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Unusual methylene-interrupted polyunsaturated fatty acids of abyssal and hadal invertebrates
Progress in Oceanography 178 (2019) 102132
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Unusual methylene-interrupted polyunsaturated fatty acids of abyssal and hadal invertebrates Vladimir I. Kharlamenkoa, Nelly A. Odintsovab,
T
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a Laboratory of Comparative Biochemistry, National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690041, Russia b Cytotechnology Laboratory, National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690041, Russia
A R T I C LE I N FO
A B S T R A C T
Keywords: Deep-sea ecosystems Megabenthic invertebrates Methylene-interrupted polyunsaturated fatty acids The Kuril-Kamchatka Trench The North-West Pacific region
The purpose of this study was to identify specific profiles of unusual methylene-interrupted polyunsaturated fatty acids (PUFAs) in megabenthic invertebrates inhabiting the Kuril-Kamchatka Trench, adjacent areas of the Pacific Ocean and the Sea of Okhotsk at the depths between 3206 and 9581 m. The analyzed taxa contained twelve of unusual methylene-interrupted PUFAs. The fatty acid (FA) 21:4n-7 was commonly found in many megabenthic invertebrates. A high level of long-chain FA 26:7n-3 was detected in abyssal ophiurioides. Some unusual n-7 FAs (possibly, derived from bacterial precursors) were found in the thyasirid bivalve Axinulus sp., which did not host chemotrophic symbionts, but could use pedal feeding. The sea urchin Kamptosoma sp. and the starfish Eremicaster sp. contained n-8 and n-5 PUFAs, typical of the Foraminifera living in the Kuril-Kamchatka Trench. The dominant species of holothurians at the bottom of the Kuril-Kamchatka Trench contained 21:4n-6 and 23:2n-6. An unusual (uncommon) very long-chain FA 32:5n-3 was found in the glass sponge Hyalonema sp. This data can be used to select model taxa for screening biologically active substances. Considering the low biomass levels of deep-sea invertebrates, the development of their cell cultures will be needed for further progress in this field.
1. Introduction Many secondary metabolites of marine invertebrates inhabiting shallow benthic communities have high antioxidant, antibacterial, antifungal, and antitumor activities (Volkman, 1999; Cooper, 2004) and have been used in medicine as remedies for 4000 years (Evans-Illidge et al., 2013). Marine-derived drugs are a chemical novelty, and the diversity of marine-derived natural products exceeds that of terrestrial sources (Bergé and Barnathan, 2005; Martins et al., 2014; Blagodatski et al., 2017). Deep-sea multicellular organisms could be novel sources of valuable biologically active substances (BAS) because they often live in extreme conditions, for example, withstanding considerable ocean pressure. In addition, the trophic resources of marine invertebrates inhabiting the abyssal (i.e., depths greater than 3000 m) and hadal (i.e., depths greater than 6000 m) zones differ from the trophic resources utilized by marine invertebrates dwelling at shallower depths. The main producer of organic matter in the euphotic zone of marine ecosystems is phytoplankton synthesizing different PUFAs (Hama, 1991) which are then transferred to its consumers. Deep-sea piezophilic bacteria could also synthesize PUFAs (Fang et al., 2004; De Carvalho and Caramujo,
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2012). Moreover, some foraminifera could contain a high level of 20:4n-6 (Larkin et al., 2014). Nematodes, participating in a trophic upgrade in deep-sea ecosystems, synthesize PUFAs too (Leduc et al., 2015; Van Campenhout and Vanreusel, 2016). As organic matter sinks down from the euphotic zone to the abyssal zone, the percentage of PUFAs is reduced, and the trophic value of the organic matter declines (Wakeham et al., 1997; Shin et al., 2002; Xu et al., 2018). However, benthic invertebrates from deep-sea ecosystems can consume food that has undergone a trophic upgrade—that is, when PUFAs are synthesized from saturated and monounsaturated FAs produced by bacteria or protozoans (DeLong and Yayanos, 1986; Breteler et al., 1999; De Carvalho and Caramujo, 2012). PUFAs, which were obtained as a result of such a trophic upgrade, may differ in structure from common n-3 and n-6 PUFAs (Ben-Mlih et al., 1992; Pranal et al., 1997; Allen et al., 2001; Saito, 2008; Colaço et al., 2009). Benthic invertebrates from deep-sea ecosystems can considerably reduce the content of n-3 and n-6 PUFAs, at the same time the levels of n-4 and n-7 PUFAs can be high. The reasons of the appearance of odd FAs 21:4n-6 and 23:2n-6 in the holothurian Prototrochus sp. and the sea cucumber Elpidia hansoni living at a maximal depth of the Kuril-Kamchatka
Corresponding author. E-mail addresses: [email protected] (V.I. Kharlamenko), [email protected] (N.A. Odintsova).
https://doi.org/10.1016/j.pocean.2019.102132
Available online 19 July 2019 0079-6611/ © 2019 Elsevier Ltd. All rights reserved.
Progress in Oceanography 178 (2019) 102132
V.I. Kharlamenko and N.A. Odintsova
Trench are still unclear. The information on the ability to produce unusual substances, such as, for example, novel FAs or glycerophospholipids or sphingolipids, can be important when choosing model taxa for screening BAS for medical purposes (Blagodatski et al., 2017). In a review (Bergé and Barnathan, 2005), sponges were mentioned as a source of numerous unusual long-chain Δ5,9 FAs (Carballeira and Reyes, 1990; Suh et al., 2012; Ruocco et al., 2016), also found in molluscs, echinoderms and several other marine invertebrates (Barnathan, 2009). Moreover, nonmethylene-interrupted FAs with different structures were detected in some Cnidaria, Mollusca, Echinodermata, and Arthropoda (Kornprobst and Barnathan, 2010). Methylene-interrupted PUFAs, for example, n-4 and n-7 PUFAs, were found in many invertebrates, including symbiotrophic bivalves (Saito, 2008). Very long methylene-interrupted PUFAs, such as 26:7n-3, were previously detected in bathyal ophiurioids and medusae living in the shelf (Svetashev and Kharlamenko, 2015; Svetashev, 2019). The tetracosapolyenoic acids 24:6n-3 and 24:5n-6 have been found in shelf octocorals (Vysotskii and Svetashev, 1991), as well as in ophiurioids and crinoids (Takagi et al., 1986). Until recently, nothing was known about unusual PUFAs in abyssal and hadal invertebrates. The idea of obtaining novel valuable substances from deep-sea organisms in the sufficient quantities has attracted the attention of scientists for long time. However, the samples of such deep-sea marine invertebrates are not readily available. Moreover, their low biomass and low growth rates are the major limitations of this research. The pursuit of such research could be connected with the cultivation of deep-sea invertebrate cells. Generally, the interest in cell cultivation has increased over the last years due to the discovery of a more and more broad spectrum of various secondary metabolites of marine origin (Ballarin et al., 2018). However, cell cultures of deep-sea organisms have never been used to obtain marine lipids, such as plasmalogens, phospholipids, glycolipids, or diverse FAs. During three expeditions to the Kuril-Kamchatka Trench, adjacent regions of the Pacific Ocean (abyssal plain) and the Sea of Okhotsk (the Kuril Basin), we collected samples of common benthic invertebrate species with sufficient biomass allowing us to analyse their FA composition. In deep-sea foraminifera, numerous PUFAs have been detected previously (Kharlamenko et al., 2017; Kharlamenko, 2018). This study presents the data on the detection of unusual FAs in deep-sea megabenthic invertebrates which are potential consumers of these foraminifera. In addition, a suggestion on the use of unusual FAs as BAS via their production in cell cultures of deep-sea organisms is also discussed.
Fig. 1. A location map of stations where megabenthic invertebrates were sampled.
was filtered, and the residue was repeatedly extracted in chloroformmethanol (2:1 v/v). The extracts were mixed and separated into layers by adding H2O and chloroform. The lower layer was evaporated, and the remaining total lipids obtained were dissolved in chloroform and stored at −80 °C. Methyl esters of FAs were prepared according to (Carreau and Dubacq, 1978) and purified by TLC in benzene. The 4,4dimethyloxazoline (DMOX) derivatives of methyl esters of FAs were prepared according to (Svetashev, 2011).
2.3. Analysis of fatty acids using gas chromatography Methyl esters of FAs were analysed on a Shimadzu GC2010 Plus gas chromatograph (Japan) using a quartz capillary column (length 30 m, internal diameter 0.25 mm; Supelcowax 10; Supelco, USA). The temperature of the column was 205 °C, and the temperature of the injector and detector was 250 °C, the analysis time was 90 min. Helium was used as a carrier gas. Mass spectrometry of methyl esters and DMOX derivatives was performed on a Shimadzu GC-MS QP5050A mass spectrometer (Japan) using an MDN-5S column (30 m long, 0.25 mm internal diameter, Supelco) or Supelcowax 10 (Supelco) column. The MDN-5S column had an initial column temperature (160 °C) that was increased to 260 °C at 2 °C/min. It remained at 260 °C for an additional 25 min. Some samples of the DMOX derivatives were performed on polar Supelcowax 10 column at 215 °C under isothermal conditions.
2. Materials and methods 2.1. Sampling
2.4. Mass spectrometry analysis
Three sampling periods were performed (July–September 2012, July-August 2015, and August-September 2016) during the joint German-Russian expeditions KuramBio, SokhoBio and KuramBio II on board the RV Sonne (cruises SO-223 and SO-250) and on board the RV Akademic M.A. Lavrentiev (cruise LV71) to the Kuril-Kamchatka Trench, abyssal plain, and the Kuril Basin of the Sea of Okhotsk. Invertebrates were collected at 30 abyssal and hadal stations (Fig. 1) at depths between 3206 and 9581 m using an Agassiz trawl and an epibenthic sledge. No experiments were conducted on animals of endangered or protected species.
The mass spectra were recorded at 70 eV. The spectra were compared with the NIST library 2.0 and an online fatty acid mass spectra archive site (http://www.lipidhome.co.uk/ms/masspec.html, 2019). The data on the FA composition is presented as percentage of total fatty acids.
2.5. Statistical analysis The statistical analysis was performed with Statistica 8 and Primer 6 statistical packages. The results were subjected to one-way analysis of variance (ANOVA) using Office Excel 2013 software (Microsoft Corporation, USA) to test whether the values of the means from each experimental group were significantly different. A p-value < 0.05 was considered statistically significant in the analysis of all data.
2.2. Total lipid extraction and preparation of FA derivatives Invertebrate specimens were rinsed in distilled water, placed in vials filled with chloroform-methanol (1:2 v/v), and stored in a freezer at −80 °C. Lipids of deep-sea invertebrates were extracted using the method described in (Bligh and Dyer, 1959). The obtained homogenate 2
Progress in Oceanography 178 (2019) 102132
V.I. Kharlamenko and N.A. Odintsova
Table 1 Invertebrates sampled from the abyssal and hadal zones of the Kuril-Kamchatka Trench, adjacent areas of the Pacific Ocean and the Sea of Okhotsk. Taxa
The KurilKamchatka Trench
Abissal plan of the Pacific Ocean
The Kuril Basin
Species*
Samples
Species
Samples
Species
Samples
3 0 16
3 0 45
1 3 9
1 5 10
9 5 2
20 22 13
12
20
0
0
3
34
3 2 9 1 0 2 11 3 1 1 1 1 0 0 66
4 2 54 1 0 4 12 6 10 3 4 5 0 0 173
2 1 5 3 4 3 9 4 3 0 1 4 1 0 53
4 2 9 3 4 6 21 8 6 0 4 6 2 1 92
0 3 5 0 0 2 9 2 3 0 0 6 0 1 50
0 12 18 0 0 9 66 6 11 0 0 34 0 3 248
Table 2 Maximal concentration of major unusual methylene-interrupted PUFAs in lipids of deep-sea megabenthic invertebrates from the abyssal and hadal zones of the Kuril-Kamchatka Trench, adjacent areas of the Pacific Ocean and the Sea of Okhotsk (only the fatty acids contributing > 0.4% were considered). Taxa
Porifera Anthozoa Polychaeta (Sedentaria and Errantia) Polychaeta (Echiuroidea) Pogonophora Sipuncula Bivalvia Gastropoda Scaphopoda Echinoidea Holothurioidea Asteroidea Ophiuroidea Crinoidea Ascidiacea Crustacea Hydrozoa Bryozoa Total
* Preliminary species identification.
Maximal concentration (% of total fatty acids) 21:4n-7
24:6n-3
26:7n-3
Anthozoa Pennatulacea Scleractinia Actiniaria
0.5 ± 0.1 2.5 ± 0.2 2.3 ± 0.2
7.0 ± 0.5 n.d. 0.8 ± 0.1
n.d. n.d. n.d.
Polychaeta Sedentaria and Errantia Echiura
5.6 ± 0.5 1.8 ± 0.1
n.d. n.d.
n.d. n.d.
Sipuncula Sipunculidea
2.0 ± 0.2
n.d.
n.d.
Mollusca Scaphopoda Bivalvia Gastropoda
3.0 ± 0.2 2.5 ± 0.2 10.6 ± 0.5
n.d. n.d. n.d.
n.d. n.d. n.d.
Echinodermata Echinoidea Holothuroidea Asteroidea Ophiuroidea Crinoidea
4.0 2.5 5.1 0.8 0.9
n.d. n.d. n.d. 20.5 ± 0.5 9.9 ± 0.5
n.d. n.d. n.d. 14.5 ± 0.5 n.d.
± ± ± ± ±
0.3 0.2 0.4 0.1 0.1
n.d. – not detected. In most cases, more than one sample per analysed specimen was used. Error bars indicate ± standard deviation; n = 3.
3. Results 3.1. Analysis of fatty acids in lipids of abyssal and hadal megabenthic invertebrates
Table 3 Minor unusual methylene-interrupted PUFAs from deep-sea megabenthic invertebrates sampled at the Kuril Basin of the Sea of Okhotsk (KB) and the KurilKamchatka Trench (KKT).
513 samples taking from 1250 collected abyssal and hadal megabenthic invertebrates were analysed. In most cases, more than one sample per analysed specimen was used (Table 1, Table 3). In total, we analysed 169 different species: 66 species from the Kuril Kamchatka Trench, 53 species from abissal plan of the Pacific Ocean and 50 species from the Kuril Basin of the Okhotsk Sea. Every species was unique for its location. There was no significant difference among all stations. In some of the analysed organisms, unusual methylene-interrupted PUFAs (21:4n-7, 24:6n-3 and 26:7n-3) made up a relatively large proportion. Only the FAs contributing > 0.4% were considered. The FA 21:4n-7 was found in many marine invertebrates analysed in this study, and its content is characteristic of some taxonomic groups (Table 2). The greatest content of 21:4n-7 was detected in gastropods (10.6 ± 0.5), but high levels of this FA were also found in polychaetes, starfish, and sea urchins (Table 2). Lipids of ophiurioids, sea lilies, and sea pens contained high levels of 24:6n-3: the percentage of 24:6n-3 in ophiurioids reached up to 20.5% (the Kuril-Kamchatka Trench). The FA 26:7n-3 was only found in six species from 13 ophiurioids studied, wherein the content was the highest (14.5%) in ophiurioids collected in the Kuril Basin (the Sea of Okhotsk). Some other unusual methyleneinterrupted PUFAs in megabenthic invertebrates were only found as minor PUFAs (Table 3). FAs, 22:4n-8 and 22:5n-5, were detected in the sea urchin Kamptosoma sp. and the starfish Eremicaster sp. The unusual FA 32:5n-3 together with non-methylene-interrupted FAs, which are typical for glass sponges, were found in the glass sponge Hyalonema sp. (Table 3). The distribution of unusual methylene-interrupted PUFAs from deep-sea megabenthic invertebrates sampled at the Kuril Basin of the Sea of Okhotsk and the Kuril-Kamchatka Trench is presented in a supplementary (Table S1). This table provides a more detailed species list (wherever possible) and the information on quantity and type of the PUFAs found.
Unusual FA
Species
32:5n-3 18:3n-7 20:3n-7 22:3n-7 22:4n-8 22:5n-5
Concentration (% of total fatty acids)
Area
Depth, m
Sponges Hyalonema sp.
2.4 ± 0.3
KB
3301
Bivalvia Axinulus sp. Axinulus sp. Axinulus sp.
2.4 ± 0.9 1.7 ± 0.7 0.6 ± 0.2
KKT KKT KKT
9293 9293 9293
1.7 ± 0.3
KKT
5109
1.7 ± 0.1
KKT
5109
Echinoidea Kamptosoma abyssale Kamptosoma abyssale
22:4n-8 22:5n-5
Asteroidea Eremicaster sp. Eremicaster sp.
1.0 ± 0.2 2.8 ± 0.5
KKT KKT
6204 6209
23:2n-6 21:4n-6
Holothuroidea Elpidia hansoni Prototrochus sp.
1.5 ± 0.1 3.8 ± 0.1
KKT KKT
9581 9581
3.2. Mass spectrometry analysis of the DMOX derivatives of FAs The bivalve mollusc Axinulus sp. that was sampled at a depth of over 9 km contained n-7 series PUFAs (Table 3 and Table S1). The mass spectra of the DMOX derivatives of some new FAs (18:3n-7, 20:3n-7 and 22:3n-7) are presented in Fig. S1. One of the notable components of the holothurian Prototrohus sp. from the hadal zone of the Kuril Kamchatka Trench was FA 21:4n-6, whereas the dominant holothurian species in this area (the sea cucumber Elpidia hansoni) contained only FA 23:2n-6 (Table 3 and Table S1). The mass spectra of the DMOX derivatives of these FAs in lipids of hadal holothurians are presented in 3
Progress in Oceanography 178 (2019) 102132
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in the species studied have previously been described for abyssal deposit feeders (Kharlamenko et al., 2018). We found n-7 PUFAs in the thyasirid bivalve Axinulus sp., which does not have host chemotrophic symbionts but may use pedal feeding (Zanzerl and Dufour, 2017). Thus, these FAs could derive from bacterial precursors. The FA 21:4n-6 is an isomer of 21:4n-7, although their biosynthesis pathways may be different. The origin of the FA 32:5n-3 in the glass sponge Hyalonema sp. is unclear. We suggest that the potential origin of this very long-chain FA may be connected with bacterial precursors derived from deep-sea bacteria living at high pressure (DeLong and Yayanos, 1986). The presence of this FA together with high levels of very long FAs 30:4n-6 and 30:5n-3 found in Cliona celata (Litchfield et al., 1979), as well as 34:5n-3 found in the sponge Petrosia pellasarca (Carballeira and Reyes, 1990), can explain the adaptation of cellular membranes of deep-sea organisms to lipid-solidifying effects, caused by low temperatures and high ocean pressure (MacDonald, 1984). The longest methylene-interrupted PUFA that is common in the sponges analysed in this study is 22:6n-3, while C. celata has been reported to be the source of many BAS (Suh et al., 2012; Ruocco et al., 2016), although so far there exists only one study where very longchain FAs could be used as potentially active substances for precursors of lipid mediators (Sassa and Kihara, 2014). There is very few data on the biological activity of unusual PUFAs found in abyssal and hadal invertebrates. For example, the PUFA 21:4n7 from the marine opisthobranch Scaphander lignarius, not isolated before in it, was shown to be a significantly more potent cytotoxic agent than common PUFAs, such as 20:4n-6 and 20:5n-3 (Van Campenhout and Vanreusel, 2016). The PUFA 24:6n-3, found in some fishes, brittle stars and jellyfish, exerted the strongest suppression on the synthesis of some lipids and the mRNA levels relating to lipid metabolism in human hepatoma HepCr cells (Nagao et al., 2014). The brittle star (the ophiurioid) Ophiura irrorata from the Kuril Basin of the Sea of Okhotsk, having 26:7n-3 in its glycerophospholipids, showed highly specific antiWnt activities targeting multiple levels within the Wnt signaling pathway, which is one of the key factors in oncogenic transformation, growth and metastasis in different cancers (Blagodatski et al., 2017). As mentioned before, from our own data (Kharlamenko et al., 2017; Kharlamenko, 2018; this study) and from the data of other authors (Koyama, 2007), it is quite challenging to study deep-sea marine invertebrates due to their low biomass levels and extremely low growth rates. Therefore, it seems too complicated to obtain the amount of material from these sources that would be sufficient for full-fledged research or practical use. This can be remedied through the cultivation of deep-sea multicellular organism cells. Primary cell cultures of different marine invertebrate species have been successfully obtained, but all studies have only been carried out on shelf species (Odintsova and Khomenko, 1991; Odintsova et al., 2005; Pomponi, 2006; Gordaliza, 2010; Odintsova et al., 2011; Rinkevich, 2011; Ageenko et al., 2014; Maiorova and Odintsova, 2015; Ryazanova et al., 2015; Odintsova et al., 2017; Ballarin et al., 2018; Boroda et al., 2019). Marine invertebrate species contain a large number of novel bioactive molecules, many of which are of significant potential interest for human health (Ballarin et al., 2018). An attempt to cultivate deep-sea multicellular organisms (from a depth of 1162 m) was undertaken by Japanese researchers; it was demonstrated, that special aquarium equipment that takes into account high hydrostatic pressures and low seawater temperatures, was necessary for cultivating living deep-sea organisms, whereas it was not needed for cultivating cells of these organisms (Koyama, 2007). Unfortunately, no comparative information on deep-sea organisms and their cells was provided in this article, and the true causes of this phenomenon remain unclear. If successful, such work will make cells of these deep-sea animal available (for example, through cell freezing in liquid nitrogen) for a further study of their various properties. Stem cell cultures of marine
Fig. S2. 4. Discussion The initial links of food webs in abyssal and hadal ecosystems can substantially change the composition of the organic matter that is deposited from the euphotic zone and is poor in PUFAs. The first provider of a trophic upgrade in deep-sea ecosystems are piezophilic bacteria dwelling in bottom sediments and synthesizing mainly n-3 PUFAs, 20:5n-3 and 22:6n-3 (Fang et al., 2004). The second provider of a trophic upgrade occupying deep-sea ecosystems are foraminifera containing elevated levels of 20:4n-6 (Kharlamenko et al., 2017). Furthermore, foraminifera could synthesize 20:4n-6 FA themselves as pointed in (Larkin et al., 2014). The third and less studied provider of a trophic upgrade in deep-sea ecosystems are nematodes (Leduc et al., 2015; Van Campenhout and Vanreusel, 2016; Mordukhovich et al., 2018), which having the ability to synthesize PUFAs, that are not present in their food sources (Leduc and Probert, 2009). Following the analysis of the FA composition of megabenthic invertebrates from the Kuril-Kamchatka Trench and adjacent regions of the Pacific Ocean, we found a wide variety of unsual FAs. For example, we detected the FA 21:4n-7 in many abyssal and hadal benthic invertebrates, thus contradicting the previous view that this FA is rare (Vasskog et al., 2012). Like other authors (Takagi et al., 1986; Vysotskii and Svetashev, 1991), we found 24:6n-3 in Anthozoa and Echinodea, but there was no noticeable concentration of the very long-chain PUFA 24:5n-6, the high levels of which were previously found in Octocorallia (Vysotskii and Svetashev, 1991). The FA 24:6n-3 could be the precursor of another rare FA: 26:7n-3 (Svetashev and Kharlamenko, 2015), found in abyssal ophiurioids at a high level (14.5 ± 0.5). However, the content of this FA (26:7n-3) did not exceed 5% in bathyal ophiurioids (Svetashev and Kharlamenko, 2015). Benthic foraminifera have previously been described as the main source of methylene-interrupted PUFAs in deep-sea benthic ecosystems (Kharlamenko, 2018). Although, the composition of PUFAs of deep-sea benthic foraminifera has been described earlier in several papers (Gooday et al., 2002; Suhr et al., 2003; Larkin, 2006; Würzberg et al., 2011), the authors indicated only major PUFAs, having omitted possible minor or rare PUFAs. The discovery of a large number of novel FAs might have been triggered by the sampling method on the one hand, and the FAs structural analysis based on the DMOX derivatives on the other. Foraminifera samples have usually been collected using a giant box corer or multiple corer (Gooday et al., 2002; Suhr et al., 2003; Larkin, 2006; Würzberg et al., 2011). We used an epibenthic sledge for the first time. The sledge catches an area of about one thousand square meters in contrast to a sampling area of a giant box corer (0.25 m2) or a sampling area of multiple corer (0.0025 m2). Thus, the sledge captures much more foraminifera. In addition, we could not previously find any novel FAs when the structure of the FAs was determined by comparison with standard kits of FAs. The use of these methods of collecting and analyzing samples allowed us to find nine novel and three rare PUFAs in just three common species of foraminifera (Kharlamenko, 2018). To continue these studies, we analysed the FA composition of benthic invertebrates who fed on foraminifera. The sea urchin Kamptosoma sp. and the starfish Eremicaster sp. were especially noticeable in this regard: two PUFAs characteristic of foraminifera were found in their tissues. The discovery of novel PUFAs provides the possibility to determine more accurately the role of foraminifera as food source for benthic invertebrates. However, we cannot exclude the fact that some unusual FAs may be of nematode origin since the data on the FA composition of deep-sea nematodes were very variable and highlighted the importance of sediment bacteria (Mordukhovich et al., 2018). Based on the results of the analysis of sediment samples, we do not exclude also that the sediments may contain the amounts of unusual PUFAs comparable with those in deep-sea nematodes. Most of the FAs 4
Progress in Oceanography 178 (2019) 102132
V.I. Kharlamenko and N.A. Odintsova
invertebrates are a novel model system characterized by a high level of physiological and synthetic processes. The production of BAS in vitro may be an alternative to the chemical synthesis and aquaculture (Odintsova, 2009).
Appendix A. Supplementary material
5. Conclusion
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Large-scale studies of the composition of FAs of marine megabenthic invertebrates of abyssal and hadal ecosystems in the North-West Pacific region have revealed twelve unusual methylene-interrupted PUFAs. Together with the previously presented data on unusual FAs of abyssal and hadal foraminifera, this data significantly complements our knowledge of marine methylene-interrupted PUFAs and can be used to select model taxa for screening BAS for medical purposes. Some further progress in this field could become possible when cell cultures of deepsea invertebrates are obtained. We assume that the next stage of the research will include holothurians, brittle stars (ophiurioids), and sponges as model taxa. This is also important to take into account the presence and abundance of unusual methylene-interrupted PUFAs found in deep-sea organisms.
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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.pocean.2019.102132.
Author contributions V.K. designed and coordinated the research, prepared the manuscript parts concerning sampled material, analyzed FA composition of invertebrates. V.K. and N.O. equally contributed to writing the MS, read and approved the final version of the MS. Funding The material was collected and sorted within the framework of several large international projects. The project SoJaBio was financially supported by the German Research Foundation (Br 1121/37-1), both KuramBio I and II projects and the SokhoBio Project by the PTJ (German Ministry for Science and Education), grant 03G0857A, KuramBio I BMBF grant 03G0223A, as well as KuramBio II BMBF grant 03G0250A to Prof. Dr. Angelika Brandt, University of Hamburg, now Senckenberg Museum, Frankfurt, Germany. The projects were also financially supported by the Russian Foundation of Basic Research (projects 13-04-02144, 16-04-01431; 16-04-01477), the Council of the President of the Russian Federation (project MК-2599.2013.4), Russian Federation Government grant No 11.G34.31.0010, grant of Presidium of the Far East Branch of RAS (12–I–P30–07), Otto Schmidt Laboratory grant (OSL-14-15). The taxonomic processing of the different taxa was supported by the Russian Science Foundation (14-50-00034); biogeography and distribution study was supported by the Russian Ministry of Science and Education (Project 14.616.21.0077, ID No. RFMEF161617X0077). Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments We are grateful to the coordinators of the Study Prof. Angelika Brandt and Dr. Marina Malyutina. We wish to thank the captains and the crews of the R/V Sonne and R/V Akademik M.A. Lavrentyev for their assistance on board. We thank all members of these expeditions including all student-helpers and technicians for support and help with sorting of the extensive expedition material. We are grateful to Dr. V.I. Svetashev and Dr. Ekaterina Ermolenko for their help in preparing the paper and understanding how to answer the Review questions. We thank Dr. A.V. Boroda and Dr. G.M. Kamenev for their help in preparing the paper. 5
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Unusual methylene-interrupted polyunsaturated fatty acids of abyssal and hadal invertebrates Vladimir I. Kharlamenkoa, Nelly A. Odintsovab,
T
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a Laboratory of Comparative Biochemistry, National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690041, Russia b Cytotechnology Laboratory, National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690041, Russia
A R T I C LE I N FO
A B S T R A C T
Keywords: Deep-sea ecosystems Megabenthic invertebrates Methylene-interrupted polyunsaturated fatty acids The Kuril-Kamchatka Trench The North-West Pacific region
The purpose of this study was to identify specific profiles of unusual methylene-interrupted polyunsaturated fatty acids (PUFAs) in megabenthic invertebrates inhabiting the Kuril-Kamchatka Trench, adjacent areas of the Pacific Ocean and the Sea of Okhotsk at the depths between 3206 and 9581 m. The analyzed taxa contained twelve of unusual methylene-interrupted PUFAs. The fatty acid (FA) 21:4n-7 was commonly found in many megabenthic invertebrates. A high level of long-chain FA 26:7n-3 was detected in abyssal ophiurioides. Some unusual n-7 FAs (possibly, derived from bacterial precursors) were found in the thyasirid bivalve Axinulus sp., which did not host chemotrophic symbionts, but could use pedal feeding. The sea urchin Kamptosoma sp. and the starfish Eremicaster sp. contained n-8 and n-5 PUFAs, typical of the Foraminifera living in the Kuril-Kamchatka Trench. The dominant species of holothurians at the bottom of the Kuril-Kamchatka Trench contained 21:4n-6 and 23:2n-6. An unusual (uncommon) very long-chain FA 32:5n-3 was found in the glass sponge Hyalonema sp. This data can be used to select model taxa for screening biologically active substances. Considering the low biomass levels of deep-sea invertebrates, the development of their cell cultures will be needed for further progress in this field.
1. Introduction Many secondary metabolites of marine invertebrates inhabiting shallow benthic communities have high antioxidant, antibacterial, antifungal, and antitumor activities (Volkman, 1999; Cooper, 2004) and have been used in medicine as remedies for 4000 years (Evans-Illidge et al., 2013). Marine-derived drugs are a chemical novelty, and the diversity of marine-derived natural products exceeds that of terrestrial sources (Bergé and Barnathan, 2005; Martins et al., 2014; Blagodatski et al., 2017). Deep-sea multicellular organisms could be novel sources of valuable biologically active substances (BAS) because they often live in extreme conditions, for example, withstanding considerable ocean pressure. In addition, the trophic resources of marine invertebrates inhabiting the abyssal (i.e., depths greater than 3000 m) and hadal (i.e., depths greater than 6000 m) zones differ from the trophic resources utilized by marine invertebrates dwelling at shallower depths. The main producer of organic matter in the euphotic zone of marine ecosystems is phytoplankton synthesizing different PUFAs (Hama, 1991) which are then transferred to its consumers. Deep-sea piezophilic bacteria could also synthesize PUFAs (Fang et al., 2004; De Carvalho and Caramujo,
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2012). Moreover, some foraminifera could contain a high level of 20:4n-6 (Larkin et al., 2014). Nematodes, participating in a trophic upgrade in deep-sea ecosystems, synthesize PUFAs too (Leduc et al., 2015; Van Campenhout and Vanreusel, 2016). As organic matter sinks down from the euphotic zone to the abyssal zone, the percentage of PUFAs is reduced, and the trophic value of the organic matter declines (Wakeham et al., 1997; Shin et al., 2002; Xu et al., 2018). However, benthic invertebrates from deep-sea ecosystems can consume food that has undergone a trophic upgrade—that is, when PUFAs are synthesized from saturated and monounsaturated FAs produced by bacteria or protozoans (DeLong and Yayanos, 1986; Breteler et al., 1999; De Carvalho and Caramujo, 2012). PUFAs, which were obtained as a result of such a trophic upgrade, may differ in structure from common n-3 and n-6 PUFAs (Ben-Mlih et al., 1992; Pranal et al., 1997; Allen et al., 2001; Saito, 2008; Colaço et al., 2009). Benthic invertebrates from deep-sea ecosystems can considerably reduce the content of n-3 and n-6 PUFAs, at the same time the levels of n-4 and n-7 PUFAs can be high. The reasons of the appearance of odd FAs 21:4n-6 and 23:2n-6 in the holothurian Prototrochus sp. and the sea cucumber Elpidia hansoni living at a maximal depth of the Kuril-Kamchatka
Corresponding author. E-mail addresses: [email protected] (V.I. Kharlamenko), [email protected] (N.A. Odintsova).
https://doi.org/10.1016/j.pocean.2019.102132
Available online 19 July 2019 0079-6611/ © 2019 Elsevier Ltd. All rights reserved.
Progress in Oceanography 178 (2019) 102132
V.I. Kharlamenko and N.A. Odintsova
Trench are still unclear. The information on the ability to produce unusual substances, such as, for example, novel FAs or glycerophospholipids or sphingolipids, can be important when choosing model taxa for screening BAS for medical purposes (Blagodatski et al., 2017). In a review (Bergé and Barnathan, 2005), sponges were mentioned as a source of numerous unusual long-chain Δ5,9 FAs (Carballeira and Reyes, 1990; Suh et al., 2012; Ruocco et al., 2016), also found in molluscs, echinoderms and several other marine invertebrates (Barnathan, 2009). Moreover, nonmethylene-interrupted FAs with different structures were detected in some Cnidaria, Mollusca, Echinodermata, and Arthropoda (Kornprobst and Barnathan, 2010). Methylene-interrupted PUFAs, for example, n-4 and n-7 PUFAs, were found in many invertebrates, including symbiotrophic bivalves (Saito, 2008). Very long methylene-interrupted PUFAs, such as 26:7n-3, were previously detected in bathyal ophiurioids and medusae living in the shelf (Svetashev and Kharlamenko, 2015; Svetashev, 2019). The tetracosapolyenoic acids 24:6n-3 and 24:5n-6 have been found in shelf octocorals (Vysotskii and Svetashev, 1991), as well as in ophiurioids and crinoids (Takagi et al., 1986). Until recently, nothing was known about unusual PUFAs in abyssal and hadal invertebrates. The idea of obtaining novel valuable substances from deep-sea organisms in the sufficient quantities has attracted the attention of scientists for long time. However, the samples of such deep-sea marine invertebrates are not readily available. Moreover, their low biomass and low growth rates are the major limitations of this research. The pursuit of such research could be connected with the cultivation of deep-sea invertebrate cells. Generally, the interest in cell cultivation has increased over the last years due to the discovery of a more and more broad spectrum of various secondary metabolites of marine origin (Ballarin et al., 2018). However, cell cultures of deep-sea organisms have never been used to obtain marine lipids, such as plasmalogens, phospholipids, glycolipids, or diverse FAs. During three expeditions to the Kuril-Kamchatka Trench, adjacent regions of the Pacific Ocean (abyssal plain) and the Sea of Okhotsk (the Kuril Basin), we collected samples of common benthic invertebrate species with sufficient biomass allowing us to analyse their FA composition. In deep-sea foraminifera, numerous PUFAs have been detected previously (Kharlamenko et al., 2017; Kharlamenko, 2018). This study presents the data on the detection of unusual FAs in deep-sea megabenthic invertebrates which are potential consumers of these foraminifera. In addition, a suggestion on the use of unusual FAs as BAS via their production in cell cultures of deep-sea organisms is also discussed.
Fig. 1. A location map of stations where megabenthic invertebrates were sampled.
was filtered, and the residue was repeatedly extracted in chloroformmethanol (2:1 v/v). The extracts were mixed and separated into layers by adding H2O and chloroform. The lower layer was evaporated, and the remaining total lipids obtained were dissolved in chloroform and stored at −80 °C. Methyl esters of FAs were prepared according to (Carreau and Dubacq, 1978) and purified by TLC in benzene. The 4,4dimethyloxazoline (DMOX) derivatives of methyl esters of FAs were prepared according to (Svetashev, 2011).
2.3. Analysis of fatty acids using gas chromatography Methyl esters of FAs were analysed on a Shimadzu GC2010 Plus gas chromatograph (Japan) using a quartz capillary column (length 30 m, internal diameter 0.25 mm; Supelcowax 10; Supelco, USA). The temperature of the column was 205 °C, and the temperature of the injector and detector was 250 °C, the analysis time was 90 min. Helium was used as a carrier gas. Mass spectrometry of methyl esters and DMOX derivatives was performed on a Shimadzu GC-MS QP5050A mass spectrometer (Japan) using an MDN-5S column (30 m long, 0.25 mm internal diameter, Supelco) or Supelcowax 10 (Supelco) column. The MDN-5S column had an initial column temperature (160 °C) that was increased to 260 °C at 2 °C/min. It remained at 260 °C for an additional 25 min. Some samples of the DMOX derivatives were performed on polar Supelcowax 10 column at 215 °C under isothermal conditions.
2. Materials and methods 2.1. Sampling
2.4. Mass spectrometry analysis
Three sampling periods were performed (July–September 2012, July-August 2015, and August-September 2016) during the joint German-Russian expeditions KuramBio, SokhoBio and KuramBio II on board the RV Sonne (cruises SO-223 and SO-250) and on board the RV Akademic M.A. Lavrentiev (cruise LV71) to the Kuril-Kamchatka Trench, abyssal plain, and the Kuril Basin of the Sea of Okhotsk. Invertebrates were collected at 30 abyssal and hadal stations (Fig. 1) at depths between 3206 and 9581 m using an Agassiz trawl and an epibenthic sledge. No experiments were conducted on animals of endangered or protected species.
The mass spectra were recorded at 70 eV. The spectra were compared with the NIST library 2.0 and an online fatty acid mass spectra archive site (http://www.lipidhome.co.uk/ms/masspec.html, 2019). The data on the FA composition is presented as percentage of total fatty acids.
2.5. Statistical analysis The statistical analysis was performed with Statistica 8 and Primer 6 statistical packages. The results were subjected to one-way analysis of variance (ANOVA) using Office Excel 2013 software (Microsoft Corporation, USA) to test whether the values of the means from each experimental group were significantly different. A p-value < 0.05 was considered statistically significant in the analysis of all data.
2.2. Total lipid extraction and preparation of FA derivatives Invertebrate specimens were rinsed in distilled water, placed in vials filled with chloroform-methanol (1:2 v/v), and stored in a freezer at −80 °C. Lipids of deep-sea invertebrates were extracted using the method described in (Bligh and Dyer, 1959). The obtained homogenate 2
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Table 1 Invertebrates sampled from the abyssal and hadal zones of the Kuril-Kamchatka Trench, adjacent areas of the Pacific Ocean and the Sea of Okhotsk. Taxa
The KurilKamchatka Trench
Abissal plan of the Pacific Ocean
The Kuril Basin
Species*
Samples
Species
Samples
Species
Samples
3 0 16
3 0 45
1 3 9
1 5 10
9 5 2
20 22 13
12
20
0
0
3
34
3 2 9 1 0 2 11 3 1 1 1 1 0 0 66
4 2 54 1 0 4 12 6 10 3 4 5 0 0 173
2 1 5 3 4 3 9 4 3 0 1 4 1 0 53
4 2 9 3 4 6 21 8 6 0 4 6 2 1 92
0 3 5 0 0 2 9 2 3 0 0 6 0 1 50
0 12 18 0 0 9 66 6 11 0 0 34 0 3 248
Table 2 Maximal concentration of major unusual methylene-interrupted PUFAs in lipids of deep-sea megabenthic invertebrates from the abyssal and hadal zones of the Kuril-Kamchatka Trench, adjacent areas of the Pacific Ocean and the Sea of Okhotsk (only the fatty acids contributing > 0.4% were considered). Taxa
Porifera Anthozoa Polychaeta (Sedentaria and Errantia) Polychaeta (Echiuroidea) Pogonophora Sipuncula Bivalvia Gastropoda Scaphopoda Echinoidea Holothurioidea Asteroidea Ophiuroidea Crinoidea Ascidiacea Crustacea Hydrozoa Bryozoa Total
* Preliminary species identification.
Maximal concentration (% of total fatty acids) 21:4n-7
24:6n-3
26:7n-3
Anthozoa Pennatulacea Scleractinia Actiniaria
0.5 ± 0.1 2.5 ± 0.2 2.3 ± 0.2
7.0 ± 0.5 n.d. 0.8 ± 0.1
n.d. n.d. n.d.
Polychaeta Sedentaria and Errantia Echiura
5.6 ± 0.5 1.8 ± 0.1
n.d. n.d.
n.d. n.d.
Sipuncula Sipunculidea
2.0 ± 0.2
n.d.
n.d.
Mollusca Scaphopoda Bivalvia Gastropoda
3.0 ± 0.2 2.5 ± 0.2 10.6 ± 0.5
n.d. n.d. n.d.
n.d. n.d. n.d.
Echinodermata Echinoidea Holothuroidea Asteroidea Ophiuroidea Crinoidea
4.0 2.5 5.1 0.8 0.9
n.d. n.d. n.d. 20.5 ± 0.5 9.9 ± 0.5
n.d. n.d. n.d. 14.5 ± 0.5 n.d.
± ± ± ± ±
0.3 0.2 0.4 0.1 0.1
n.d. – not detected. In most cases, more than one sample per analysed specimen was used. Error bars indicate ± standard deviation; n = 3.
3. Results 3.1. Analysis of fatty acids in lipids of abyssal and hadal megabenthic invertebrates
Table 3 Minor unusual methylene-interrupted PUFAs from deep-sea megabenthic invertebrates sampled at the Kuril Basin of the Sea of Okhotsk (KB) and the KurilKamchatka Trench (KKT).
513 samples taking from 1250 collected abyssal and hadal megabenthic invertebrates were analysed. In most cases, more than one sample per analysed specimen was used (Table 1, Table 3). In total, we analysed 169 different species: 66 species from the Kuril Kamchatka Trench, 53 species from abissal plan of the Pacific Ocean and 50 species from the Kuril Basin of the Okhotsk Sea. Every species was unique for its location. There was no significant difference among all stations. In some of the analysed organisms, unusual methylene-interrupted PUFAs (21:4n-7, 24:6n-3 and 26:7n-3) made up a relatively large proportion. Only the FAs contributing > 0.4% were considered. The FA 21:4n-7 was found in many marine invertebrates analysed in this study, and its content is characteristic of some taxonomic groups (Table 2). The greatest content of 21:4n-7 was detected in gastropods (10.6 ± 0.5), but high levels of this FA were also found in polychaetes, starfish, and sea urchins (Table 2). Lipids of ophiurioids, sea lilies, and sea pens contained high levels of 24:6n-3: the percentage of 24:6n-3 in ophiurioids reached up to 20.5% (the Kuril-Kamchatka Trench). The FA 26:7n-3 was only found in six species from 13 ophiurioids studied, wherein the content was the highest (14.5%) in ophiurioids collected in the Kuril Basin (the Sea of Okhotsk). Some other unusual methyleneinterrupted PUFAs in megabenthic invertebrates were only found as minor PUFAs (Table 3). FAs, 22:4n-8 and 22:5n-5, were detected in the sea urchin Kamptosoma sp. and the starfish Eremicaster sp. The unusual FA 32:5n-3 together with non-methylene-interrupted FAs, which are typical for glass sponges, were found in the glass sponge Hyalonema sp. (Table 3). The distribution of unusual methylene-interrupted PUFAs from deep-sea megabenthic invertebrates sampled at the Kuril Basin of the Sea of Okhotsk and the Kuril-Kamchatka Trench is presented in a supplementary (Table S1). This table provides a more detailed species list (wherever possible) and the information on quantity and type of the PUFAs found.
Unusual FA
Species
32:5n-3 18:3n-7 20:3n-7 22:3n-7 22:4n-8 22:5n-5
Concentration (% of total fatty acids)
Area
Depth, m
Sponges Hyalonema sp.
2.4 ± 0.3
KB
3301
Bivalvia Axinulus sp. Axinulus sp. Axinulus sp.
2.4 ± 0.9 1.7 ± 0.7 0.6 ± 0.2
KKT KKT KKT
9293 9293 9293
1.7 ± 0.3
KKT
5109
1.7 ± 0.1
KKT
5109
Echinoidea Kamptosoma abyssale Kamptosoma abyssale
22:4n-8 22:5n-5
Asteroidea Eremicaster sp. Eremicaster sp.
1.0 ± 0.2 2.8 ± 0.5
KKT KKT
6204 6209
23:2n-6 21:4n-6
Holothuroidea Elpidia hansoni Prototrochus sp.
1.5 ± 0.1 3.8 ± 0.1
KKT KKT
9581 9581
3.2. Mass spectrometry analysis of the DMOX derivatives of FAs The bivalve mollusc Axinulus sp. that was sampled at a depth of over 9 km contained n-7 series PUFAs (Table 3 and Table S1). The mass spectra of the DMOX derivatives of some new FAs (18:3n-7, 20:3n-7 and 22:3n-7) are presented in Fig. S1. One of the notable components of the holothurian Prototrohus sp. from the hadal zone of the Kuril Kamchatka Trench was FA 21:4n-6, whereas the dominant holothurian species in this area (the sea cucumber Elpidia hansoni) contained only FA 23:2n-6 (Table 3 and Table S1). The mass spectra of the DMOX derivatives of these FAs in lipids of hadal holothurians are presented in 3
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in the species studied have previously been described for abyssal deposit feeders (Kharlamenko et al., 2018). We found n-7 PUFAs in the thyasirid bivalve Axinulus sp., which does not have host chemotrophic symbionts but may use pedal feeding (Zanzerl and Dufour, 2017). Thus, these FAs could derive from bacterial precursors. The FA 21:4n-6 is an isomer of 21:4n-7, although their biosynthesis pathways may be different. The origin of the FA 32:5n-3 in the glass sponge Hyalonema sp. is unclear. We suggest that the potential origin of this very long-chain FA may be connected with bacterial precursors derived from deep-sea bacteria living at high pressure (DeLong and Yayanos, 1986). The presence of this FA together with high levels of very long FAs 30:4n-6 and 30:5n-3 found in Cliona celata (Litchfield et al., 1979), as well as 34:5n-3 found in the sponge Petrosia pellasarca (Carballeira and Reyes, 1990), can explain the adaptation of cellular membranes of deep-sea organisms to lipid-solidifying effects, caused by low temperatures and high ocean pressure (MacDonald, 1984). The longest methylene-interrupted PUFA that is common in the sponges analysed in this study is 22:6n-3, while C. celata has been reported to be the source of many BAS (Suh et al., 2012; Ruocco et al., 2016), although so far there exists only one study where very longchain FAs could be used as potentially active substances for precursors of lipid mediators (Sassa and Kihara, 2014). There is very few data on the biological activity of unusual PUFAs found in abyssal and hadal invertebrates. For example, the PUFA 21:4n7 from the marine opisthobranch Scaphander lignarius, not isolated before in it, was shown to be a significantly more potent cytotoxic agent than common PUFAs, such as 20:4n-6 and 20:5n-3 (Van Campenhout and Vanreusel, 2016). The PUFA 24:6n-3, found in some fishes, brittle stars and jellyfish, exerted the strongest suppression on the synthesis of some lipids and the mRNA levels relating to lipid metabolism in human hepatoma HepCr cells (Nagao et al., 2014). The brittle star (the ophiurioid) Ophiura irrorata from the Kuril Basin of the Sea of Okhotsk, having 26:7n-3 in its glycerophospholipids, showed highly specific antiWnt activities targeting multiple levels within the Wnt signaling pathway, which is one of the key factors in oncogenic transformation, growth and metastasis in different cancers (Blagodatski et al., 2017). As mentioned before, from our own data (Kharlamenko et al., 2017; Kharlamenko, 2018; this study) and from the data of other authors (Koyama, 2007), it is quite challenging to study deep-sea marine invertebrates due to their low biomass levels and extremely low growth rates. Therefore, it seems too complicated to obtain the amount of material from these sources that would be sufficient for full-fledged research or practical use. This can be remedied through the cultivation of deep-sea multicellular organism cells. Primary cell cultures of different marine invertebrate species have been successfully obtained, but all studies have only been carried out on shelf species (Odintsova and Khomenko, 1991; Odintsova et al., 2005; Pomponi, 2006; Gordaliza, 2010; Odintsova et al., 2011; Rinkevich, 2011; Ageenko et al., 2014; Maiorova and Odintsova, 2015; Ryazanova et al., 2015; Odintsova et al., 2017; Ballarin et al., 2018; Boroda et al., 2019). Marine invertebrate species contain a large number of novel bioactive molecules, many of which are of significant potential interest for human health (Ballarin et al., 2018). An attempt to cultivate deep-sea multicellular organisms (from a depth of 1162 m) was undertaken by Japanese researchers; it was demonstrated, that special aquarium equipment that takes into account high hydrostatic pressures and low seawater temperatures, was necessary for cultivating living deep-sea organisms, whereas it was not needed for cultivating cells of these organisms (Koyama, 2007). Unfortunately, no comparative information on deep-sea organisms and their cells was provided in this article, and the true causes of this phenomenon remain unclear. If successful, such work will make cells of these deep-sea animal available (for example, through cell freezing in liquid nitrogen) for a further study of their various properties. Stem cell cultures of marine
Fig. S2. 4. Discussion The initial links of food webs in abyssal and hadal ecosystems can substantially change the composition of the organic matter that is deposited from the euphotic zone and is poor in PUFAs. The first provider of a trophic upgrade in deep-sea ecosystems are piezophilic bacteria dwelling in bottom sediments and synthesizing mainly n-3 PUFAs, 20:5n-3 and 22:6n-3 (Fang et al., 2004). The second provider of a trophic upgrade occupying deep-sea ecosystems are foraminifera containing elevated levels of 20:4n-6 (Kharlamenko et al., 2017). Furthermore, foraminifera could synthesize 20:4n-6 FA themselves as pointed in (Larkin et al., 2014). The third and less studied provider of a trophic upgrade in deep-sea ecosystems are nematodes (Leduc et al., 2015; Van Campenhout and Vanreusel, 2016; Mordukhovich et al., 2018), which having the ability to synthesize PUFAs, that are not present in their food sources (Leduc and Probert, 2009). Following the analysis of the FA composition of megabenthic invertebrates from the Kuril-Kamchatka Trench and adjacent regions of the Pacific Ocean, we found a wide variety of unsual FAs. For example, we detected the FA 21:4n-7 in many abyssal and hadal benthic invertebrates, thus contradicting the previous view that this FA is rare (Vasskog et al., 2012). Like other authors (Takagi et al., 1986; Vysotskii and Svetashev, 1991), we found 24:6n-3 in Anthozoa and Echinodea, but there was no noticeable concentration of the very long-chain PUFA 24:5n-6, the high levels of which were previously found in Octocorallia (Vysotskii and Svetashev, 1991). The FA 24:6n-3 could be the precursor of another rare FA: 26:7n-3 (Svetashev and Kharlamenko, 2015), found in abyssal ophiurioids at a high level (14.5 ± 0.5). However, the content of this FA (26:7n-3) did not exceed 5% in bathyal ophiurioids (Svetashev and Kharlamenko, 2015). Benthic foraminifera have previously been described as the main source of methylene-interrupted PUFAs in deep-sea benthic ecosystems (Kharlamenko, 2018). Although, the composition of PUFAs of deep-sea benthic foraminifera has been described earlier in several papers (Gooday et al., 2002; Suhr et al., 2003; Larkin, 2006; Würzberg et al., 2011), the authors indicated only major PUFAs, having omitted possible minor or rare PUFAs. The discovery of a large number of novel FAs might have been triggered by the sampling method on the one hand, and the FAs structural analysis based on the DMOX derivatives on the other. Foraminifera samples have usually been collected using a giant box corer or multiple corer (Gooday et al., 2002; Suhr et al., 2003; Larkin, 2006; Würzberg et al., 2011). We used an epibenthic sledge for the first time. The sledge catches an area of about one thousand square meters in contrast to a sampling area of a giant box corer (0.25 m2) or a sampling area of multiple corer (0.0025 m2). Thus, the sledge captures much more foraminifera. In addition, we could not previously find any novel FAs when the structure of the FAs was determined by comparison with standard kits of FAs. The use of these methods of collecting and analyzing samples allowed us to find nine novel and three rare PUFAs in just three common species of foraminifera (Kharlamenko, 2018). To continue these studies, we analysed the FA composition of benthic invertebrates who fed on foraminifera. The sea urchin Kamptosoma sp. and the starfish Eremicaster sp. were especially noticeable in this regard: two PUFAs characteristic of foraminifera were found in their tissues. The discovery of novel PUFAs provides the possibility to determine more accurately the role of foraminifera as food source for benthic invertebrates. However, we cannot exclude the fact that some unusual FAs may be of nematode origin since the data on the FA composition of deep-sea nematodes were very variable and highlighted the importance of sediment bacteria (Mordukhovich et al., 2018). Based on the results of the analysis of sediment samples, we do not exclude also that the sediments may contain the amounts of unusual PUFAs comparable with those in deep-sea nematodes. Most of the FAs 4
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invertebrates are a novel model system characterized by a high level of physiological and synthetic processes. The production of BAS in vitro may be an alternative to the chemical synthesis and aquaculture (Odintsova, 2009).
Appendix A. Supplementary material
5. Conclusion
References
Large-scale studies of the composition of FAs of marine megabenthic invertebrates of abyssal and hadal ecosystems in the North-West Pacific region have revealed twelve unusual methylene-interrupted PUFAs. Together with the previously presented data on unusual FAs of abyssal and hadal foraminifera, this data significantly complements our knowledge of marine methylene-interrupted PUFAs and can be used to select model taxa for screening BAS for medical purposes. Some further progress in this field could become possible when cell cultures of deepsea invertebrates are obtained. We assume that the next stage of the research will include holothurians, brittle stars (ophiurioids), and sponges as model taxa. This is also important to take into account the presence and abundance of unusual methylene-interrupted PUFAs found in deep-sea organisms.
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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.pocean.2019.102132.
Author contributions V.K. designed and coordinated the research, prepared the manuscript parts concerning sampled material, analyzed FA composition of invertebrates. V.K. and N.O. equally contributed to writing the MS, read and approved the final version of the MS. Funding The material was collected and sorted within the framework of several large international projects. The project SoJaBio was financially supported by the German Research Foundation (Br 1121/37-1), both KuramBio I and II projects and the SokhoBio Project by the PTJ (German Ministry for Science and Education), grant 03G0857A, KuramBio I BMBF grant 03G0223A, as well as KuramBio II BMBF grant 03G0250A to Prof. Dr. Angelika Brandt, University of Hamburg, now Senckenberg Museum, Frankfurt, Germany. The projects were also financially supported by the Russian Foundation of Basic Research (projects 13-04-02144, 16-04-01431; 16-04-01477), the Council of the President of the Russian Federation (project MК-2599.2013.4), Russian Federation Government grant No 11.G34.31.0010, grant of Presidium of the Far East Branch of RAS (12–I–P30–07), Otto Schmidt Laboratory grant (OSL-14-15). The taxonomic processing of the different taxa was supported by the Russian Science Foundation (14-50-00034); biogeography and distribution study was supported by the Russian Ministry of Science and Education (Project 14.616.21.0077, ID No. RFMEF161617X0077). Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments We are grateful to the coordinators of the Study Prof. Angelika Brandt and Dr. Marina Malyutina. We wish to thank the captains and the crews of the R/V Sonne and R/V Akademik M.A. Lavrentyev for their assistance on board. We thank all members of these expeditions including all student-helpers and technicians for support and help with sorting of the extensive expedition material. We are grateful to Dr. V.I. Svetashev and Dr. Ekaterina Ermolenko for their help in preparing the paper and understanding how to answer the Review questions. We thank Dr. A.V. Boroda and Dr. G.M. Kamenev for their help in preparing the paper. 5
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