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

Central European Journal of Biology

Abdominal setae and midgut Bacteria of the mudshrimp Pestarella tyrrhena

Research Article Alexandra Demiri1, Alexandra Meziti2, Sokratis Papaspyrou1#, Maria Thessalou-Legaki1, Konstantinos Ar. Kormas2*

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

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 (: 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. Keywords: Bacteria • Setal tuft • Midgut • 16S rRNA diversity • Thalassinidea • Decapoda • Pestarella tyrrhena © Versita Warsaw and Springer-Verlag Berlin Heidelberg.

1. Introduction Initially, symbiotic prokaryotes were examined through their relationships with the host, however Symbiosis between prokaryotes and eukaryotes nowadays the efforts are made toward independent are widespread throughout many ecosystems and examination, encompassing either culture-independent determines many aspects of the physiology, ecology or culturing approaches [1]. Since a culture based approach and evolution of the hosts [1]. Prokaryotes can dwell has significant limitations, a 16S rRNA gene analysis is in internal (e.g. digestive tract) or external (mouth the most widely used method to analyse the microbial parts, gills or special organs) body parts of a variety of diversity of a heterogeneous sample [6]. The majority invertebrates, and are involved in different metabolic of the studies on bacteria associated with , activities of their hosts either as symbionts or parasites have been focused either on the hindgut where symbiotic [2-4]. In particular, gut microbiota has been one of the bacteria are most likely to be found since there they most ancient and widespread types of symbiosis and can access food remnants without competing with their has offered evolutionary opportunities to the hosts host for nutrients absorption [4,7-9] or on epibionts based on nutritional alternatives [5]. [e.g. 10,11].

*E-mail: [email protected] 558 Abdominal setae and midgut Bacteria of the mudshrimp Pestarella tyrrhena

Pestarella tyrrhena is a deposit feeding thalassinid The same were used for the investigation shrimp that lives in complex burrows in marine sediments of the resident midgut bacterial community. Based on along the coast of the Mediterranean Sea and the Atlantic knowledge from previous studies [14, Thessalou-Legaki Ocean [12]. The mudshrimp feeds primarily on detritus & Papaspyrou, unpublished data], these animals were encountered during its burrowing activities [13]. It has left for 24 hours in individual aquaria kept in the dark (with also been observed that the mudshrimp accumulates a screen mesh near the bottom to avoid coprophagy, detritus, mainly seagrass detritus, in burrowing chambers which has been observed in this species [14]) in order where decomposition occurs. As this detritus is often of to empty their guts and avoid bacteria associated with poor quality, it has been suggested that the mudshrimp faecal pellets. The emptied midgut from each of the five uses this detritus to “garden” bacteria, with a higher C:N individuals was detached aseptically after incision under ratio. The mudshrimp later feeds on this detritus and any a microscope, midguts were pooled in order to have bacteria that have grown on it [13-16]. The mudshrimp adequate amounts of amplifiable prokaryotic DNA, and bears tufts of setal hair on its dorsal side. Although the stored at -80°C until DNA extraction. Although we did not exact functions of the setal tufts is unknown, they seem analyse individual specimens as is the common practice to play a role in the sense of touch when P. tyrrhena in fingerprinting techniques (e.g. T-RFLP, DGGE), our comes into contact with the burrow walls [14]. In order to clone library average (see below) allowed us to reveal investigate whether this tuft harbours a specific bacterial as many as possible of the occurring phylotypes, since community of a possibly specialized ecophysiological our intention was not to investigate individual differences process we investigated its 16S rRNA gene diversity. in gut microbial communities. Recently, one of the phylotypes of the current study was In this paper we used clone libraries of the bacterial found to occur in the setae of the deep-sea hydrothermal 16S rRNA genes. Our data are indicative of the vent decapod Kiwa hirsuta [15] suggesting a universal structure of the gut bacteria communities of the decapod specific association in marine decapods. Since the Pestarella tyrrhena and adds information to the existing feeding behaviour of the genus Pestarella suggests that literature. DNA was extracted using the Ultra Clean Soil mouth parts are only for mechanical process of food [14] DNA Isolation Kit (MO BIO Laboratories, Inc., USA) and thus it is not expected to have resident symbionts, according to the manufacturer’s instructions. From both we did not investigate the epibionts on the mouthparts. DNA samples c.a. 1500 bp of the 16S rRNA gene were In the same individuals, we also investigated the amplified by PCR using the bacterial primers BAC8f resident bacterial community in the midgut of P. tyrrhena (5’-AGAGTTTGATCCTGGCTCAG-3’) and BAC1492r using the same technique in order to infer benefits to (5’-CGGCTACCTTGTTACGACTT-3’) on a thermal the animals. We also aimed at investigating the bacterial cycler (MyCycler, Bio-Rad Inc., USA). After PCR community of the hindgut but no amplifiable DNA was optimisation, the conditions were 1 min pre-PCR hold at retrieved. Some of our retrieved sequences were also 94°C, followed by 30 or 27 cycles for the setal tuft and closely related to gut bacterial phylotypes of the deep- midgut samples, respectively, of denaturation at 94°C for sea hydrothermal vent shrimp Rimicaris exoculata (M-A 45 sec, annealing at 52.5°C for 45 sec and elongation Cambon-Bonavita, personal communication). Such at 72°C for 2min, and a final 7 min post-PCR elongation co-occurrence patterns are the first step for defining at 72°C. The PCR products were stained with ethidium potentially interacting organisms and interaction bromide and visualized on a 1.2% agarose gel under networks in highly complex systems, like the invertebrate UV light. – bacteria associations. Bands from both samples were excised and purified with the NucleoSpin®ExtractII kit (Macherey-Nagel, Germany) according to the manufacturer’s instructions. 2. Experimental Procedures Purified products were A-tailed in order to improve cloning efficiency. For the setal tuft sample A-tailing was Pestarella tyrrhena specimens were collected from performed by mixing 25 µl of purified PCR product, 11.9 µl

Vravrona Bay (eastern Attica, Greece) using a handheld purified PCR water, 3 µl of MgCl2 (25 mM), 5 µl of bait pump in September 2005 and the animals were deoxynucleoside triphosphate (2 mM), 5 µl of Buffer10x transported to the laboratory in less than 6 h. Immediately (200 M m Tris, pH=8.55) and 0.1 µl Taq polymerase. upon return to the laboratory, whole setal tufts –with no The midgut sample was A-tailed by mixing 7 µl of purified

cuticle included– from five mudshrimps were detached PCR product, 0.8 µl purified PCR water, 0.4 µl of MgCl2 from the third, fourth and fifth abdominal body part using (25 mM), 1 µl of deoxynucleoside triphosphate (2 mM), a sterilized scalpel, and frozen immediately at -80°C 0.6 µl of Buffer10x (200 mM Tris, pH=8.55) and 0.2 µl until further analysis. Taq polymerase. The samples were incubated for 10 min

559 A. Demiri et al.

at 72°C in the thermal cycler. For the cloning procedure a singleton Actionabacteria-related phylotype (pt174) material and protocols of the TOPO XL PCR Cloning Kit was also found. We did not calculate the C estimator (Invitrogen Co., USA) were used. The resulting clones for the setal tuft nor did we proceed to analysing were checked for the right insert (c.a. 1500 bp) by PCR more clones since all phylotypes were singletons, i.e. using the primers M13f (5’-GTAAAACGACGGCCAG-3’) suggesting that this clone library was consisted solely and M13r (5’-CAGGAAACAGCTATGAC-3’), that by unique phylotypes, pointing towards an unspecific consisted of a pre-PCR hold for 2 min at 94°C, then 30 bacterial community. 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 3.2 Emptied midgut post-PCR hold at 72°C. Clones were grown in liquid A total of 16 unique phylotypes were retrieved out of cultures and then the plasmids were purified using 64 clones analysed (Table 1). According to the Good’s NucleoSpin®Plasmid QuickPure (Macherey-Nagel, C estimator for the midgut clone coverage [18,19], an Germany) according to the manufacturer’s instructions. asymptotic curve above 0.90 was reached (Figure 2). Sequencing of the inserts in the purified plasmids The majority (9/16) of the phylotypes belonged to the was performed by Macrogen Inc. (Korea) using capillary Proteobacteria, and in particular to the γ- and ε- sub- electrophoresis and the BigDye Terminator kit (Applied phyla. The dominant (25.0%) phylotype (pt21) was Biosystems Inc., USA) with the primers M13f or M13r. closely related to Vibrio fischeri. The second most For each clone, forward and reverse sequences were abundant (17.2%) phylotype (pt9m) was related to assembled and afterwards checked for chimeras on an epibiont of the hydrothermal vent polychaete CHIMERA_CHECK function of the Ribosomal Database Paralvinella palmiformis. The rest of the phylotypes Project II [17]. The assembled sequences were belonged to the phyla of Spirochaetes, Bacteroidetes, compared to already known sequences in GenBank Firmicutes, Actinobacteria and Tenericutes, while one for identification of closest relatives through the BLAST phylotype was similar to a pine tree chloroplast. The function (http://www.ncbi.nlm.nih.gov/BLAST/). They closest relatives of these phylotypes were either from were aligned using the ARB FastAligner utility (www.arb- different marine habitats or were associated with other home.de), distances were identified through Neighbour- invertebrates. Joining method and checked with the Jukes Cantor. All analyses used minimum evolution and parsimony methods carried out in PAUP programme. The GenBank 4. Discussion accession numbers of the phylotypes found in this study are DQ890421-DQ890445. 4.1 Setal tuft Library clone coverage was calculated by the formula We were able to retrieve only nine clones from the setal [1 − (n /N)] [18], where n is the number of operational 1 1 tuft of the decapod Pestarella tyrrhena. In the case taxonomic units (OTU) represented by only one clone where a specialized community existed on the tuft, and N is the total number of the clones examined in the first clones analysed would reveal the dominant each library. 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 3. Results the retrieved phylotypes were singletons. This does not enable us to securely estimate the setal tuft’s bacterial 3.1 Setal tuft diversity. However, the inferred ecophysiology of the Nine unique phylotypes were retrieved from nine retrieved phylotypes offers new valuable information on clones, suggesting that the diversity of this sample was the possible role of these bacteria. inexhaustible. Six (pt171, pt172, pt 176, pt177, pt181, The majority of the phylotypes belonged to one of pt182) out of the nine unique phylotypes belonged to the most abundant phyla in the marine ecosystem, the the Bacteroidetes phylum (Table 1, Figure 1) and were Bacteroidetes, however no firm evidence exists yet on related (≥94% similarity) to other members of the phylum their symbiotic role in thalassinids. Cultivated members originating from a variety of marine habitats, including of this phylum are affiliated with the gut of numerous epibionts of an alga, a sponge and an octocoral. Two termite species [20,21]. Some of the phylotypes we clones belonged to the α-Proteobacteria and were found were closely related to a newly isolated genus related to a hydrothermal vent phylotype (pt183) and a of Flavobacteria with relatives belonging to the genera phototropic Erythrobacter-like phylotype (pt173). Finally, Winogradskyella and Maribacter isolated from sea

560 Abdominal setae and midgut Bacteria of the mudshrimp Pestarella tyrrhena

# of similar Closest phylotype Closest organism Putative Clone clones (%) Description (%) affiliation (≥98%) [GenBank #] [GenBank #]

Se t a l Tu f t

Clone pItb-vmat-24 Roseobacter sp. SYOP1 Shallow submarine pt183 1 α-Proteobacteria (97) (97) hydrothermal system [AB294940] [DQ659418] Maribacter polysiphoniae Isolated from a red pt182 1 Bacteroidetes (97) alga [AM497875] Psychroserpens sp. MEBiC05023 Isolated from a pt181 1 Bacteroidetes (96) marine sponge [EU581702] Clone DOKDO 020 Isolated from Dokdo pt177 1 Bacteroidetes (94) Island, East Sea of - [DQ191181] Korea Gaetbulibacter sp. EM39 Sea water on the pt176 1 Bacteroidetes (97) eastern coast in [EU443204] Jeju, Korea Clone MD04G08 pt174 1 Actinobacteria (93) House dust - [FM874521] Clone JL991 Erythrobacter ishigakiensis pt173 1 α-Proteobacteria (99) Marine environments (98) [DQ985050] [AB363004] Clone LC1408B-77 Lost City pt172 1 Bacteroidetes (96) - hydrothermal Field [DQ270634] Clone EC64 On the octocoral pt171 1 Bacteroidetes (94) Erythropodium - [DQ889917] caribaeorum

Mi d g u t Vibrio fischeri ES114 pt21 14 γ-Proteobacteria (99) [CP000020] From the Clone P. palm C 43 hydrothermal Sulfurimonas autotrophica pt9m 11 ε-Proteobacteria (94) vent polychaete (92) [AJ441198] Paralvinella [AB088432] palmiformis Spirochaeta sp. B pt19 6 Spirochaetes (90) [AY337320] Sulfurimonas denitrificans pt62m 6 ε-Proteobacteria (94) [CP000153] Vibrio sp. V794 Vibrio probioticus pt50m 5 γ-Proteobacteria (98) Aquatic animals (98) [DQ146993] [AJ345063] From the deep-sea Clone 42 Alvinocarid shrimp pt7m 4 Tenericutes (91) - Rimicaris exoculata [AJ515720] gut Clone SGUS483 On the caribbean Vibrio parahaemolyticus pt22 4 γ-Proteobacteria (92) coral Montastraea (91) [FJ202988] faveolata [EU660363] Pinus thunbergii plastid pt29 4 Chloroplast (99) - [D17510] Clone WF16S_276 pt46 3 Bacteroidetes (83) Wild pygmy loris - [EU939430]

Table 1. Bacterial 16S rDNA phylotypes occurring on the setal tuft and in the emptied midgut of the decapod Pestarella tyrrhena.

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# of similar Closest phylotype Closest organism Putative Clone clones (%) Description (%) affiliation (≥98%) [GenBank #] [GenBank #] Pseudomonas sp. CF14-10 Pseudomonas stutzeri pt28 1 γ-Proteobacteria (98) deep-sea sediment (98) [FJ170038] [EU855213] Clone Oh3137A10B Propionibacterium acnes (98) pt45m 1 Actinobacteria (98) From fleas [AE017283] [EU137440] Pseudomonas sp. BWDY-9 Pseudomonas mendocina pt48 1 γ-Proteobacteria (98) Estuary (99) [DQ200852] [DQ178221] Anoxybacillus flavithermus pt59 1 Firmicutes (99) [CP000922] Sulfurimonas denitrificans pt66 1 ε-Proteobacteria (88) [CP000153] Vibrio sp. CH-291 pt101 1 γ-Proteobacteria (82) Surface Baltic Sea [AJ580582] Spirochaeta sp. B pt236 1 Spirochaetes (90) [AY337320] continuedTable 1. Bacterial 16S rDNA phylotypes occurring on the setal tuft and in the emptied midgut of the decapod Pestarella tyrrhena. organisms such as the sponge Lissodendoryx isidictyalis Ruegeria sp. are associated to the dinoflagellates carter [22] and some red algae [23]. One Cytophagales Scrippsiella and Lingulodinium, and to Gymnodinium phylotype had a relative isolated from the products of the catenatum respectively [26]. Most likely the presence of octocoral Erythropodium caribaeorum while another was these ubiquitous water column free-living phototrophs closely related to the Psychroserpens genus. In addition, [27] is a result of water transport from the water column a phylotype of the cluster had a close relative isolated to the burrow via irrigation. Finally, phylotype pt174 is from the Lost City hydrothermal field [24], and this shows a putative member of the Actinobacteria, an important the wide functional diversity of this group. None of the group in the marine environment, but it was distantly phylotypes were related to other epibionts. related to any other phylotypes or cultured species. The ecophysiology of the Bacteroidetes phylotypes found, renders them excellent potential epibionts as their 4.2 Emptied midgut closest relatives have the ability to adhere on surfaces The clone coverage in the midgut was satisfactory, [9] and they have been detected on external surfaces of indicating that the majority of the midgut’s existing other marine animals. Additionally, the aerobic life mode bacterial diversity was revealed (Figure 2). Approximately of these phylotypes suggests that they might originate 44% of the phylotypes found (pt21, pt50m, pt29, pt28, from the oxic burrow lining as the mudshrimp moves pt45m, pt48, pt59) was identified to the species level. along the burrow. Our dataset and existing ones [7,8] The emptied midgut was dominated by members of the for other decapods should be cautiously compared as γ- and ε-Proteobacteria. it is not realistic to directly compare either culture-based The dominant phylotype (pt21, 21.9%) was identical or SEM (Scanning Electron Microscopy)-based studies to Vibrio fischeri whereas one more phylotype (pt50m, with 16S rRNA gene libraries when they do not originate 7.9%) was closely related to other vibrios found in aquatic exactly from the same sample. Today, the 16S rRNA animals. Vibrio spp. are often considered as symbionts approach is considered the best practice to reveal as in healthy shrimps’ digestive tract [28]. Representatives much as possible about the prokaryotic diversity of a of the genus take up dissolved organic matter (e.g. natural, complex natural sample. Culturing, on the other polyunsaturated organic acids) which many sea hand is very selective and SEM provides information animals cannot compose de novo. In addition, they can on morphology, which has little taxonomical and decompose chitin [29], rendering them significant to the identification use for the majority of the prokaryotes. host’s nutrition. The ability of vibrios to metabolise chitin Two phylotypes belonged to α-Proteobacteria, in crustaceans is well documented [30]. The rest of the specifically to aerobic sea anoxygenic photobacteria γ-Proteobacteria phylotypes were related to the genera [25]. Their closest relatives, Erythrobacter sp. and Vibrio and Pseudomonas. Members of these two genera

562 Abdominal setae and midgut Bacteria of the mudshrimp Pestarella tyrrhena

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.

are the most abundant strains isolated from other The second most dominant phylotype (pt9m, 17.2%) crustaceans and their role, based on enzymatic activity, and also phylotype pt62m (9.4%) belonged to the is connected to the degradation of proteins, lipids and ε-Proteobacteria and were possibly related to the closely chitin [7,8,31]. related genera Sulfurimonas and Thiomicrospira [32].

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1.0

0.8

0.6

Good's C 0.4

0.2

0.0 0 10 20 30 40 50 60 70 Library size

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

564 Abdominal setae and midgut Bacteria of the mudshrimp Pestarella tyrrhena

In conclusion, the bacteria found on the setal tuft Acknowledgments were rather common dwellers of the water column or epibionts of marine biota and did not indicate any specific Part of this work was supported by the Special Account community composition. This suggests that Bacteria on for Research, University of Athens. 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.

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