A new species of Protodrilus (Annelida, Protodrilidae), covering bone surfaces bright red, in whale-fall ecosystems in the northwest Pacific.
Since the first discovery of a whale-fall community in 1987 in the Santa Catalina Basin (Smith et ai, 1989), many whale-fall communities around sunken whale carcasses have been reported (Fujioka et ai, 1993; Wada, 1993; Naganuma et al, 1996; Smith and Baco, 2003; Glover et al, 2005; Braby et ai, 2007; Fujiwara et al, 2007). Whale-fall communities are characterized as chemosynthesis-based assemblages, similar to hydrothermal vent and hydrocarbon seep communities. The energy source initially derives from large concentrations of organic materials in the whale car-casses, which decompose, producing substantial amounts of sulfide. Many species belonging to a variety of taxa, such as Anthosactis (Cnidaria; Daly and Gusmao, 2007), Osedax (Annelida; Rouse et al, 2004; Fujikura et al, 2006), and Asymmetron (Chordata; Nishikawa, 2004), have been described as new to science from these unique environments.
A mass stranding of Physeter macrocephalus Linnaeus, 1758, occurred on the southwestern coast of Kyushu Island, southern Japan, in 2002. The sperm whale bodies were submerged in the waters off Cape Nomamisaki, Kagoshima, Kyushu, at depths of between 200 and 260 m (31-32 N[degrees], 129-130 E[degrees]), where continual observations were conducted between 2003 and 2012 (Fujiwara et al, 2007; Kinoshita et al, 2010). Over our successive ecological studies of the faunal composition and the community succession associated with the whale carcasses, we found numerous annelid worms of a single morphotype that covered the exposed whale-bone surfaces. These surfaces appeared bright red due to the coloration of the worm bodies, 2.5 y after the carcasses had been deposited. These red worms are a new species belonging to the genus Protodrilus (Annelida, Pro-todrilidae), and are described in this paper. This dominant new species has few morphological characteristics; its description is based on body size, coloration, ciliary distribution, male lateral organs, fertile region, and posterior lobes.
Protodrilus Hatschek, 1880, and Astomus Jouin, 1979, are two genera belonging to the family Protodrilidae Czerniavsky (1881) (Hatschek, 1878, 1888, 1893). Protodrilidae has been grouped with Protodriloidae and Saccocirridae in the clade Protodrilida (Purschke and Jouin, 1988; Purschke, 1990, 1993), but has never been confirmed by molecular data (Struck, 2011). Thus, in spite of the monophyly of Protodrilida, the position of the three families in Annelida is still unresolved. Until today, 37 species have been described in the family Protodrilidae. Species of the genus Protodrilus are small, slender annelid worms, and are known only to be interstitial inhabitants of shallow waters (Remane, 1926, 1932; von Nordheim, 1983, 1989; Domenico et ai, 2013; Martinez et al, 2013). From the lack of any hard structures and accessory morphological features such as chaetae and jaws, there is a systematic difficulty not only in the genus Protodrilus but also in the family Protodrilidae and associated families (Westheide, 1985; Fauchald and Rouse, 1997; Rouse and Fauchald, 1997). Therefore, information on this small-sized annelid group remains incomplete. This study provides new information about the diversity of the Pro-todrilus group, which, until now, has been known only to distribute in shallow, interstitial environments.
Materials and Methods
Twelve carcasses of the sperm whale Physeter macrocephalus Linnaeus, 1758, were deposited in the waters off Cape Namomisaki, Kyushu Island, at depths of between 219-254 m in February 2002 (31[degrees]18.515' N-31[degrees]23.865' N, 129[degrees]58.766' E-129[degrees]59.520' E). The whales were 12-16 m in total length and weighed an estimated 21-39 tons (Fujiwara et ai, 2007). Dives of the ROV Hyper-Dolphin, belonging to the Japan Agency for Marine-Earth Science and Technology (JAMSTEC), were conducted from July to August 2004, or 2.5 y after the carcasses were deposited. The seafloor sediment was sandy, and the water temperature was 12 [degrees]C. In situ observations were made using a digital, high-definition TV camera mounted on the ROV.
Whale bones were collected using the ROV manipulators. The bones were kept on board ship in aquaria with filtered seawater at 12 [degrees]C, and Protodrilus annelids were sampled from the surfaces of the vertebrae. With a stereo-microscope (MZ95; Leica, Tokyo, Japan), observations were made of the morphological characteristics, state of sexual maturity, presence and condition of oocytes, and behavioral characteristics of the live specimens. Worms were fixed with 10% neutral formalin in seawater, and observations were conducted using both the stereo-microscope MZ95 and a scanning electron microscope (SEM S-4200; Hitachi, Hitachi, Japan). Samples for the scanning electron microscope were placed in 70% ethanol after fixation, then dried and coated with Pt-Pd (electrically conducting metal, preventing charging of the specimen during scanning electron microscopy) using standard procedures.
Deep-frozen specimens were used for DNA sequencing. DNA was extracted from worm whole bodies. To eliminate surface contaminants, each specimen was thoroughly washed in autoclaved and filtered seawater. DNA extraction was conducted separately using the DNeasy Tissue Kit (Qiagen Japan, Tokyo, Japan).
The eukaryotic 18S ribosomal RNA (rRNA) gene was amplified by polymerase chain reaction (PCR) using the Ex Taq PCR Kit (Takara, Kyoto, Japan). Two oligonucleotide primers (1 ([micro]mol-1 each) and < 1 ([micro]g of DNA template were added to the reaction mixtures. Thermal cycling was as follows: denaturing at 96 [degrees]C for 20 s, annealing at 55 [degrees]C for 45 s, and extending at 72 [degrees]C for 2 min, for a total of 35 cycles. The oligonucleotide primer sequences used for this amplification were IN and 2N (Grzebyk et al., 1998). The molecular size of the PCR products was checked with 1.2% Agarose S (Nippon Gene, Toyama, Japan) gel electrophoresis.
DNA sequencing of the amplified 18S rRNA genes was performed using the BigDye Terminator Cycling Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA). Six universal 18S rRNA gene-specific primers were used in sequencing reactions. Sequencing was performed using an ABI PRISM 3100 genetic analyzer (Applied Biosystems). The sequences reported here have been deposited in the DNA Data Bank of Japan (DDBJ) under accession number LC004911.
A nearly complete sequence of the 18S rRNA gene ( 1671 bp) was analyzed using the gapped-BLAST search algorithm (Altschul et ai, 1997; Benson et al, 2000) to estimate the degree of similarity to other 18S rRNA gene sequences. The database used for similarity analyses was the non-redundant nucleotide sequence database from GenBank. All reported 18S rRNA gene sequences from the taxonomic group "Canalipalpata incertae sedis" were manually aligned, and phylogenetic analysis was restricted to nucleotide positions that were unambiguously alignable in all sequences. The alignments of 18S rRNA genes were tested for optimal fit of various nucleotide substitution models using MEGA 5.2.2 software (Molecular Evolutionary Genetics Analysis; Tamura et al., 2011 ). The base frequencies, proportion of invariable sites and a gamma distribution were estimated from the dataset. The TN model (Tamura and Nei, 1993), incorporating variable sites (TN + I), was selected by MEGA. Maximum likelihood (ML) analysis of this dataset with MEGA was performed using an input tree generated by the BIONJ algorithm (Gascuel, 1997) with the models. Bootstrap trees (1000 replicates) were constructed using the same parameters as the ML tree.
The typing materials were deposited in the National Museum of Nature and Science, Tokyo (NSMT) and JAMSTEC.
Description of the species
Family Protodrilidae Czerniavsky, 1881
Genus Protodrilus Hatschek, 1880
Protodrilus puniceus sp. nov.
(Hiiro-mukashigokai in Japanese) (Figs. 1-4)
A medium-to-large sized Protodrilus, usually longer than 10 mm, bright red to pink in color when alive. Eyes absent, no nuchal organs, no so-called statocysts dorso-anteriorly; salivary glands inconspicuous; thick, double-circular band of cilia around mouth, which continues as a ventral ciliary band to the last segment, male paired, separated lateral organs in segments 6-11. Pygidium with two developed lobes with cilia located on the dorso-central part of the anus.
Description of holotype
Slender, filiform body, 18 mm long and 0.4 mm wide while alive, and 10 mm long and 0.25 mm wide after formalin fixation; with 87 segments. A pair of transparent palps is located terminally and nearly ventrally on the prostomium about 0.5 mm long while alive and 0.3 mm long after formalin fixation. Prostomium rounded. Eyes absent; no nuchal organs; no so-called statocysts in the dorso-anterior part of the prostomium. No transversal ciliary rings on the peristomium. Color in life, bright red to pink; tan after formalin fixation. Male.
Ciliation of the body is developed on the ventral side; weakly developed on the dorsal side. Ciliary rings are absent on the body segments. Not numerous, but short, stiff sensory cilia can be found all over the body, usually arranged in small tufts. Mouth and pharyngeal bulb opaque, mouth opens on the ventral side, intestine follows. Salivary glands inconspicuous, connected to gut. The mouth located on the anterior ventral side is surrounded by a thick, double band of cilia and continues to the ventral ciliary band that runs from the first body segment to the last. A row of ciliation can be observed located ventrally to each palp. Some tufts of cilia are found on each palp.
Neither chaeta nor parapodium is present in any segment, although clear dissepiments can be recognized. Paired, separated lateral organs exist from segments 6-11; ciliated inside. No other special characteristics are present throughout the elongated body.
A pygidium with two well-developed adhesive lobes. Cilia are located on the dorso-central part of the anus.
Paratypes and variation: Up to 20 mm long and 0.4 mm wide when alive; with 90 segments. Most specimens are longer than 10 mm. A pair of transparent palps, up to approximately 0.5 mm in length, is located terminally and nearly ventrally on the prostomium (Fig. 1 A, B; Fig. 2A-C). Body dimensions, including width of the base of the anterior head, width of the fertile region, and length of palps, were, for females with 30 segments, as follows: anterior head, 175 pm; fertile region, 200 ([micro]m; and palps, 300 ([micro]m. For males with 72 segments, measurements were anterior head, 275 pm; fertile region, 325 ([micro]m; and palps, 500 ([micro]m. For females with 73 segments, the anterior head was 250 ([micro]m; the fertile region was 375 ([micro]m; and palps were 500 ([micro]m. The live animal is bright red to pink (Fig. 1 A, B; Fig. 4A-F, H); after formalin fixation, the color was whitish tan.
Sensory organs are absent (Fig. 1C, D; Fig. 2A-C). Instead, short, stiff sensory cilia and ciliary tufts can be found all over the body. One row of numerous cilia is observed along the ventral side of each palp (Fig. 2B-D). Mouth and pharyngeal bulb are opaque, mouth opens on the ventral side, followed by dark brown intestine (Fig. IB). Salivary glands inconspicuous. Thick, double-circular band of cilia around the mouth (Fig. 2D, E), and the ventral ciliary band continues transversely until the end of the segments (Fig. 2B, D, E, G, H). Cilia in ventral band, palp, and pygidium are 8-10 ([micro]m long. Clear dissepiments can be recognized (Fig. 1A; Fig. 2F, G; Fig. 3A), although some dissepiments in anterior segments 1-4 are inconspicuous (Fig. IF). Paired, separated lateral organs in males exist from segment 6 to 11 (Fig. 3A-C). Many cilia are present in each groove (Fig. 3B, C). No other special characteristics are apparent on the elongated body.
A pygidium with two well-developed adhesive lobes and numerous cilia are located on the dorso-central part of the anus (Fig. IG; Fig. 2H, I).
Habitat and ecology
Only known from the external surface and internal area of sperm whale bones. A dense cluster of Protodrilus puniceus sp. nov. was distributed in patches in a maxilla and a vertebra of a sperm whale at a depth of about 220 m off Cape Nomamisaki (Fig. 4A, B). The worms were located in innumerable small pores in bones (Fig. 4C-E). Some were completely buried in bones, while others were found with part of their bodies emerging from pores, such as the anterior end, pygidium, or middle part of the body (Fig. 4D, E).
The siboglinid polychaete Osedax japonicus and the bathymodiolid mussel Adipicola pacifica (Okutani et ai, 2003), both endemic to the bone surfaces, were also found on the same blocks of bones with P. puniceus sp. nov. However, the three species did not coexist, and habitat segregation was observed in the maxilla (Fig. 4A). Spatial partitioning was seen between P. puniceus sp. nov. and A. pacifica on the surface of both the vertebra and the epiphysis (Fig. 4B). On the other hand, P. puniceus was frequently observed to aggregate with A. pacifica on a vertebra (Fig. 4F). Many worms were seen elongating their bodies on the surface of an ulna in an aquarium. A pair of transparent palps of an emerged worm was observed to wave or attach to the surface of the ulna (Fig. 4G). Small particles were seen being conveyed toward the mouth by the ventral ciliary band, and water appeared to be moved toward the anterior part of the body. The worms sometimes left the bones by themselves, aggregated with each other immediately, and formed a mass in aquaria (Fig. 4H). They were adhesive, with self-secreted mucous, and appeared to attach their ventral sides to each other.
High regenerative ability was observed in living worms when cut in pieces in laboratory.
Reproduction: Females contained oval oocytes in the coelom beginning from segment 6 in August (Fig. IE, F). Oocyte size was about 45 ([micro]m in the long axis and 35([micro]m in the short axis (Fig. 2H). One segment contained about 6 to 8 oocytes, and up to 18 oocytes were observed in a large female. Free sperm (approximately 130-140 ([micro]m) with elongated heads were observed externally under a microscope (Fig. 1H).
Distribution: Only collected from sperm whale bones deposited at depths of between 200 and 260 m off Cape Nomamisaki, Kyushu, Japan.
Although almost all of the species belonging to the genus Protodrilus are reported to be transparent, whitish-opaque, or yellowish in body color when alive, the new species described here is a bright, conspicuous red to pink throughout the body. Some species show a pink, reddish, or red color, but only partially, that is, restricted to the pharynx (pharyngeal bulb), such as Protodrilus haurakiensis von Nordheim, 1989; P. jagersteni von Nordheim, 1989; P. submersus von Nordheim, 1989; P. litoralis von Nordheim, 1989; P. ciliatus Jagersten, 1952; P. leuckarti Hatschek, 1881; P. affinis Jouin, 1968; P. hatscheki Pierantoni, 1908; P. rubropharyngeus Jagersten, 1940; P. flavocapitatus Ul-janin, 1877; P. robustus Jagersten, 1952; and P. hochbergi Martinez, Di Domenico, Jorger, Norenburg & Worsaae, 2013. A particularly intense red color has been reported in P. purpureus Schneider, 1868 (von Nordheim, 1989; Martinez et al, 2013). The present species is eyeless, with no so-called statocysts or nuchal organs as sensory organs located antero-dorsally, and is a morphologically exceptional finding when compared with other known Protodrilus species. Other species that were recently reported to be without eyespots are Protodrilus jagersteni; P. submersus; P. litoralis; P.jouinae von Nordheim, 1989; P. gracilis von Nordheim, 1989; P. adhaerens Jagersten, 1952; P. similis Jouin, 1970; P. hypoleucus Armenante, 1903; P. helgolandicus von Nordheim, 1983; P. purpureus; P. ciliatus; P. leuckarti; P. affinis; P. hatscheki, P. albicans Jouin, 1970; P. pierantonii Aiyar & Alikuhni, 1944; P. indicus Aiyar & Alikuhni, 1944; P. infundibuliformis Schmidt & Westheide, 1977; P. spongioides Pierantoni, 1903; P. robustus; P. flabelliger Wieser, 1957; and P. minutas Kirsteuer, 1966 (von Nordheim, 1989). Still other species that were observed without eyespots include Protodrilus corde roi Marcus, 1948; P. ovarium Di Domenico, Martinez, Lana & Worsaae, 2013; P. smithsoni Martinez, Di Domenico, Jorger, Norenburg & Worsaae, 2013; P. draco Martinez, Di Domenico, Jorger, Norenburg & Worsaae, 2013; and P. hochbergi (Domenico et al, 2013; Martinez et al, 2013). A lack of statocysts has been reported in Protodrilus jouinae, P. gracilis, P. similis, P. purpureus, P. corderoi, and P. ovarium (von Nordheim, 1989; Domenico et al, 2013). However, no other species has been described as being without nuchal organs. Although other species have conspicuous salivary glands, these glands seem to be connected to the gut and are difficult to distinguish in Protodrilus puniceus sp. nov. From the morphological characteristics of body color--of the pharynx only--and lack of eyespots and statocysts, the new species described here mostly resembles the already known P. gracilis and P. purpureus, but differs largely in the lack of nuchal organs and inconspicuous salivary glands. Moreover, the size of the new species is almost more than double the total length, width, and number of segments of the previous known species. Body and palps are elastic while alive, but are strongly contracted after fixation. This is the first description of this genus from an organically enriched environment in the deep sea.
Etymology: The species name puniceus derives from the Latin for pink and tan colors, originating from its conspicuous color when alive.
Type series: Holotype (NSMT-Pol H-598) and 20 paratypes (NSMT-Pol H-599) were extracted from vertebrate bone by W. Sato-Okoshi, K. Okoshi, and Y. Fujiwara on 19 August, 2004.
Phylogenetic analysis of the 18S rRNA gene sequence of Protodrilus puniceus sp. nov.
Phylogenetic analysis using the maximum likelihood (ML) method placed the 18S rRNA gene sequence of Protodrilus puniceus sp. nov. within the family Protodrilidae (Fig. 5). This worm formed a monophyletic clade with other Protodrilus annelids in ML, supported by a bootstrap value of 100%, and was the closest species to Protodrilus ciliatus, supported by a bootstrap value of 100.
Thirty-seven species were previously recorded and described in the family Protodrilidae, mainly from waters around the Atlantic and New Zealand (Langerhans, 1880; Wieser, 1957; von Nordheim, 1983, 1989; Reiser, 1997; Domenico et al, 2013; Martinez et al, 2013). They were all collected from coarse to fine sandy bottom sediments in shallow waters, such as intertidal beaches or subtidal areas. Some species live in the top 5 cm of the sediments, while others live 30-60 cm from the bottom surface (von Nordheim, 1983, 1989). There is no biological or ecological information on Protodrilus species inhabiting deep-sea oceans, organically enriched sediments, or whale-fall environments. Because there are too many differences between sandy, interstitial, shallow-water habitats and those with organically enriched and/or sulfide-enriched sediments in the deep sea, it is difficult--and premature--to discuss Protodrilus species characteristics. The genus Protodrilus may have high diversity, and may have adapted to many more types of environments than what we already know. It is also uncertain whether the abundant Protodrilus species reported here lives exclusively in whale-fall environments or is capable of living in other environments as well. An example of Saccocirrus (of the so-called related family Saccocirridae) was reported to migrate to decaying fish and feed on it (Westheide, 2008). So it is possible that this newly described species is not an obligate feeder on whale carcasses as an Osedax species, and the relationship may be facultative.
Almost all of the Protodrilus species described previously from sandy, shallower waters possess sensory organs, that is, eyes, nuchal organs, so-called statocysts, and transversal ciliary rings on the surface of the body. However, the new species described here is simple in morphology and has no conspicuous sensory organs or special accessories except for palps. One of the most noticeable features of this species is the well-developed, wide, ventral ciliary band that surrounds a mouth and continues longitudinally throughout the body (Fig. 2D, E, G, H). This characteristic ventral ciliary band seems to be helpful in obtaining and conveying food, such as particulate organic substances originating from whale carcasses and bacteria. Particulate matter was transported to the mouth using the ventrally elongated ciliary band, then swallowed. Previously known species that live interstitially in the sand of shallower waters also have a similar ventral ciliary band and are known to be deposit feeders. Protodrilus helgolandicus, inhabiting subtidal sands off Helgoland, feeds presumably on both bacteria and small diatoms covering sand grains. Protodrilus puniceus sp. nov. seems also to be an infaunal deposit feeder, but it lives in the small pores of whale bones and is assumed to feed on bone-derived organic substances and associated bacteria.
Another noticeable character of the species is the conspicuous bright red to pink body color of the living animals. Until now, none of the species belonging to Protodrilus has been described as having a deep-colored body. It is uncertain whether this red body color is derived from pigments, hemoglobin in blood vessels, or other sources. Some red-bodied animals that inhabit whale-fall environments, such as Osedax spp. and vestiferans, owe their bright coloration to hemoglobin-containing blood vessels. In fact, it may be advantageous for those animals living in anoxic conditions as the result of organically enriched environments, such as a whale-fall community, to have hemoglobin.
We collected and observed off Cape Nomamisaki four different types of whale bone: maxilla, vertebra, epiphysis, and ulna. Protodrilus puniceus sp. nov. inhabited all of these bones in high density from both 2.5-y-old (2004) and 3.5-y-old (2005) carcasses. Whereas a dense cluster of Osedax japonicus (Fujikura et al., 2006) was first observed on the surface of a maxilla 1.5 y after deposit, Protodrilus worms were seen distributing in patches, sometimes partly associated with the whale-fall mussel Adipicola pacifica, on all types of bone surfaces from 2004 onward. It is unclear when P. puniceus first appeared on whale bones. The worms might have settled there just before the deepsea diving cruise in 2004, and robust population growth might have occurred rapidly thereafter. It is also possible that the worms colonized the whale bones much earlier than 2.5 y after the carcasses were deposited. Due to their infaunal inhabitance and small size, they might have been invisible in the early successional stage of the carcasses. Overpopulated worms would have lost their burrows and nutrients in the bones, and appeared on the bone surfaces in later years.
Purschke and Jouin (1988) and Purschke (1993) demonstrated that the three families, Protodrilidae, Protodriloidae, and Saccocirridae, each possess a pair of palps (not tentacles) and form a distinct order, Protodrilida, as a sister group to Spionida. However, the position of the three families in Annelida should be regarded as uncertain. Molecular phylogenetic analysis clearly showed that the worms investigated here were a member of the genus Protodrilus, which was consistent with our morphological observations (Fig. 5). However, the phylogenetic topology of annelids remains problematic (Rousset et al., 2007; Struck et al, 2008; Struck, 2011). Therefore, we could reach no conclusion about the higher taxonomic position of Protodrilidae, even though the bootstrap value of each clade was substantially high.
We are interested in determining why and how this Protodrilus species, which belongs to the Protodrilidae and which is widely reported to be dispersed in sandy intertidal areas, occurred predominantly in a whale-fall community. When and where did the species originate? How did the animals establish their populations? There is a report of the occurrence of Protodrilus n. sp. from a whale fall in Monterey Bay, California (Braby et al., 2007). Compared with hydrothermal vent and hydrocarbon seep ecosystems, whale-fall ecosystems have received far less investigative attention. It is highly possible that other species belonging to the genus Protodrilus or higher taxonomic groups inhabit sunken whale car-casses in the deep sea. On the other hand, no Protodrilus worm was reported to be collected from sperm whale carcasses that were implanted at a depth of 920 m off Hatsushima Island in Sagami Bay, Japan. We have only opened the door to this unfamiliar annelid group. Further investigation of its biology, ecological function, and relationships between the species and whale carcasses is expected to increase our knowledge--not only of this unique annelid group--but also of whale-fall ecosystems.
We wish to express our sincere thanks to Dr. Akira Taniguchi for his continuous encouragement and support for this research; Dr. Kaoru Kubokawa for organizing cruise NT05-12; and Mr. Masaru Kawato for molecular analyses. We thank the operation team of the ROV Hyper-Dolphin and the captain and crew of the R/V Natsushima. Thanks are due also to Ms. Kumiko Ito of Tohoku University for her technical assistance in the scanning electron microscope observations. We also wish to thank Dr. Katrine Worsaae.
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WAKA SATO-OKOSHI (1*), KENJI OKOSHI (2), AND YOSHIHIRO FUJIWARA (3)
(1) Laboratory of Biological Oceanography, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan; (2) Department of Environmental Science, Faculty of Science, Toho University, Chiba 274-8510, Japan; and (3) 'Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka 237-0061, Japan
Received June 17, 2013; accepted 16 June 2015.
(*) To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
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|Author:||Sato-Okoshi, Waka; Okoshi, Kenji; Fujiwara, Yoshihiro|
|Publication:||The Biological Bulletin|
|Date:||Oct 1, 2015|
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