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. IntroductionSymbiosis 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].

Initially, symbiotic prokaryotes were examined 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].

Cent. Eur. J. Biol. • 4(4) • 2009 • 558–566DOI: 10.2478/s11535-009-0053-x

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Pestarella tyrrhena is a deposit feeding thalassinid 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.

2. Experimental ProceduresPestarella 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.

The same animals were used for the investigation 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

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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’), that 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 (Macherey-Nagel, 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.

3. Results

3.1 Setal tuftNine 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 midgutA 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 tuftWe 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%)

Putativeaffiliation

Closest phylotype(%)

[GenBank #]Description

Closest organism(%)

[GenBank #]

Setal tuft

pt183 1 α-ProteobacteriaClone pItb-vmat-24

(97)[AB294940]

Shallow submarine hydrothermal system

Roseobacter sp. SYOP1(97)

[DQ659418]

pt182 1 BacteroidetesMaribacter polysiphoniae

(97)[AM497875]

Isolated from a red alga

pt181 1 BacteroidetesPsychroserpens sp. MEBiC05023

(96)[EU581702]

Isolated from a marine sponge

pt177 1 BacteroidetesClone DOKDO 020

(94)[DQ191181]

Isolated from Dokdo Island, East Sea of Korea

-

pt176 1 BacteroidetesGaetbulibacter sp. EM39

(97)[EU443204]

Sea water on the eastern coast in Jeju, Korea

pt174 1 ActinobacteriaClone MD04G08

(93)[FM874521]

House dust -

pt173 1 α-ProteobacteriaClone JL991

(99)[DQ985050]

Marine environmentsErythrobacter ishigakiensis

(98)[AB363004]

pt172 1 BacteroidetesClone LC1408B-77

(96)[DQ270634]

Lost City hydrothermal Field -

pt171 1 BacteroidetesClone EC64

(94)[DQ889917]

On the octocoral Erythropodium caribaeorum

-

Midgut

pt21 14 γ-ProteobacteriaVibrio fischeri ES114

(99)[CP000020]

pt9m 11 ε-ProteobacteriaClone P. palm C 43

(94)[AJ441198]

From the hydrothermal vent polychaete Paralvinella palmiformis

Sulfurimonas autotrophica(92)

[AB088432]

pt19 6 SpirochaetesSpirochaeta sp. B

(90)[AY337320]

pt62m 6 ε-ProteobacteriaSulfurimonas denitrificans

(94)[CP000153]

pt50m 5 γ-ProteobacteriaVibrio sp. V794

(98)[DQ146993]

Aquatic animalsVibrio probioticus

(98)[AJ345063]

pt7m 4 TenericutesClone 42

(91)[AJ515720]

From the deep-sea Alvinocarid shrimp Rimicaris exoculata gut

-

pt22 4 γ-ProteobacteriaClone SGUS483

(92)[FJ202988]

On the caribbean coral Montastraea faveolata

Vibrio parahaemolyticus(91)

[EU660363]

pt29 4 ChloroplastPinus thunbergii plastid

(99)[D17510]

-

pt46 3 BacteroidetesClone WF16S_276

(83)[EU939430]

Wild pygmy loris -

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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 midgutThe 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%)

Putativeaffiliation

Closest phylotype(%)

[GenBank #]Description

Closest organism(%)

[GenBank #]

pt28 1 γ-ProteobacteriaPseudomonas sp. CF14-10

(98)[FJ170038]

deep-sea sedimentPseudomonas stutzeri

(98)[EU855213]

pt45m 1 ActinobacteriaClone Oh3137A10B

(98)[EU137440]

From fleas Propionibacterium acnes (98)[AE017283]

pt48 1 γ-ProteobacteriaPseudomonas sp. BWDY-9

(98)[DQ200852]

EstuaryPseudomonas mendocina

(99)[DQ178221]

pt59 1 FirmicutesAnoxybacillus flavithermus

(99)[CP000922]

pt66 1 ε-ProteobacteriaSulfurimonas denitrificans

(88)[CP000153]

pt101 1 γ-ProteobacteriaVibrio sp. CH-291

(82)[AJ580582]

Surface Baltic Sea

pt236 1 SpirochaetesSpirochaeta sp. B

(90)[AY337320]

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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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].

The second most dominant phylotype (pt9m, 17.2%) and also phylotype pt62m (9.4%) belonged to the ε-Proteobacteria and were possibly related to the closely related genera Sulfurimonas and Thiomicrospira [32].

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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0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50 60 70Library size

Goo

d'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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References

In conclusion, the bacteria found on the setal tuft 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.

AcknowledgmentsPart of this work was supported by the Special Account for Research, University of Athens.

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