ncbi.nlm.nih.gov/BLAST/). They
were aligned using the ARB FastAligner utility (www.arb-
home.de), distances were identified through Neighbour-
Joining method and checked with the Jukes Cantor.
All analyses used minimum evolution and parsimony
methods carried out in PAUP programme. The GenBank
accession numbers of the phylotypes found in this study
are DQ890421-DQ890445.
Library clone coverage was calculated by the formula
[1 − (n1/N)] [18], where n1 is the number of operational
taxonomic units (OTU) represented by only one clone
and N is the total number of the clones examined in
each library.
that
(Macherey-Nagel,
3. Results
3.1 Setal tuft
Nine unique phylotypes were retrieved from nine
clones, suggesting that the diversity of this sample was
inexhaustible. Six (pt171, pt172, pt 176, pt177, pt181,
pt182) out of the nine unique phylotypes belonged to
the Bacteroidetes phylum (Table 1, Figure 1) and were
related (≥94% similarity) to other members of the phylum
originating from a variety of marine habitats, including
epibionts of an alga, a sponge and an octocoral. Two
clones belonged to the α-Proteobacteria and were
related to a hydrothermal vent phylotype (pt183) and a
phototropic Erythrobacter-like phylotype (pt173). Finally,
a singleton Actionabacteria-related phylotype (pt174)
was also found. We did not calculate the C estimator
for the setal tuft nor did we proceed to analysing
more clones since all phylotypes were singletons, i.e.
suggesting that this clone library was consisted solely
by unique phylotypes, pointing towards an unspecific
bacterial community.
3.2 Emptied midgut
A total of 16 unique phylotypes were retrieved out of
64 clones analysed (Table 1). According to the Good’s
C estimator for the midgut clone coverage [18,19], an
asymptotic curve above 0.90 was reached (Figure 2).
The majority (9/16) of the phylotypes belonged to the
Proteobacteria, and in particular to the γ- and ε- sub-
phyla. The dominant (25.0%) phylotype (pt21) was
closely related to Vibrio fischeri. The second most
abundant (17.2%) phylotype (pt9m) was related to
an epibiont of the hydrothermal vent polychaete
Paralvinella palmiformis. The rest of the phylotypes
belonged to the phyla of Spirochaetes, Bacteroidetes,
Firmicutes, Actinobacteria and Tenericutes, while one
phylotype was similar to a pine tree chloroplast. The
closest relatives of these phylotypes were either from
different marine habitats or were associated with other
invertebrates.
4. Discussion
4.1 Setal tuft
We were able to retrieve only nine clones from the setal
tuft of the decapod Pestarella tyrrhena. In the case
where a specialized community existed on the tuft,
the first clones analysed would reveal the dominant
phylotypes by revealing identical sequences. This is the
reason why the clone library coverage, based on Good’s
C estimator [18] was very low (data not shown) as all
the retrieved phylotypes were singletons. This does not
enable us to securely estimate the setal tuft’s bacterial
diversity. However, the inferred ecophysiology of the
retrieved phylotypes offers new valuable information on
the possible role of these bacteria.
The majority of the phylotypes belonged to one of
the most abundant phyla in the marine ecosystem, the
Bacteroidetes, however no firm evidence exists yet on
their symbiotic role in thalassinids. Cultivated members
of this phylum are affiliated with the gut of numerous
termite species [20,21]. Some of the phylotypes we
found were closely related to a newly isolated genus
of Flavobacteria with relatives belonging to the genera
Winogradskyella and Maribacter isolated from sea
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Table 1. Bacterial 16S rDNA phylotypes occurring on the setal tuft and in the emptied midgut of the decapod Pestarella tyrrhena.
Clone
# of similar
clones
(≥98%)
Putative
affiliation
Closest phylotype
(%)
[GenBank #]
Description
Closest organism
(%)
[GenBank #]
Setal tuft
pt1831
α-Proteobacteria
Clone pItb-vmat-24
(97)
[AB294940]
Maribacter polysiphoniae
(97)
[AM497875]
Psychroserpens sp. MEBiC05023
(96)
[EU581702]
Clone DOKDO 020
(94)
[DQ191181]
Gaetbulibacter sp. EM39
(97)
[EU443204]
Clone MD04G08
(93)
[FM874521]
Clone JL991
(99)
[DQ985050]
Clone LC1408B-77
(96)
[DQ270634]
Clone EC64
(94)
[DQ889917]
Shallow submarine
hydrothermal system
Roseobacter sp. SYOP1
(97)
[DQ659418]
pt1821Bacteroidetes
Isolated from a red
alga
pt1811 Bacteroidetes
Isolated from a
marine sponge
pt1771Bacteroidetes
Isolated from Dokdo
Island, East Sea of
Korea
Sea water on the
eastern coast in
Jeju, Korea
-
pt1761Bacteroidetes
pt1741ActinobacteriaHouse dust-
pt1731
α-Proteobacteria
Marine environments
Erythrobacter ishigakiensis
(98)
[AB363004]
pt1721Bacteroidetes
Lost City
hydrothermal Field
-
pt1711 Bacteroidetes
On the octocoral
Erythropodium
caribaeorum
-
Midgut
pt21 14
γ-Proteobacteria
Vibrio fischeri ES114
(99)
[CP000020]
pt9m11
ε-Proteobacteria
Clone P . palm C 43
(94)
[AJ441198]
From the
hydrothermal
vent polychaete
Paralvinella
palmiformis
Sulfurimonas autotrophica
(92)
[AB088432]
pt196Spirochaetes
Spirochaeta sp. B
(90)
[AY337320]
Sulfurimonas denitrificans
(94)
[CP000153]
Vibrio sp. V794
(98)
[DQ146993]
pt62m6
ε-Proteobacteria
pt50m5
γ-Proteobacteria
Aquatic animals
Vibrio probioticus
(98)
[AJ345063]
pt7m4 Tenericutes
Clone 42
(91)
[AJ515720]
From the deep-sea
Alvinocarid shrimp
Rimicaris exoculata
gut
On the caribbean
coral Montastraea
faveolata
-
pt224
γ-Proteobacteria
Clone SGUS483
(92)
[FJ202988]
Pinus thunbergii plastid
(99)
[D17510]
Clone WF16S_276
(83)
[EU939430]
Vibrio parahaemolyticus
(91)
[EU660363]
pt294 Chloroplast-
pt463 BacteroidetesWild pygmy loris-

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A. Demiri et al.
organisms such as the sponge Lissodendoryx isidictyalis
carter [22] and some red algae [23]. One Cytophagales
phylotype had a relative isolated from the products of the
octocoral Erythropodium caribaeorum while another was
closely related to the Psychroserpens genus. In addition,
a phylotype of the cluster had a close relative isolated
from the Lost City hydrothermal field [24], and this shows
the wide functional diversity of this group. None of the
phylotypes were related to other crustacean epibionts.
The ecophysiology of the Bacteroidetes phylotypes
found, renders them excellent potential epibionts as their
closest relatives have the ability to adhere on surfaces
[9] and they have been detected on external surfaces of
other marine animals. Additionally, the aerobic life mode
of these phylotypes suggests that they might originate
from the oxic burrow lining as the mudshrimp moves
along the burrow. Our dataset and existing ones [7,8]
for other decapods should be cautiously compared as
it is not realistic to directly compare either culture-based
or SEM (Scanning Electron Microscopy)-based studies
with 16S rRNA gene libraries when they do not originate
exactly from the same sample. Today, the 16S rRNA
approach is considered the best practice to reveal as
much as possible about the prokaryotic diversity of a
natural, complex natural sample. Culturing, on the other
hand is very selective and SEM provides information
on morphology, which has little taxonomical and
identification use for the majority of the prokaryotes.
Two phylotypes belonged to α-Proteobacteria,
specifically to aerobic sea anoxygenic photobacteria
[25]. Their closest relatives, Erythrobacter sp. and
Ruegeria sp. are associated to the dinoflagellates
Scrippsiella and Lingulodinium, and to Gymnodinium
catenatum respectively [26]. Most likely the presence of
these ubiquitous water column free-living phototrophs
[27] is a result of water transport from the water column
to the burrow via irrigation. Finally, phylotype pt174 is
a putative member of the Actinobacteria, an important
group in the marine environment, but it was distantly
related to any other phylotypes or cultured species.
4.2 Emptied midgut
The clone coverage in the midgut was satisfactory,
indicating that the majority of the midgut’s existing
bacterial diversity was revealed (Figure 2). Approximately
44% of the phylotypes found (pt21, pt50m, pt29, pt28,
pt45m, pt48, pt59) was identified to the species level.
The emptied midgut was dominated by members of the
γ- and ε-Proteobacteria.
The dominant phylotype (pt21, 21.9%) was identical
to Vibrio fischeri whereas one more phylotype (pt50m,
7.9%) was closely related to other vibrios found in aquatic
animals. Vibrio spp. are often considered as symbionts
in healthy shrimps’ digestive tract [28]. Representatives
of the genus take up dissolved organic matter (e.g.
polyunsaturated organic acids) which many sea
animals cannot compose de novo. In addition, they can
decompose chitin [29], rendering them significant to the
host’s nutrition. The ability of vibrios to metabolise chitin
in crustaceans is well documented [30]. The rest of the
γ-Proteobacteria phylotypes were related to the genera
Vibrio and Pseudomonas. Members of these two genera
Clone
# of similar
clones
(≥98%)
Putative
affiliation
Closest phylotype
(%)
[GenBank #]
Pseudomonas sp. CF14-10
(98)
[FJ170038]
Clone Oh3137A10B
(98)
[EU137440]
Pseudomonas sp. BWDY-9
(98)
[DQ200852]
Anoxybacillus flavithermus
(99)
[CP000922]
Sulfurimonas denitrificans
(88)
[CP000153]
Vibrio sp. CH-291
(82)
[AJ580582]
Spirochaeta sp. B
(90)
[AY337320]
Description
Closest organism
(%)
[GenBank #]
Pseudomonas stutzeri
(98)
[EU855213]
pt281
γ-Proteobacteria
deep-sea sediment
pt45m1ActinobacteriaFrom fleas
Propionibacterium acnes (98)
[AE017283]
pt481
γ-Proteobacteria
Estuary
Pseudomonas mendocina
(99)
[DQ178221]
pt591 Firmicutes
pt661
ε-Proteobacteria
pt1011
γ-Proteobacteria
Surface Baltic Sea
pt2361Spirochaetes
continuedTable 1. Bacterial 16S rDNA phylotypes occurring on the setal tuft and in the emptied midgut of the decapod Pestarella tyrrhena.
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Abdominal setae and midgut Bacteria
of the mudshrimp Pestarella tyrrhena
are the most abundant strains isolated from other
crustaceans and their role, based on enzymatic activity,
is connected to the degradation of proteins, lipids and
chitin [7,8,31].

and also phylotype pt62m (9.4%) belonged to the
ε-Proteobacteria and were possibly related to the closely
related genera Sulfurimonas and Thiomicrospira [32].
The second most dominant phylotype (pt9m, 17.2%)
Figure 1. Phylogenetic tree of relationships of bacterial 16S rDNA (ca. 1500 bp) of the representative clones found in the emptied midgut of the
decapod Pestarella tyrrhena, based on the neighbour-joining method as determined by distance Jukes-Cantor analysis. One thousand
bootstrap analyses (distance) were conducted, and percentages greater than 50% are indicated at the nodes. Groups (bold letters)
that have ≥98% similar nucleic acid sequences are represented by a single sequence, with the number of clones out of the total in
parentheses. The numbers in brackets are GenBank accession numbers. Scale bar represents 2% estimated distance.

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A. Demiri et al.
0.0
0.2
0.4
0.6
0.8
1.0
0102030
Library size
4050 6070
Good's C
Figure 2. Clone library coverage based on Good’s C estimator of the bacterial 16S rDNA clone library from the emptied midgut of the decapod
Pestarella tyrrhena.
Both of them are frequently observed in hydrothermal vent
environments [33]. In the present study, it is rather unsafe
to attribute the found phylotypes as true symbionts since
their closest relatives were found on external secretion of
a deep sea polychaete [34] and not in the midgut of the
mudshrimp. However, Thiomicrospira representatives are
likely symbionts since some of its members have been
found in the head-food region of Alviniconcha gastropods
along with Sulfurimonas spp. [35]; although the closest
relative available for phylotype pt66 was Sulfurimonas
denitrificans, we cannot speculate on its possible role as
a denitrifies due to their genetic distance. In general, the
three ε-Proteobacteria phylotypes we found represent
sulfur-oxidizers that can use CO2 as a carbon source.
In the possible case of symbiosis with the mudshrimp
they can supply energy to the animal by oxidizing sulfur
compounds originating from the decomposition of ingested
food particles or from the ingestion of anoxic sediment.
Two phylotypes (pt19, pt236) belonged distantly to
the genus Spirochaeta. This genus occurs in aquatic
environments, especially in sites with intense decay of
organic compounds [36] (e.g. parts of the mudshrimp’s
burrows with buried plant particles [16]). Additionally, they
have been found as symbionts in termite guts [37]. This
evidence renders them possibly metabolically active in
the gut as it is also a habitat where intense decomposition
occurs. In addition, members of this taxon are well known
symbionts with a key role in rumens’ nitrate metabolism
[41], whereas others are found in and on body parts
of different invertebrates as fermenters co-working
with ε-Proteobacteria [38,39]. In addition, with their
saccharolytic metabolism Spirochaeta-like bacteria could
assist the mudshrimp’s digestion of complex sugars or act
as energy suppliers by fermentative metabolism to other
co-existing bacteria, such as the three ε-Proteobacteria
phylotypes.
Phylotype pt7m possibly represents a new phylotype
in the Entomoplasmatales (Tenericutes phylum) as it was
distantly related only to an uncultivated clone originating
from the digestive tract of the deep-sea hydrothermal vent
shrimp Rimicaris exoculata. Although it is very difficult to
infer its ecophysiology, it is possible that it belongs to
the “R. exoculata’s gut microbial community” clade [4].
Entomoplasmatales are obligate parasitic heterotrophic
bacteria lacking a cell wall [36] and they access organic
food particles through their hosts.
Phylotype pt59 was practically identical to Anoxybacillus
flavithermus, first isolated from a whale skeleton in a deep-
sea trench [40]. It performs denitrification, hydrolyses
starch and consumes many sugars [40] but its symbiotic
nature has not been proven yet.
The actinobacterial phylotype pt45m was related
to Propionibacterium acnes. Its immediate closest
relative is related to the Oropsylla hirsuta flea [41].
Propionibacterium acnes-like phylotypes have been
found in the esophagus of the sea urchin Paracentrotus
lividus and in the stomach of the sea squirt Microcosmus
sp. [42], suggesting a possible association of this species
in marine invertebrates.
Phylotype pt46 belonged to the Bacteroidetes but
it seems to represent a new clade in this phylum as it
was not related to any known sequence or cultured
representative. Thus, it is possible symbiotic role remains
to be found. Finally, phylotype pt29 represents a plant
chloroplast of pine trees, which are very abundant around
the sampling area. Its occurrence on the mudshrimp’s
midgut is probably due to food remnants or transferred
sediment particles.

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of the mudshrimp Pestarella tyrrhena
References

were rather common dwellers of the water column or
epibionts of marine biota and did not indicate any specific
community composition. This suggests that Bacteria on
the setal tuft were possibly not involved in any specific
microbial processes. On the contrary, Bacteria in the
midgut constituted a structured community dominated by
phylotypes representing potential true symbionts, mostly
belonging to the γ- and ε-Proteobacteria. In addition, the
phylotypes that were not likely to be true symbionts (i.e.
transient bacteria via food or water) appeared in very low
abundances, indicative of their accidental occurrence.
In conclusion, the bacteria found on the setal tuft
Acknowledgments
Part of this work was supported by the Special Account
for Research, University of Athens.
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