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59Russian Journal of Nematology, 2013, 21 (1), 1 - 12
Trophomera conchicola sp. n. (Nematoda: Benthimermithidae) from chemosymbiotic bivalves Idas modiolaeformis and Lucinoma kazani (Mollusca: Mytilidae and Lucinidae) in Eastern Mediterranean
1 2 3 2
Oleksandr Holovachov , Clara F. Rodrigues ' , Magali Zbinden
2
and Sébastien Duperron
'Department of Zoology, Swedish Museum of Natural History, Box 50007, SE-104 05 Stockholm, Sweden; e-mail: [email protected] 2Université Pierre et Marie Curie, UMR 7138 (UPMC CNRS IRD MNHN), Systématique, Adaptation, Evolution, 7,
quai St. Bernard, bâtiment A, 75005 Paris, France 3Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitârio de Santiago,
3810-193 Aveiro, Portugal
Accepted for publication 6 December 2012
Summary. A new species of Trophomera Rubtzov & Platonova, 1974 is described from the chemosymbiotic bivalves Idas modiolaeformis and Lucinoma kazani collected at Amon mud volcano in the eastern part of the Central Nile Province. Trophomera conchicola sp. n. is particularly characterised by 23.4 mm long body; finely annulated cuticle; 13-14 ^m long cephalic setae; small amphid located 54 ^m from anterior end; trophosome beginning 170 ^m from anterior end; straight vulva; short conoid tail 73 ^m long without terminal spike. This is the first record of the genus in bivalves, in hypoxic hydrogen-sulphide rich environment, and in the Mediterranean Sea.
Key words: Amon mud volcano, deep sea, molecular phylogeny, parasitic nematodes.
The genus Trophomera is a member of the family Benthimermithidae and is an uncommon parasite of marine invertebrates. Only 41 nominal species of the genus Trophomera are described to date (Miljutin & Miljutina, 2009; Miljutin, 2011; Tchesunov & Rozenberg, 2011), most of which are known on the basis of only few, and often just a single, adult individual. These adult specimens are most commonly found free-living in sediment samples. Juveniles are rarely found, and when they are discovered by themselves, without adult individuals in the same sample, they are impossible to assign to a certain species. Due to the fact that benthimermithids parasitise as juveniles, while adults are free-living, little is known about their hosts. So far, host were established only for three species of this genus. Trophomera australis (Petter, 1983) Miljutin, 2006 is known to parasitise free-living nematodes of the genus Deontosoma (Enoplida,
Leptosomatidae) (Tchesunov, 1988), T. bathycola (Rubtsov, 1980) Miljutin, 2006 was described from the body cavity of the priapulid Chaetostephanus cirratus (Rubtsov, 1980), and T. granovitchi Tchesunov & Rozenberg, 2011 was found in several different species of crustaceans, i.e. in Isopoda, Amphipoda and Ostracoda (Tchesunov & Rozenberg, 2011). Other records of benthimermithids from polychaetes, sipunculids, crustaceans and echinoderms (Hope, 1977; Petter, 1980, 1983; Tchesunov, 1997) are based on unidentifiable juvenile stages only.
Several adult and juvenile specimens of the genus Trophomera were found inside chemosymbiotic bivalves Idas modiolaeformis and Lucinoma kazani, which were collected in 2006 during the BIONIL cruise exploring cold seep sites north of the Nile Deep-Sea Fan in the eastern Mediterranean. They are described below as a new species Trophomera conchicola sp. n.
MATERIALS AND METHODS
Locality and habitat. Chemosymbiotic bivalves Idas modiolaeformis and Lucinoma kazani were collected at Amon mud volcano (N 32°22, E 31°42, 1153 m depth) located in the eastern part of the Central Nile Province during the BIONIL cruise (2006; chief scientist A. Boetius). Idas modiolaeformis is a small mytilid found attached to several substrates (e.g. authigenic carbonates, tubes of the siboglinid annelid Lamellibrachia sp., carbonate crusts) at cold seeps in the eastern Mediterranean (Olu-Le Roy et al., 2004; Lorion et al., 2012). It harbours six distinct bacterial symbionts in its gills that include five Gammaproteobacteria (one methane- and two sulphur-oxidizers, a methylotroph, and a bacterium of unknown metabolism) and a representative of the Bacteroidetes (Duperron et al., 2008). Lucinoma kazani is an infaunal clam bivalve that lives in the reduced sediment of several mud volcanoes from the Eastern Mediterranean. This bivalve associates with a sulphur-oxidizing bacterial symbiont (Salas & Woodside, 2002; Duperron et al., 2007). Bivalve specimens were originally preserved in ethanol (for molecular characterisation) and subsequently fixed in 4% formaldehyde (4°C, 2-4 h) and dehydrated in an ethanol series for fluorescence in situ hybridisation (FISH). Subsequently, nematode specimens were extracted from three Idas and two Lucinoma specimens and were used for molecular studies, studies by light microscopy (LM) and scanning electron microscopy (SEM).
Light microscopy (LM). For LM, specimens were transferred to pure glycerin using Seinhorst's (1959) rapid method as modified by De Grisse (1969) and mounted on permanent slides in glycerin with paraffin wax as support for the coverslip. All measurements given in the description are in ^m except for body length in mm. Abbreviations are as follows: a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; c', tail length/anal body diameter; V, distance from the anterior end of body to vulva/body length as %.
Semi-thin sections. One specimen was dehydrated in ethanol and propylene oxide series and then embedded in an epoxy resin (Serlabo). Semi-thin sections (600 nm) were cut using an ultramicrotome and were stained with toluidine blue for observations by LM (using an Olympus BX61 microscope).
Scanning electron microscopy (SEM). Samples for SEM were dehydrated through an ethanol series and dried in a critical-point dryer (CPD7501). They
were coated with gold (scan coat six sputter coater) and observed under a SEM (Cambridge S260 at 10 kV).
Molecular analysis. The DNEASY kit (Qiagen, Valencia, CA) was used to extract total DNA from three nematode specimens. About 900 bp of 18S were amplified using the primer combination MN18F and Nem_18S_R (Bhadury et al., 2006). PCR mixtures contained (total 20 ^l, molecular-grade water) 0.4 pmol ^l-1 primers, 1 ^l of DNA template, 1 x reaction buffer (Bioline), 1.5 mM MgCl2, 1.5 U BioTaq DNA polymerase (Bioline) and 0.25 mM each dNTP. Reactions were held at 95°C for 5 min, followed by 37 cycles of 95°C for 1 min, 54°C for 1 min and 72°C for 2 min, and finally one cycle of 2 min at 55°C with a final extension of 8 min at 72°C. PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN, Valencia CA) and sequenced biodirectionally at GATC Biotech (Germany). Only 802 and 831 bases of the 5' end of the 18S rDNA gene were sequenced successfully.
Phylogenetic analysis. The newly determined SSU rDNA sequences of T. conchicola n. sp. (GenBank accession numbers HE820056 and HE820057) were analysed against a background of selected sequence data available in public databases for the phylum Nematoda (Table 1). The secondary structure alignment from Holovachov et al. (2009) was modified by adding sequences from the public databases and newly generated ones and edited by eye with the JAVA-based secondary structure alignment editor 4SALE (Seibel et al, 2006) to maximise apparent homology and minimise gaps. The resulting alignment was analysed with Bayesian phylogenetic inference using the mcmcphase program of the PHASE package (Version 2.0; Gowri-Shankar & Jow, 2006). The alignment was partitioned using nucleotide substitution model REV (Tavare, 1986) for non-paired sites, and RNA7A (Higgs, 2000) for paired sites. For Bayesian inference, chains were allowed to burn in for 1 million generations, followed by 10 million generations during which tree topologies, branch length and model parameters were sampled every 200th generation. Two datasets were analysed, one dataset was 880 bases long with sequences trimmed to the size of T. conchicola sp. n. sequences, and another dataset was 1784 bases long (nearly complete 18S rDNA). Placement of Trophomera was identical in both analyses; however, the general support of the tree topology was lower for the trimmed dataset. Therefore, the discussion in this paper is based on the phylogenetic analysis of the nearly complete 18S rDNA dataset.
Table 1. List of nematode SSU ribosomal RNA gene sequences obtained from GenBank.
Order Species name Accession № Habitat
Araeolaimida Axonolaimus sp. EF591331 marine, brackish
Araeolaimida Cylindrolaimus communis AY593939 terrestrial
Araeolaimida Cylindrolaimus sp. FJ969121 terrestrial
Araeolaimida Diplopeltula sp. EF591329 marine
Araeolaimida Odontophora sp. FJ040459 marine
Araeolaimida Sabatieria pulchra EF591335 marine
Araeolaimida Sabatieria pulchra FJ040466 marine
Chromadorida Achromadora ruricola AY593941 freshwater
Chromadorida Choanolaimus psammophilus FJ040467 terrestrial
Chromadorida Chromadorita leuckarti FJ969119 freshwater
Chromadorida Ethmolaimus pratensis AY593942 freshwater
Chromadorida Ethmolaimus pratensis FJ040475 freshwater
Chromadorida Halichoanolaimus sp. EF591338 marine
Chromadorida Paracanthonchus caecus AF047888 marine
Chromadorida Paracyatholaimus intermedius FJ969133 freshwater
Chromadorida Ptycholaimellus sp. FJ040472 marine
Chromadorida Punctodora ratzeburgensis FJ969138 freshwater
Chromadorida Synonchiella sp. FJ040468 marine
Desmodorida Desmodora ovigera Y16913 marine
Desmodorida Draconema japonicum FJ182217 marine
Desmodorida Epsilonema sp. FJ182218 marine
Desmodorida Eubostrichus topiarius Y16917 marine
Desmodorida Laxus oneistus Y16919 marine
Desmodorida Metachromadora sp. EF591339 marine
Desmodorida Paradraconema jejuen.se FJ182220 marine
Desmodorida Stilbonema majum Y16922 marine
Desmoscolecida (?) Cyartonema elegans AY854203 marine
Desmoscolecida Paratricoma sp. FJ460255 marine
Desmoscolecida Tricoma sp. 1 FJ460256 marine
Desmoscolecida Tricoma sp. 2 FJ460257 marine
Dorylaimida Cryptonchus sp. FJ040479 freshwater
Dorylaimida Cryptonchus tristis EF207244 freshwater
Monhysterida Astomonema sp. DQ408759 marine
Monhysterida Daptonema procerum AF047889 marine
Monhysterida Desmolaimus sp. EF591333 marine
Monhysterida Diplolaimella dievengatensis AJ966482 marine
Monhysterida Diplolaimelloides meyli AF036611 marine
Monhysterida Eumonhystera filiformis AY593937 freshwater
Monhysterida Geomonhystera villosa EF591334 terrestrial, freshwater
Monhysterida Halomonhystera disjuncta AJ966485 marine
Monhysterida Monhystera riemanni AY593938 freshwater
Monhysterida Theristus acer AJ966505 marine
Table 1. List of nematode SSU ribosomal RNA gene sequences obtained from GenBank (continued).
Order Species name Accession № Habitat
Monhysterida Theristus agilis AY284695 marine
Plectida Anaplectus grandepapillatus AY284697 terrestrial, freshwater
Plectida Camacolaimus sp. EF591325 marine
Plectida (?) Ceramonema altogolfi JN815320 marine
Plectida (?) Ceramonema inguinispina JN815319 marine
Plectida (?) Ceramonema reticulatum FJ474095 marine
Plectida Chronogaster boettgeri AY593931 freshwater
Plectida Chronogaster typica FJ040456 freshwater
Plectida (?) Creagrocercus drawidae HM064453 parasite of terrestrial Oligochaeta
Plectida Domorganus macronephriticus FJ040454 symbiont of terrestrial Oligochaeta
Plectida Leptolaimus sp. EF591323 marine
Plectida P araplectonema pedunculatum EF591320 freshwater
Plectida Plectus acuminatus AF037628 terrestrial
Plectida Plectus aquatilis AF036602 terrestrial, freshwater
Plectida Procamacolaimus sp. EF591326 marine
Plectida Setostephanolaimus spartinae EF591321 marine
Rhabditida Acrobeloides sp. AF034391 terrestrial
Rhabditida Anguillicola crassus DQ118535 parasite of aquatic Vertebrata
Rhabditida Ascaris lumbricoides U94366 parasite of terrestrial Vertebrata
Rhabditida Brumptaemilius justini AF036589 parasite of terrestrial Diplopoda
Rhabditida Gnathostoma turgidum Z96948 parasite of terrestrial Vertebrata
Rhabditida Leidynema portentosae EF180073 parasite of terrestrial Arthropoda
Rhabditida Oxyuris equi EF180062 parasite of terrestrial Vertebrata
Rhabditida Rhigonema thysanophora EF180067 parasite of terrestrial Diplopoda
Rhabditida Teratocephalus lirellus AF036607 terrestrial
Rhabditida Truttaedacnitis truttae EF180063 parasite of freshwater Vertebrata
Triplonchida Tobrilus gracilis AJ966506 freshwater
DESCRIPTION
Trophomera conchicola sp. n. (Figs 1-3)
Female from Idas (Figs 1A, D). Body very long (L = 23.4 mm) and thin (a = 113), cylindrical over most of its length, tapering anteriorly in pharyngeal region and posteriorly on tail. Body 208 ^m wide at vulva. Cuticle thick, 7 ^m thick at midbody, with fine transverse striation most distinctly visible along the anterior end (Fig. 2B). External lateral alae (lateral field) absent. Somatic sensilla small papilliform, most prominent along the pharyngeal body region; arranged in four longitudinal rows, one dorsal, one ventral and one lateral on each body side (Figs 2B-C). Somatic sensilla are connected to large epidermal glands. Lip region rounded, continuous with body contour, lips absent/undeveloped. Inner
labial sensilla indistinct. Outer labial sensilla small, arranger in a circle close to the oral opening, their nerve endings are visible penetrating the head cuticle on the sides of the cheilostom. Cephalic sensilla setiform, 13-14 ^m long, arranged in a circle 14 ^m from anterior end (Fig. 2F). Subcephalic and cervical sensilla and ocelli absent. Amphidial fovea small transversely oval slit, 2 ^m wide, situated 54 ^m from anterior end. Nerve ring surrounding pharynx at about its posterior third. Secretory-excretory system not seen. Buccal cavity small cylindrical, 9 ^m long. Cheilostom undifferentiated, clearly demarcated by the cuticle. Pharyngostom undifferentiated and very short. Pharynx short (b = 138) and vestigial, 170 ^m long, subcylindrical anteriorly, gradually widening posteriorly; devoid of musculature; with weakly developed lumen visible only in its anterior part.
Fig. 1. Trophomera conchicola sp. n.: A: holotype female, anterior end (arrowhead points to amphid); B: pre-adult juvenile, anterior end (arrows point to nerve endings of cephalic sensillae, arrowhead points to amphid); C: early stage juvenile, anterior end, D: holotype female, posterior end. Scale bars: A-C = 20 ^m; D = 25 ^m.
Fig. 2. Trophomera conchicola sp. n. (SEM): A: anterior end; B: somatic sensilla; C-D: labial region; E: posterior end; F: cephalic setae. Scale bars: A, E = 50 цт; B-D = 10 цт; E = 1 цт.
Pharyngeal glands indistinct. Trophosome with distinct cell borders but with rudimentary internal lumen (Figs 3A and 3C). There is a distinct cuticular rectal tube 43 ^m long at the posterior end of the trophosome. Reproductive system didelphic, amphidelphic; not fully developed. Vulva located at about the mid body (V = 52.3 %). Vagina straight, equal to 1/4 of the vulval body diameter. Intrauterine eggs not seen. Tail 73 ^m long, conoid, very short (c = 321, c' = 0.56), still enveloped in the cuticle from the previous developmental stage. Caudal glands and spinneret absent.
Pre-adult parasitic juveniles from Idas (Fig. 1B). Body very long and thin (cannot be measured), cylindrical over most of its length, tapering anteriorly in pharyngeal region and posteriorly on tail. Body 205 ^m wide. Cuticle thinner than in adults, appears smooth under the light microscope. External lateral alae (lateral field) absent. Somatic sensilla and epidermal glands absent. Lip region rounded, continuous with body contour, lips absent/undeveloped. Inner and outer labial sensilla indistinct. Cephalic sensilla indistinct but their nerve endings are very distinct, extending along the sides of the pharynx towards the nerve ring. Subcephalic and cervical sensilla and ocelli absent. Amphidial fovea small porelike, situated 59-61 ^m from anterior end; amphidial nerves also very distinct, extending towards the nerve ring. Nerve ring surrounding pharynx at about its posterior fifth. Secretory-excretory system not seen. There are four sets of large glandular cells located just under cuticle of pharyngeal region: one ventral one dorsal and two (left and right) lateral. Their function is unclear but they appear to be connected to the pharyngeal region (stoma). Buccal cavity narrow, tubular, spear-like. Pharynx short and vestigial, 130155 ^m long, fusiform anteriorly, narrowing at the level of nerve ring and pyriform posteriorly; devoid of musculature; pharyngeal lumen indistinct. Dorsal pharyngeal gland strongly developed. Intestine with distinct internal lumen. Rectum 22 ^m long. Primordia of the anterior and posterior genital branches are developed, one of the juveniles with developing vagina. Tail 135 ^m long, conoid, with subcylindrical terminal part (c' = 1.3). Caudal glands poorly developed, spinneret present.
Early stage parasitic juvenile from Lucinoma (Fig. 1C). Body very long (L = 10 mm) and thin (a = 77), cylindrical over most of its length, tapering anteriorly in pharyngeal region and posteriorly on tail. Body 130 ^m wide. Cuticle thinner than in adults, 1.5 ^m thick at midbody, appears smooth under the light microscope. External lateral alae (lateral field) absent. Somatic sensilla and epidermal
glands absent. Lip region rounded, continuous with body contour, lips absent/undeveloped. Inner and outer labial sensilla indistinct. Cephalic sensilla and amphids indistinct. Subcephalic and cervical sensilla and ocelli absent. Nerve ring surrounding pharynx at about its posterior fifth. Secretory-excretory system not seen. Buccal cavity narrow, tubular, spear-like. Pharynx short (b = 94) and vestigial, 106 ^m long, fusiform anteriorly, narrowing at the level of nerve ring and pyriform posteriorly; devoid of musculature; pharyngeal lumen indistinct. Dorsal pharyngeal gland strongly developed. Intestine with distinct internal lumen. Rectum indistinct. Primordia of the anterior and posterior genital branches are obscure. Tail 136 ^m long, conoid, with subcylindrical terminal part (c = 74, c' = 1.5). Caudal glands poorly developed, spinneret present.
Diagnosis. Trophomera conchicola sp. n. is particularly characterised by 23.4 mm long body; finely annulated cuticle; 13-14 ^m long cephalic setae; small amphid located 54 ^m from anterior end; trophosome beginning 170 ^m from anterior end; straight vulva; short conoid tail 73 ^m long without terminal spike.
Relationships. Trophomera conchicola sp. n. is most similar to T. iturupiensis (Rubtzov & Platonova, 1974) Miljutin, 2006 in large body size (23.4 mm in T. conchicola vs 26-32 mm in T. iturupiensis); however, the new species differs from T. iturupiensis in having shorter cephalic setiform sensilla (13-14 ^m long vs 30-32 ^m in T. iturupiensis), narrower amphid (2 ^m wide vs 5 ^m wide in T. iturupiensis) and somatic sensilla arranged in four rows only (vs eight rows in T. iturupiensis). The new species should also be compared with other large Trophomera species with short conoid or hemispherical tail, such as T. granovitchi Tchesunov & Rozenberg, 2011 and T. longiovaris Miljutin, 2011. From T. granovitchi the new species differs in having somewhat longer body (23.4 mm vs 2.6-7.0 mm in T. granovitchi), longer cephalic setae (13-14 ^m long vs 4.5-5.5 ^m long in T. granovitchi), narrower amphid (2 ^m wide vs 6-8 ^m wide in T. granovitchi) and tail without acute spike (vs with spike in T. granovitchi). From T. longiovaris the new species differs in having somewhat longer body (23.4 mm vs 7.9-15.4 mm in T. longiovaris), longer cephalic setae (13-14 ^m long vs 7 ^m long in T. longiovaris) and shorter tail.
Molecular phylogenetic analysis. The first phylogenetic analyses of Benthimermithidae were based on the nearly complete 18S rDNA sequence of T. granovitchi (Tchesunov et al., 2009) and partial 28S rDNA sequence of Trophomera sp. (Mardashova et al., 2011) and support a close relationship with
Fig. 3. Semi-thin sections of Trophomera conchicola sp. n. (A-B) and dissected Lucinoma kazani showing cavities (arrows) from which nematodes were extracted (D): A: Mid-sagittal section showing vestigial pharynx (arrow) and rudimentary lumen of the trophosome (arrowhead); B: Para-sagittal plane showing longitudinal muscules (arrowhead) and epidermal glands (arrows); C: Transverse section at the level of trophosome showing epidermal gland (arrow) and rudimentary lumen of the trophosome (arrowhead). Not to scale.
Plectida, but the complete data were not yet published. Our phylogenetic analysis supports the idea of close relationships between Plectida and Trophomera; however, the length of the Trophomera sequences available to us is not sufficient (Fig. 4). It is not impossible that the use of full-length 18S rRNA sequence of Trophomera in future phylogenetic analysis will alter the current topology of the tree. Therefore, we consider our results preliminary and recommend that they are treated with caution.
Type material. Holotype female (slide № 8411) deposited in the invertebrate type collection of the Department of Zoology, Swedish Museum of Natural History, Stockholm, Sweden. Holotype female was sectioned.
Additional material. Four parasitic juveniles deposited in the general invertebrate collection of the Department of Zoology, Swedish Museum of Natural History, Stockholm, Sweden. Three of the juveniles were collected in Idas. They are not complete: two individuals have their posterior ends missing, and one female is without the anterior end. A single complete juvenile specimen was found in Lucinoma.
Type locality and habitat. Idas modiolaeformis and Lucinoma kazani, Amon mud volcano (N 32°22', E 31°42'), eastern Mediterranean Sea. Specimens that were extracted during the cruise occurred in the pallial cavity of Idas and were alive and active after the dissection. The specimen from Lucinoma was located between the mantle tissue and the shell, and was partly trapped into a cavity inside the shell (Fig. 3D).
Etymology. Species name is derived from a combination of Latin words "concha" (mollusc) and "colo" (inhabit) and means "one who lives in the mollusc".
DISCUSSION
Types of association between marine nematodes and marine molluscs are insufficiently studied. The majority of known cases include the symbiotic or parasitic relationship between nematodes and marine bivalves, with only one case of commensalism between nematodes and deep-sea gastropod (Holovachov et al., 2011). Moreover, most of the nematode species found in association with molluscs belong to the taxa with mostly free-living representatives, who either seek shelter in the host shell or feed on its dead tissues, and the effects of nematodes on their host remain unstudied. This is the first report of a species of the highly specialised parasitic genus Trophomera associated with bivalve
molluscs. Other members of this group are known to infest marine nematodes, priapulids, polychaetes, sipunculids, crustaceans and echinoderms (Hope, 1977; Petter, 1980, 1983; Rubtsov, 1980; Tchesunov, 1988, 1997; Tchesunov & Rozenberg, 2011). Further investigation might thus extend the range of metazoan groups parasitised by Trophomera species.
All T. conchicola specimens were collected from a single site, the Amon mud volcano, during a single cruise in 2006. Other cruises exploring this and similar sites in the eastern Mediterranean took place in 2003, 2007 and 2009, and no additional specimens of Trophomera were ever collected. This suggests that the nematodes are rare, and possibly that localised outbreaks of infestation can occur during certain periods. Whether infestation is detrimental to the hosts is not clear, as no tissue lesion was visible. However, given the small size of Idas modiolaeformis (around 1 cm shell length, infested or not), and the rather large size of Trophomera, one can expect that T. conchicola extracts a significant fraction of resources from the hosts, yet it remains to be investigated how the host is affected (for example, growth, health, reproduction etc.).
The mussel I. modiolaeformis lives attached to carbonate crusts at cold seeps in the eastern Mediterranean while L. kazani lives buried in the sediment (Duperron et al., 2007, 2008). In both cases, the environment is hypoxic, and reduced compounds such as methane and hydrogen sulphide are more enriched compared to normal seawater. This indicates that T. conchicola can withstand relatively low levels of oxygen, and high levels of the toxic compound hydrogen sulphide, similarly to for example the polychaete Branchipolynoe seepensis, which lives inside the pallial cavity of mussels at many seeps and vents, and probably experiences similar conditions. It is not clear whether the relationship between polychaete and mussels is commensal or parasitic (Jollivet et al., 1998). As far as it is known, this is the first record of Trophomera from this type of habitat.
Both host bivalve species harbour chemosynthetic bacteria in their gill tissue, which ensure a significant part of their nutrition (Duperron et al., 2007, 2008). They benefit from bacterial primary production, which favour higher growth rates and biomasses than expected for other organisms at similar depths. Because of this, they appear as excellent niches for parasites. The fact that the Trophomera specimen found in L. kazani was partly trapped into a cavity in the thickness of the shell is intriguing (Fig. 3D), since other species of this
Fig. 4. Majority-rule consensus tree of the Bayesian phylogenetic analysis of Chromadoria including Trophomera conchicola sp. n. rooted using Tobrilus gracilis. Branch length represent the mean posterior estimates of the expected number of substitutions per site. Animal parasitic taxa are shaded.
genus were found inside the body cavity of their hosts. It could result from the specimen digging inside the shell, which would require that specific substances (such as acids) are produced, but most probably it is a consequence of the shell carbonate precipitating around the worm as a possible defence response of the host.
Like certain free-living gutless marine nematodes of the genus Astomonema (Ott et al., 1982), Trophomera does not have a well developed pharynx for most of its life. However, unlike free-living Astomonema that thrives with the help of bacterial symbionts (Giere et al., 1995; Tchesunov et al., 2012), it is generally assumed that parasitic stages of Trophomera feed by absorbing nutrients through cuticle (osmotrophy), while free-living adults do not feed at all and rely on the reserves accumulated in the trophosome (Tchesunov, 1997). This model is further supported by the fact that we failed to amplify any bacterial marker genes from the tissues of the nematode (16S rRNA) despite several attempts, and we did not observe bacterial cells in the nematode sections (Fig. 3C).
ACKNOWLEDGEMENT
The authors would like to thank Dr V.V. Aleoshin for sharing with us his unpublished sequences of T. granovitchi and valuable comments about the molecular systematics of the genus Trophomera. We are also grateful to Prof. A.V. Tchesunov for his constructive criticism of the manuscript. Thanks are due to the pilots, crew and scientific party on cruise BIONIL 2006 (chief scientist A. Boetius), ROV Meteor, ROV Quest. SEM was performed at the 'Service de Microscopie Electronique' IFR 83 (UPMC). This research was supported by the HERMES project EC contract GOCE-CT-2005-511234, funded by the European Commission Sixth Framework Programme under the priority 'Sustainable Development, Global Change and Ecosystems' and the EU French CNRS. CFR was also supported by a post-doctoral fellowship (SFRH/BPD/64154/2009) from the Fundaçâo para a Ciência e a Tecnologia (FCT, Portugal).
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O. Holovachov, C.F. Rodrigues, M. Zbinden, S. Duperron. Trophomera conchicola sp. n. (Nematoda: Benthimermithidae) из хемосимбиотических двустворчатых моллюсков Idas modiolaeformis и Lucinoma kazani (Mollusca: Mytilidae и Lucinidae) из восточной части Средиземного моря. Резюме. Новый вид рода Trophomera Rubtzov & Platonova, 1974 описан из хемосимбиотических двустворчатых моллюсков Idas modiolaeformis и Lucinoma kazani, собранных на грязевом вулкане Амон в восточной части Центральной Нильской провинции Средиземного моря. Trophomera conchicola sp. n. характеризуется длиной тела, равной 23.4 мм, тонкой кольцевой исчерченностью кутикулы, головными щетинками длиной 13-14 мкм, небольшими амфидами, расположенными в 54 мкм сзади от головного конца, трофосомой, начинающейся в 170 мкм от переднего конца, прямой вульвой и коротким коническим хвостом длиной 73 мкм без терминального шипика. Это первое сообщение о паразитировании нематод этого рода в двустворчатых моллюсках, в среде с недостатком кислорода и избытком H2S, и из Средиземного моря.
