Journal of
The Malacological Society of London
Molluscan Studies
Journal of Molluscan Studies (2016) 82: 391– 402. doi:10.1093/mollus/eyw004
Advance Access publication date: 23 March 2016
Thyasirid bivalves from the methane seep community off Paramushir Island
(Sea of Okhotsk) and their nutrition
Vladimir I. Kharlamenko, Gennady M. Kamenev, Alexander V. Kalachev,
Serguei I. Kiyashko and Victor V. Ivin
A.V. Zhirmunsky Institute of Marine Biology of the Far Eastern Branch of the Russian Academy of Sciences, Palchevskogo 17, Vladivostok 690041, Russia
Correspondence: V.I. Kharlamenko; e-mail: [email protected]
(Received 22 January 2015; accepted 15 December 2015)
ABSTRACT
The present study focuses on two apparent species: the giant thyasirid Conchocele bisecta (Conrad, 1849),
which is the dominant species of the benthic community in a gas hydrate area with cold-water
methane-rich vents at a depth of about 800 m on the slope off Paramushir Island (Kuril Islands, Sea of
Okhotsk) and small unidentified thyasirid bivalves from this same community. An examination of the
shell morphology of these thyasirids showed that the small bivalves were in fact young specimens of C.
bisecta, characterized by a high individual and age variability. A transmission electron microscopic
study of C. bisecta revealed gills with ‘Type 3’ filaments, which were extended abfrontally and had a distinct bacteriocyte zone with extracellular symbionts. The symbiotic bacteria found were spherical,
similar to thiotrophic symbionts of other thyasirids. The isotopic d13S values of C. bisecta soft tissues
(from 239.6 to 233.8‰) were much heavier than those of methane in the Paramushir gas-hydrate
area and matched the range characteristic of symbiotrophic bivalves harbouring sulphur-oxidizing
chemoautotrophic bacteria. The variations in d13S and d15N recorded for large and small C. bisecta can
be related to ontogenetic differences in life habit: small individuals are totally buried in the sediment,
while large ones are half-buried. Data from fatty acid (FA) analysis indicate that sulphur-oxidizing
symbionts constitute almost the entire nutrition of C. bisecta, with no significant contribution of symbiotic or free-living methanotrophs. Furthermore, neither FA nor isotopic compositions provided evidence
for photosynthetic sources as food items for C. bisecta through filter feeding.
INTRODUCTION
The discovery of deep-sea hydrothermal communities (Lonsdale,
1977) radically changed our understanding of food resources and
trophic relationships in deep-sea benthos. The extremely high
biomass of benthos in these communities is supported by the
chemosynthesis performed by bacterial symbionts inhabiting
large vent clams and tube worms. Similar chemosynthetic communities were subsequently found in fields of cold-water seeps
(Paull et al., 1984) and near methane vents (Kulm et al., 1986).
Cold-seep communities are now known to be distributed worldwide in the deep-sea environment at active and passive continental margins (Subiet & Olu, 1998; Levin et al., 2010).
Chemoautotrophic vesicomyid and mytilid bivalve molluscs, as
well as sibogliniid tube worms, usually dominate the biomass in
cold seeps, providing microhabitats for diverse benthic communities (Levin & Michener, 2002; Becker et al., 2014).
Thyasirid bivalves are also widespread in modern and especially ancient cold-seep sites (Majima, Nobuhara & Kitazaki,
2005; Little et al., 2015). However, in modern seep communities
these bivalves play only a minor part and are usually represented by small-sized species (Subiet & Olu, 1998; Duperron
et al., 2013). To date, there is one well known exception among
recent cold-seep communities—the benthic community discovered in a gas hydrate area with cold-water methane-rich vents at
a depth of about 800 m on the continental slope off Paramushir
Island (Kuril Islands, Sea of Okhotsk) during surveying by a
manned underwater vehicle in 1986 (Kuznetsov, Galkin &
Rass, 1987; Zonenshayn et al., 1987). The dominant species of
this community were a giant thyasirid, Conchocele sp., and what
appeared to be a new unidentified species of small thyasirid
bivalve (shell length L less than 25 mm) (Kuznetsov, Rass &
Galkin, 1989). Modern thyasirid-dominated seep communities
may be more widespread. Large aggregations of Conchocele comprising both live individuals and dead shells have also been
observed by means of an underwater camera in another region
of the Okhotsk Sea (northeastern slope of Sakhalin Island at
depths of 385– 750 m) at gas hydrates sites in bottom sediments
(Biebow & Hütten, 1999; Kamenev, Nadtochy & Kuznetsov,
2001).
# The Author 2016. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved
V. I. KHARLAMENKO ET AL.
The giant Conchocele sp. found at the Paramushir cold-seep site
had L of 150 –180 mm and a biomass of 2.5–3 kg/m2
(Kuznetsov et al., 1989). Like many bivalves containing endosymbiotic bacteria (Kuznetsov et al., 1989), these giant thyasirids
had massive gills and light isotopic d13C values were reported for
their soft tissues (Strizhov, Kuznetsov & Gurina, 1990;
Kuznetsov, Strizhov & Kijashko, 1991). On the basis of the positive tests for methane and CO2 assimilation, it was assumed that
Conchocele sp. from Paramushir cold seeps has symbiotic relationships with both methanotrophic and thiotrophic bacteria
(Galchenko et al., 1988). A supposition that the mode of nutrition of Conchocele sp. was mixotrophic (i.e. combining filtration of
particulate organic matter and methanotrophic bacterial symbionts) was based on the intermediate values of d13C and the
morphology of gills and digestive tract (Kuznetsov et al., 1991).
However, the high sulphur content of the gills also supported
the presence of thiotrophic bacterial symbionts (Kuznetsov et al.,
1989, 1991). Evidence of methanotrophy in Conchocele sp. was
recognized as weak (Imhoff et al., 2003) and more data are
needed to interpret the nutrition of Conchocele sp.
The Conchocele sp. from Paramushir cold-seep site was later
identified as C. bisecta (Conrad, 1849) (Kamenev et al., 2001), a
species that is sporadically found in deep waters of the North
Pacific. Some individuals of this species have been collected from
other modern cold seeps (Biebow & Hütten, 1999; Coan, Scott
& Bernard, 2000; Okutani, 2000; Sasaki, Okutani & Fujikura,
2005) and it is abundant in fossil seep deposits from Japan
(Majima et al., 2005). So far, the small thyasirids from
Paramushir cold seeps have been considered as a separate unidentified species (Galkin & Sagalevich, 2012) and no data on
their feeding exist.
The purpose of this study was to reinvestigate the thyasirids
from the Paramushir seep site using live specimens of both giant
and small forms obtained by ROV Sub-Atlantic during the revisiting of the site in 2013. The aims were: (1) to resolve the taxonomy of small and giant thyasirids, based on morphological
comparison of newly obtained specimens with those collected in
1986; (2) to investigate the thyasirid bacterial symbionts by
using the new high-quality material for transmission electron
microscopy (TEM); (3) to clarify the role of bacterial symbiosis
and filter-feeding in the nutrition of the thyasirids, based on
analysis of carbon and nitrogen stable isotopes and of fatty acid
(FA) composition.
Figure 1. A. Sampling area. B. Stations where thyasirids were found
during cruise of RV Akademik M.A. Lavrentyev.
nos 28446– 28456). A total of 44 live specimens, 32 shells and 51
separate valves of variously sized thyasirids (L¼4.8 –104.4 mm)
were examined from the methane-rich vents of Paramushir
Island (Table 1).
For comparison, the following material was examined: C.
bisecta: 16 specimens and 4 valves, Sea of Okhotsk (MIMB
4067–4070, 4298, 4572, 28635); 1 valve, Sea of Japan (MIMB
4573); 2 specimens and 2 valves, Pacific Ocean (MIMB 4071;
Royal British Columbia Museum, Victoria, Canada, RBCM
006-00076-001); Channelaxinus excavata (Dall, 1901):(1 specimen,
Gulf of California, Pacific Ocean (RBCM 990-590-1); C. novaeguinensis Okutani, 2002: holotype (National Museum of Nature
and Science, Tsukuba, Japan; NMNS Mo 73192); C. koyamai
Habe, 1981: holotype (NMNS Mo 58903); C. scarlatoi Ivanova
& Moskaletz, 1984: holotype (MIMB 1/31326); paratype
(MIMB 2/31327); 1 specimen, Sea of Japan (MIMB 4423).
MATERIAL AND METHODS
Material and sampling
The material was collected from silty sand and microbial mats in
the area of methane-rich vents at a depth of 769 –805 m, northwest of Paramushir Island on 26 –31 May 2013, during the expedition of the A.V. Zhirmunsky Institute of Marine Biology
(Far Eastern Branch of the Russian Academy of Sciences,
Vladivostok; IMB FEB RAS) (on RV Akademik M.A. Lavrentyev,
cruise LV-61), by using the remotely-operated vehicle
Sub-Atlantic or an Ocean-50 grab sampler (0.25 m22) (Fig. 1).
Samples were fixed in precooled 96% ethanol and in 4% buffered formalin. The shell morphology of all the specimens of
Conchocele bisecta and the small thyasirids collected in the same
area by the expedition of the P.P. Shirshov Institute of
Oceanology (Russian Academy of Sciences, Moscow; IO RAS)
(on RV Akademik Mstislav Keldysh, cruise 11-A), from 25 June to
6 July 1986, was also studied. During the IO RAS expedition,
the sampling was carried out by means of an Ocean-50 grab
sampler (0.25 m22), a Sigsbee trawl and manned submersibles
(Pisces-VII and Pisces-XI) and live specimens were fixed in 70%
ethanol. The bivalves were counted and stored in 70% ethanol
at the IO RAS and Museum of the IMB FEB RAS (MIMB reg.
Morphological analysis
Shell morphology was studied in live-collected individuals and
fresh valves connected by the ligament. Shell measurements
were taken for right valves only, as they were more often undamaged than left valves. The following parameters were measured
(Fig. 2): length (L), height (H), width of valve (W) (not shown
in figure), length of anterior adductor scar (A) and length of
nymph (N). The ratios H/L, W/L, A/L and N/L were calculated
as indices of shape. All measurements were made using a calliper
or an ocular micrometer, with an accuracy of 0.1 mm.
For comparative analysis of large and small thyasirids, from
the entire sample of specimens (L ¼ 4.8–104.4 mm) we selected
two groups, of large (L ¼ 57.5 –104.4 mm) and small thyasirids
(L ¼ 4.8 –22.0 mm), following the groupings defined by
Kuznetsov et al. (1989). Statistical analysis was performed on
392
Table 1. List of stations sampled by the IO RAS and IMB FEB RAS expeditions in the methane-rich vents area off Paramushir Island (Kuril Islands, Sea of Okhotsk) where thyasirid bivalves (shells and
live specimens) were found.
Station
Date
Start-end
Latitude, N
AL61
393
AMK11-A
13-1
26.05.2013
Depth (m)
Gear
Material examined
Longitude, E
′
155818.399′ – 155818.369′
777
ROV
4 shells, 3 large and 5 small live specimens
′
155818.408′
778
ROV
5 live small specimens
50830.935 –50830.955
′
13-5
28.05.2013
50830.872
13-6
30.05.2013
50830.938′
155818.373′
779
ROV
1 live small specimen
13-7
31.05.2013
50830.917′
155818.421′
778
ROV
2 shells
14-1
27.05.2013
50830.811′
155818.565′
786
ROV
2 shells, 1 live large specimen
15-1
28.05.2013
50830.973′
155818.350′
787
OG
1 shell
15-2
28.05.2013
50830.942′
155818.288′
792
OG
1 shell
16-1
28.05.2013
50830.948′
155818.480′
785
OG
9 live small specimens
17-3
30.05.2013
50831.002′
155818.444′
785
OG
1 shell
18-1
28.05.2013
50830.890′
155818.329′
790
OG
2 shells
18-3
28.05.2013
50830.893′
155818.373′
790
OG
14 shells
1383
25.06.1986
50830.76′
155819.81′
794
OG
Fragments of valves
1389
26.06.1986
50831.20′ – 50831.60′
155818.33′ – 155821.60′
784 – 787
Pisces-XI
Fragments of 1 valve
1391/5
27.06.1986
50830.79′ – 50830.30′
155818.01′ – 155817.77′
784 – 787
ST
14 valves
′
155818.20′
–
Pisces-VII
6 valves
1394/2
28.06.1986
50831.81
1395/2
29.06.1986
50830.90′
155818.12′
787
OG
Fragments of 1 valve
1396
29.06.1986
–
–
–
Pisces-XI
2 shells of small specimens
1405
01.07.1986
50830.82′ – 50830.87′
′
155818.12′ – 155818.11′
770 – 769
Pisces-XI
1 shell, 1 live small specimen
′
155818.04′ – 155818.71′
–
Pisces-VII
Fragments of 1 valve
1406
01.07.1986
50831.33 – 50830.69
1 413
04.07.1986
50830.88′ – 50830.81′
155818.14′ – 155819.30′
792 – 804
ST
17 valves, 2 shells, 1 large and 18 small live specimens
1424
06.07.1986
50830.77′
155818.10′
786
OG
10 valves
1426
06.07.1986
50830.80′ – 50830.43′
155818.29′ – 155817.75′
788 – 805
ST
4 valves
Abbreviations: AMK11-A, cruise 11-A on RV Akademik Mstislav Keldysh; AL61, cruise 61 on RV Akademik M.A. Lavrentyev; OG, Ocean-50 grab; ST, Sigsbee trawl; ROV, remotely operated vehicle; Pisces-VII and
Pisces-XI, manned underwater vehicles.
THYASIRIDS FROM METHANE SEEPS IN SEA OF OKHOTSK
Cruise
V. I. KHARLAMENKO ET AL.
esters (FAMEs) were prepared from the total lipid extract
according to standard procedure (Carreau & Dubacq, 1978)
and purified by thin-layer chromatography in benzene. FAMEs
were analysed in a Shimadzu GC 2010 chromatograph, using a
fused quartz capillary (30 m 0.25 mm) SUPELCOWAX 10
(Supelco) column. The temperature of the column was 210 8C
and that of both injector and detector was 250 8C. The
4,4-dimethyloxazoline (DMOX) derivatives of the FAs were
prepared according to Svetashev (2011). Mass spectrometry was
performed in a Shimadzu GCMS QP5050A spectrometer by
using a MDN-5S column (Supelco). The initial temperature of
the column was 190 8C, raised to 290 8C at 2 8C/min, and this
temperature was maintained for 25 min. All spectra were
obtained at 70 eV and were compared with the NIST library
and FA mass spectra archive (AOCS, 2016). The FAs were identified from the gas chromatography –mass spectrometry of the
FAMEs and the DMOX derivatives.
For stable isotope analysis, the dried samples were ground to a
fine powder using an agate mortar and pestle; 0.5-mg subsamples
were packaged into tin caps. Because of the low lipid content in
most of the isotopic samples analysed, neither lipid extraction nor
lipid correction was performed. The isotopic analysis was conducted at the Stable Isotope Laboratory (Far Eastern Geological
Institute, FEB RAS, Vladivostok) using a FlashEA 1112 elemental analyser coupled to a MAT 253 isotope mass spectrometer
(ThermoQuest, Germany) via a ConFlo IV interface. Sample isotopic ratios were expressed in the conventional ‘d’ signature as
parts per thousand (‰) according to the equation:
ðRsample Rstandard Þ
1; 000
d13 C or d15 N ¼
Rstandard
Figure 2. Shell measurements. Abbreviations: A, length of anterior
adductor scar; H, shell height; L, shell length; N, length of nymph.
the shape indices. All data were tested with a Kolmogorov test
and, since some showed deviation from normality, analyses were
performed on log10 transformed variables. Indices of large and
small thyasirid groups were compared using the parametric
Student t test and one-way analysis of variance, using a significance level of P , 0.05. Analysis was carried out using the software package STATISTICA and Data Analysis Module of MS
Excel 97-2003.
For SEM, shells were cleaned of soft tissue and periostracum
in diluted bleach, washed in distilled water and dried. They
were mounted with adhesive tape on aluminium stubs and
coated with gold before examination using a Zeiss EVO 40XVP.
where R ¼ 13C/12C or 15N/14N. The d values were expressed relative to the international reference standards: Pee Dee Belemnite
for carbon and atmospheric N2 for nitrogen. In order to control
the data quality the internal laboratory standard was measured
after every sixth sample during the analysis. The internal precision, based on the SD of the replicates of the laboratory standard,
was + 0.1‰ for both d13C and d15N.
Transmission electron microscopy
RESULTS
Small pieces of gills were prefixed in 2.5% glutaraldehyde in
0.2 M cacodylate buffer ( pH 7.4) for 2 h at 4 8C. The specimens
were then rinsed in the same buffer and postfixed in 2% osmium
tetroxide buffered with 0.2 M cacodylate buffer ( pH 7.4) for 1 h
at room temperature. The fixed tissues were dehydrated in a
series of ethanol and acetone solutions and then embedded in
Epon-Araldite. Semithin (c. 1 mm) and ultrathin (c. 75 nm) sections were cut using a Leica UC 6 ultramicrotome equipped
with glass and diamond knives respectively. For light microscopic studies, the specimens were stained with methylene blue-azure
II-basic fuchsin (Humphrey & Pittman, 1974) and examined
with a Keyence BZ9000 microscope. For TEM, the specimens
were stained with 2% alcoholic uranyl acetate and Reynolds’
lead citrate (Reynolds, 1963) and examined with a Zeiss Libra
200FE microscope (Far East Centre of Electron Microscopy,
IMB FEB RAS) operated at 200 kV.
Morphological analysis
Comparative morphological analysis of the thyasirids from the
IO RAS and IMB FEB RAS expeditions (L ¼ 4.8 –104.4 mm)
showed that all belong to a single species, Conchocele bisecta
(Conrad, 1849). All specimens lack the submarginal sulcus
typical of Thyasira, Parathyasira, Channelaxinus and Ascetoaxinus
and do not have the auricle typical of Thyasira (Oliver & Kileen,
2002; Oliver & Sellanes, 2005; Zelaya, 2009, 2010; Coan &
Valentich-Scott, 2012; Oliver & Frey, 2014). Moreover, they do
not have the long and deeply impressed lunule characteristic of
Channelaxinus and Ascetoaxinus; neither do they have the long,
narrow, deep channel that holds the ligament of Channelaxinus or
the scalloped edges adjacent to the lunule as in Ascetoaxinus
(Coan & Valentich-Scott, 2012; Oliver & Frey, 2014). All the
studied specimens have a subquadrate or obliquely oval shell, a
distinct, long, radial posterior sulcus of the shell, a weakly
impressed lunule, a partly sunken, opisthodetic ligament
attached to a strong and broad nymph, and a greatly elongated
anterior adductor scar (Figs 3, 4). All these characters are
typical of the genus Conchocele Gabb, 1866 (Coan et al., 2000;
Kamenev et al., 2001; Oliver & Sellanes, 2005; Coan &
Valentich-Scott, 2012; Oliver & Frey, 2014). A comparison with
specimens, descriptions and images of all species of Conchocele
(C. bisecta, ‘Conchocele’ koyamai Habe, 1981, C. scarlatoi Ivanova &
Moskaletz, 1984 and C. novaeguinensis Okutani, 2002; Fig. 5) has
FAs and stable isotope analysis
The tissues of thyasirids, or a whole animal in the case of small
specimens, were washed with seawater and distilled water. One
part of each sample was placed in a mixture of equal parts of
chloroform and methanol and the other part was dried at 60 8C
for 24 h.
For analysis of FAs, lipids were extracted using the method of
Bligh & Dyer (1959) and stored at 218 8C. Fatty acid methyl
394
THYASIRIDS FROM METHANE SEEPS IN SEA OF OKHOTSK
Figure 3. Variously sized specimens of Conchocele bisecta, Paramushir Island (Kuril Islands, Sea of Okhotsk; see Table 1), depth 777– 792 m. A, B.
Exterior of left valve and interior of right valve (MIMB 28453), Stn 14-1, L ¼ 104.4 mm. C, D. Exterior of left valve and dorsal view (MIMB 28446),
Stn 13-1, L ¼ 77.0 mm. E, F. Exterior and interior of left valve (MIMB 28450), Stn 15-1, L ¼ 51.3 mm. G. Exterior of left valve (MIMB 28452), Stn
18-1, L ¼ 36.8 mm. H, I. Exterior and interior of left valve (MIMB 28448), Stn 18-3, L ¼ 30.5 mm. Scale bars ¼ 10 mm.
shown that our specimens correspond to C. bisecta (Ivanova &
Moskaletz, 1984; Coan et al., 2000; Kamenev et al., 2001;
Okutani, 2002; Zelaya, 2009; Oliver & Frey, 2014). In contrast
to C. bisecta, C. novaeguinensis has a strongly curved posterodorsal
shell margin with a very thick, long and broad nymph, a more
dorsally situated flexure, and a greatly concave anterior shell
margin. The shallow-water species C. scarlatoi differs from C.
bisecta in having a more pointed umbo and an apical angle of
less than 908, which are characters that do not change with age.
The features of ‘C.’ koyamai are not typical of Conchocele and its
generic placement is currently uncertain (Okutani, 2002; Oliver
& Frey, 2014).
The small specimens from Paramushir Island (L , 23 mm)
have a relatively thinner, higher and more orbicular shell (H/L ¼
0.881 + 0.012), with a larger apical angle, a weaker posterior
sulcus, a less elongated anterior adductor scar and nymph
(A/L ¼ 0.380 + 0.013; N/L ¼ 0.329 + 0.010), as compared with
large specimens (L . 57 mm) (H/L ¼ 0.740 + 0.007; A/L ¼
0.465 + 0.012; N/L¼ 0.404 + 0.013) (Supplementary Material
Tables S1, S2). The means and variances of the shape indices
H/L, A/L and N/L differ significantly in the two groups of small
and large specimens (Table 2). However, examination of the complete size range has shown a gradual change in these characters
with an increase in shell size (Figs 3, 4), the shell becoming more
elongate, less rounded and thicker, while the apical angle and
relative lengths of the nymph and anterior adductor scar decrease. Nevertheless, the main diagnostic characters (shape and
proportions of shell, shape of anterior and posterior margins, relative length of nymph and apical angle) in all studied specimens
are consistent with those of C. bisecta.
Transmission electron microscopy
On their lateral surface, gills of C. bisecta bear numerous bacteriocytes, with less numerous intercalary cells interspersed among
them (Fig. 6A, B). Both cell types rest on the basal lamina that
separates them from the haemocoel. The bacteriocytes are columnar cells up to 15 –18 mm in height (Fig. 6B, C), connected
to one another by desmosome-like junctions. These junctions
also appear between bacteriocytes and intercalary cells. On
their apical surface, bacteriocytes have deep invaginations,
so-called ‘bacterial chambers’, containing symbiotic bacteria
(Fig. 6C). A bacteriocyte may have several chambers, up to
6 mm in depth, with apertures covered by numerous microvilli
(Fig. 6C). Ectosymbiotic bacteria are up to 0.5 mm in length
and variously shaped, e.g. spherical, ovoid or bottle-like
395
V. I. KHARLAMENKO ET AL.
Figure 4. Small specimens of Conchocele bisecta, Paramushir Island (Kuril Islands, Sea of Okhotsk), depth 777–792 m. A, B. Exterior of left valve and
interior of right valve (MIMB 28448), Stn 18-3, L ¼ 20.3 mm. C. Dorsal view (MIMB 28446), Stn 13-1, L ¼ 8.4 mm. D. Exterior of right valve
(MIMB 28446), Stn 13-1, L ¼ 7.0 mm. E –G. Interior of left and right valves and prodissoconch (MIMB 28446), Stn 13-1, L ¼ 7.5 mm. H, I. Exterior
and interior of left valve (MIMB 28456), Stn 13-5, L ¼ 5.5 mm. Scale bars: A, B ¼ 10 mm; C–F, H, I ¼ 1 mm; G ¼ 100 mm.
(Fig. 6C). Their cytoplasm contains electron-dense inclusions
adjacent to the cell membrane, but no concentric stacks of intracellular membranes were observed (Fig. 6C). The cytoplasm of
bacteriocytes contains abundant glycogen granules, electronlucent vacuoles, mitochondria, Golgi complexes and lysosomes,
often filled with whorls of membranes. The large nucleus, up to
5 mm in diameter with several nucleoli, is located at the base
(Fig. 6C –E).
Intercalary cells are elongate, with electron-dense cytoplasm
(Fig. 6B). They often have a narrow base and an enlarged
apical part. The round or elongated nucleus is located either
basally or apically. Cellular organelles include mitochondria,
numerous electron-lucent vacuoles of various sizes and electrondense globules.
Stable isotopes
Tissue samples from large and small C. bisecta showed d13S values
in the range 239.6 to 233.8‰ (Table 3). The range of d15N
values was broader, from 21.1 to 8.4‰. The gills inhabited by
symbiotic bacteria were depleted in 13C and 15N as compared with
other tissues in both large and small individuals (Table 3). Tissueaveraged (or whole tissue) d13S values of small individuals were more
depleted, as compared with those of the large specimens (Fig. 7),
but the d15N values were similarly variable in both size groups.
Fatty acids
In the lipids of the gills of C. bisecta, monounsaturated fatty acids
(MUFAs) dominated, composing 80.3% of the total FAs
396
THYASIRIDS FROM METHANE SEEPS IN SEA OF OKHOTSK
Figure 5. Species of Concocele. A– D. C. scarlatoi Ivanova & Moskaletz, 1984, holotype (MIMB 1/31326), Vityaz Bight, Peter the Great Bay, Sea of
Japan, depth 30 m, L ¼ 70.9 mm. E –G. C. scarlatoi, paratype (MIMB 2/31327), exterior of right valve, dorsal view showing posterior sulcus and
ligament, dorsal view of both valves, Furugelm Island, Peter the Great Bay, Sea of Japan, depth 33 m, L ¼ 23.0 mm. H–J. C. scarlatoi, valves of small
specimen (MIMB 4423), Chazma Bight, Strelok Bay, Sea of Japan, depth 10 m, L ¼ 26.0 mm. K, L. C. novaeguinensis Okutani, 2002, holotype (NMNS
Mo 73192), L ¼ 73.9 mm. M. ‘C.’ koyamai Habe, 1981, holotype (NMNS Mo 58903), L ¼ 21.0 mm. Scale bars ¼ 10 mm.
(Table 4). The main FA was 16:1 (n –7), making up 56.1%;
a high content of 20:1 (n–13) was also found. The content of
polyunsaturated fatty acids (PUFAs), mainly C18 PUFA (n –4
series), was 2.2%. The content of C20 and C22 PUFAs was extremely low (0.5%). The content of nonmethylene-interrupted
(NMI) FAs in the gills was 6.3% and of saturated FAs 9.7%. In
397
V. I. KHARLAMENKO ET AL.
Sellanes, 2005), resulting in errors in diagnoses. Here we demonstrate that the numerous small specimens with L of up to
2.0–2.5 cm collected at methane seeps off Paramushir Island
(Kuznetsov et al., 1987, 1989; Galkin & Sagalevich, 2012) are in
fact young C. bisecta.
Like many species of bivalves containing endosymbiotic bacteria (Kuznetsov et al., 1989), C. bisecta has massive gills, said to
have a structure unique among bivalves (Kuznetsov et al., 1991).
Both intra- and extracellular bacteria have been reported in the
gill tissue of C. bisecta, and mixed cultures of methanotrophic
and thiotrophic bacteria from the gills were obtained
(Galchenko et al., 1988). However, the evidence for methanotrophic bacteria in the gills is weak (Imhoff et al., 2003), because
the structures typical of methanotrophs were not evident in electron microscope photographs. Our TEM examination of C.
bisecta has shown that it has gills with the ‘Type 3’ filaments
described by Dufour (2005). The filaments of this type are
extended abfrontally and have a distinct bacteriocyte zone with
extracellular symbionts (Dufour, 2005). We did not detect any
intracellular symbionts or symbionts with the stacked internal
membranes typical of Type I methanotrophs. The symbiotic
bacteria were mainly spherical (or ovoid) in shape like the thiotrophic symbionts of other thyasirids (Brissac et al., 2011).
The carbon of C. bisecta soft tissues was anomalously depleted
in 13S as compared with particulate organic matter produced
through photosynthesis in the euphotic zone of the sea (d13S
values vary from 224 to 218‰; Fry & Sherr, 1984), but this
species showed much heavier d13S signatures than the methane
in their environment in the Paramushir gas-hydrate area (d13S
value 254.6‰; Lein et al., 1989). The d13S values of C. bisecta in
this study (from 239.6 to 233.7‰) fall within the range characteristic of symbiotrophic bivalves from hydrothermal vents
and cold seeps, feeding predominantly upon sulphur-oxidizing
chemoautotrophic bacteria (Kennicutt et al., 1992; Becker et al.,
2014; Zapata-Hernandez et al., 2014). Considering the isotopic
data, we conclude that the diet of C. bisecta depends mainly on
sulphur-oxidizing symbionts.
Previously reported d13S data for C. bisecta from the Sea of
Okhotsk (Sahling et al., 2003), including that from the Paramushir
cold-seep area (Kuznetsov et al., 1991), were obtained only from
large individuals and were in a narrow range, from 234.6 to
233.2‰. The broader variations of isotopic signatures d13S and
d15N recorded from the newly studied C. bisecta individuals
(Fig. 7), may be related to ontogenetic differences in habitats:
small individuals are totally buried in the sediment, while adults
are only half-buried (Fig. 8). The lower d13S values of small individuals could result from the fact that their bacterial symbionts assimilate more 13C-depleted CO2, produced from anaerobic
methane oxidation in deeper sediments and dissolved in interstitial
water (Schmaljohann et al., 1990). Similarly, the broad variations
of d15N values in small individuals could result from the assimilation by bacterial symbionts of various 15N-depleted forms of dissolved inorganic nitrogen, which are produced in the sediment
(Lee & Childress, 1994).
All the specimens of C. bisecta studied are characterized by a
high content of MUFAs and very low content of PUFAs
(Table 4). This is a characteristic feature of the lipid composition
of all bivalves that feed upon chemoautotrophic symbionts.
Most such bivalves contain sulphur-oxidizing symbiotic bacteria
(Taylor & Glover, 2010) and their gills are characterized by a
high content of (n– 7) MUFAs. In the gills of bivalve molluscs of
the family Solemyidae high contents of 18:1 (n –7) have been
found; in the Lucinidae the contents of 16:1 (n–7) and 18:1
(n–7) were both high, while the families Thyasiridae and
Vesicomyidae contain high concentrations of 16:1 (n–7) in their
gills (Conway & McDowell Capuzzo, 1991; Ben-Mlih, Marty &
Fiala-Medioni, 1992; Fullarton et al., 1995; Saito, Murata &
Hashimoto, 2014). The gills of bivalves of the subfamily
Table 2. Results of comparison of the means (t test) and variances
(ANOVA) of shell shape indices of small and large groups of Conchocele
bisecta.
Indices
t test
t
ANOVA
P-value
F
,0.001
105.22
n
P-value
H/L*
210.57
W/L
1.32
.0.05
1.74
.0.05
19/17
A/L*
4.87
,0.001
23.28
,0.001
19/17
N/L*
4.61
,0.001
21.6
,0.001
19/17
,0.001
19/17
L, shell length; H, height; W, width; A, length of anterior adductor scar; N,
length of nymph; n, number of valves in compared groups.
*P , 0.05.
the lipids of the digestive diverticula, MUFAs also predominated, making up 79.5% of the total FAs. The dominant
MUFA was 16:1 (n– 7), but the level of 18:1 (n –7) was similar.
The content of PUFAs in the digestive diverticula was even
lower than in the gills (1.9%), but the concentrations of C20 and
C22 PUFAs were twofold higher than in the gills. In the foot the
level of MUFAs was much lower (74.8%) than in the gills, while
the contents of saturated FAs (10.9%) and PUFAs (6.3%) were
higher. The main FA was 16:1 (n –7) and C18 PUFAs predominated among the PUFAs. The FAs found in the small thyasirids
were the same as in the large specimens. In the small thyasirids,
the content of MUFAs was 77.9% and the main FA was 16:1
(n– 7) at 53.9%. The FA profiles of the small individuals were
similar to that of the gills of the large thyasirids.
DISCUSSION
The genus Conchocele is currently represented by three Recent
species (C. bisecta, C. scarlatoi and C. novaeguinensis). However, in
the World Register of Marine Species (WoRMS) database
(www.marinespecies.org; accessed 16 February 2016) only two
species (C. bisecta and C. novaeguinensis) are listed, following the
revision of the genus by Oliver & Frey (2014). Apparently, these
authors omitted C. scarlatoi, because it was described in Russian
in a regional publication that is not readily available. Coan et al.
(2000) supposed that C. scarlatoi could be a synonym of C. bisecta.
However, study of the type and additional material of C. scarlatoi
showed that it is a distinct species, characterized by weak individual and age variability (Fig. 5) (Kamenev et al., 2001). In
general, Recent species of Conchocele are poorly represented in
museum collections; for example, only five valves of C. novaeguinensis and three specimens of C. scarlatoi are known (Okutani,
2002). Furthermore, available collections of large thyasirids, including C. bisecta, contain only adult shells in which the characters of the early shell are lost through erosion (Oliver & Frey,
2014). This has probably hampered the identification of the
small specimens of C. bisecta previously found off Paramushir
Island and led to the suggestion that they belong to a different
thyasirid species (Kuznetsov et al., 1989). The MIMB collection
now contains a large number of undamaged valves and shells of
different-sized specimens of C. bisecta, allowing investigation of
both age and individual variability.
A high variability of some morphological characteristics of
thyasirids has been reported by many authors. It has been
shown that shell proportions and shape vary between similarlysized individuals and between different sizes of C. bisecta
(Kamenev et al., 2001). A similar variability is also typical of
various species of Thyasira, Mendicula, Axinopsida and Adonthorina
(Scott, 1986; Payne & Allen, 1991; Kamenev, 1996, 2013;
Kamenev & Nadtochy, 2000; Oliver & Kileen, 2002; Oliver &
398
THYASIRIDS FROM METHANE SEEPS IN SEA OF OKHOTSK
Figure 6. Conchocele bisecta. A. Longitudinal section through the zone in gill filament, light microscopy. B. Bacteriocytes in gill filament, TEM. C.
Enlarged view of bacteriocytes. D. Microvilli covering symbiotic bacteria within bacterial chamber. E. Golgi complex in cytoplasm of bacteriocyte. F.
Residual bodies in cytoplasm of bacteriocyte. Abbreviations: bc, bacteriocyte; bs, bacterial symbionts; hc, haemocoel; ic, intercalary cells; g, Golgi
complex; mv, microvilli; nu, nucleus; ly, lysosome. Scale bars: A ¼ 50 mm; B ¼ 20 mm; C–D ¼ 2 mm; E ¼ 0.5 mm; F ¼ 2 mm.
399
V. I. KHARLAMENKO ET AL.
Table 3. Carbon and nitrogen stable isotope compositions of tissues of
Conchocele bisecta.
Table 4. Fatty acids of Conchocele bisecta (% of total FA).
Fatty acids
Specimens
Shell length L (mm)
Tissue
d13C (‰)
d15N (‰)
Large
104
Gill
235.46
2.04
Foot
234.19
4.28
Large
39
Small
15
9
specimens
(n ¼ 4)
Large specimens
Gill
Digestive
Foot
(n ¼ 2)
diverticula
(n ¼ 2)
(n ¼ 2)
Adductor
233.78
6.46
Gill
236.76
1.09
12:0
2.9 + 1
1.2 + 0.8
4.3 + 3.0
0.0 + 0.0
Foot
234.76
5.30
14:0
4.1 + 0.4
4.2 + 0.9
2.4 + 0.7
1.5 + 0.4
5.81
16:0
4.3 + 0.2
3.6 + 0.3
5.8 + 0.6
6.8 + 0.8
53.9 + 0.8
56.1 + 5.3
29.1 + 2.8
25.2 + 2.8
Adductor
Small
Small
234.59
mantle
234.45
4.61
16:1 (n –7)
Gill
238.51
2.18
18:0
0.8 + 0
0.7 + 0
0.4 + 0.1
2.6 + 0.0
Foot
236.66
4.13
18:1 (n –13)
4.6 + 0.9
3.9 + 0.9
2.0 + 0.6
4.2 + 0.2
6.77
18:1 (n –9)
0.6 + 1.1
1.8 + 0.2
4.1 + 0.6
6.4 + 0.8
8.38
18:1 (n –7)
6.4 + 0.9
5.3 + 0.5
24.2 + 0.9
10.8 + 1.1
Gill
239.65
Foot
236.96
Small
5– 7
Whole
238.11
0.55
18:2 (5, 11)
1.5 + 0.1
2.0 + 0.3
1.8 + 0.2
1.8 + 0.3
Small
5– 7
Whole
238.16
21.10
18:2 (n –7)
0.4 + 0.3
0.0 + 0
0.1 + 0.1
1.6 + 0.1
Small
5– 7
Whole
237.74
2.51
18:2 (n –4)
1.9 + 0.1
1.7 + 0.1
0.8 + 0.3
1.6 + 0.2
19:1 (n –12)
1.0 + 0.1
1.5 + 0.2
0.9 + 0.4
2.8 + 0.2
18:3 (5, 11, 14)
0.9 + 0.5
1.5 + 0.3
0.3 + 0.3
0.7 + 0.0
19:2 (5, 14)
1.1 + 0.1
1.4 + 0.2
0.7 + 0.4
1.2 + 0.2
20:1 (n –13)
9.4 + 0.2
10.1 + 1.7
12.1 + 3.9
23.4 + 0.4
20:1 (n –7)
3.0 + 0.1
3.1 + 0.9
8.0 + 0.1
4.6 + 1.6
20:2 (5, 13)
1.6 + 0.2
1.4 + 0.2
2.0 + 1
1.5 + 0.9
20:4 (n –6)
0.4 + 0.3
0.0 + 0
0.2 + 0.2
0.7 + 0.3
20:5 (n –3)
1.0 + 0.1
0.4 + 0.1
0.6 + 0.5
1.6 + 0.5
22:6 (n –3)
0.2 + 0.1
0.1 + 0.0
0.2 + 0.1
0.8 + 0.5
Saturated FAs
12.1
9.7
12.9
10.9
MUFAs
77.9
80.3
79.5
74.8
PUFAs
3.9
2.2
1.9
6.3
n– 3 + n– 6
1.6
0.5
1
3.1
n– 4 PUFAs
2.3
1.7
0.9
3.2
NMI
5.1
6.3
4.8
5.2
PUFAs
For small specimens (n ¼ 4), data are mean + SD; for large specimens (n ¼
2), mean + range.
Abbreviation: NMI, nonmethylene-interrupted fatty acids.
Figure 7. Stable carbon and nitrogen isotope compositions of small
(open symbols) and large (solid symbols) individuals of Conchocele bisecta
from methane-rich vents area off Paramushir Island. Average values of
all soft tissue samples analysed for each individual are shown. Shell
lengths (mm) of individuals are given as numeric labels.
Bathymodiolinae that contain only sulphur-oxidizing bacteria
are also characterized by a high content of n– 7 MUFAs
(Ben-Mlih et al., 1992), whereas Bathymodiolinae that harbour
only methanotrophs in most cases also contain n –8 MUFA
(Raggi et al., 2013).
The majority of bivalves are rich in n –3 and n– 6 C20 and
C22 PUFAs, obtained from planktonic by filter feeding. These
FAs are considered essential for normal vital functions in invertebrates (Parrish, 2009 and references therein), but chemosynthetic bacteria do not synthesize them. Deep-sea bivalves with
chemoautotrophic symbionts compensate for the lack of essential
n–3 and n–6 PUFAs by synthesis of high levels of unusual
n–4 PUFAs (Saito, 2008). Those Bathymodiolinae with
methane-oxidizing bacteria have methylene-interrupted n– 4
PUFAs, while Vesicomyidae containing sulphur-oxidizing bacteria have NMI n–4 PUFAs (Saito, 2008).
We found high concentrations of 16:1 (n–7) in the gills and
up to 3.2% NMI n –4 PUFAs in the foot of C. bisecta (Table 4).
Figure 8. Large specimens and empty shells of Conchocele bisecta and a
commercially valuable triangle tanner crab, Chionoecetes angulatus, in the
methane-rich vents area off Paramushir Island (depth 777 m).
The n –8 MUFAs characteristic of symbiotic and free-living
methanotrophs were absent. According to these indicators,
C. bisecta was most similar to bivalves containing sulphuroxidizing symbionts, such as Vesicomyidae. However, the concentrations of n –4 PUFAs in C. bisecta were much lower than in
deep-sea symbiont-containing bivalves, where n –4 PUFAs constituted 24–35% of the total FA content and up to 100% of
PUFAs (Saito, 2008).
400
THYASIRIDS FROM METHANE SEEPS IN SEA OF OKHOTSK
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SUPPLEMENTARY MATERIAL
Supplementary material is available at Journal of Molluscan
Studies online.
ACKNOWLEDGEMENTS
We are grateful to the captain and the crew of the RV Akademik
M.A. Lavrentyev. We thank all members of the expedition for
their cooperation. This project was supported by the Russian
Science Foundation (grant no. 14-14-00232 to A.V. Adrianov).
GMK is very grateful to A.V. Gebruk, E.M. Krylova, T.N.
Molodtsova, all members of the Laboratory of Ocean Bottom
Fauna (IO RAS), H. Saito (NMNS), M.A. Frey, H. Gartner
(RBCM), as well as to I.A. Dyachenko and N.V. Kameneva
(MIMB) for their help with bivalve collections in their care. The
morphological study by GMK was supported by the Russian
Foundation for Basic Research (grant no. 14-04-00872-a).
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