Tetrodotoxins in Tissues and Cells of Different Body Regions of Ribbon Worms Kulikovia alborostrata and K. manchenkoi from Spokoynaya Bay, Sea of Japan.

Nemerteans, or ribbon worms, possess tetrodotoxin and its analogues (TTXs), neurotoxins of bacterial origin, which they presumably use for capturing prey and self-defense. Most TTXs-containing nemertean species have low levels of these toxins and, therefore, have usually been neglected in studies of TTXs functions and accumulation. In the present study, Kulikovia alborostrata and K. manchenkoi, two closely related species, were analyzed for TTXs distribution in the body using the HPLC-MS/MS and fluorescence microscopy methods. The abundance of TTXs-positive cells was determined in the proboscis, integument, and digestive system epithelium. As a result, six TTXs-positive cell types were identified in each species; however, only four were common. Moreover, the proportions of the toxins in different body parts were estimated. According to the HPLC-MS/MS analysis, the TTXs concentrations in K. alborostrata varied from 0.91 ng/g in the proboscis to 5.52 ng/g in the precerebral region; in K. manchenkoi, the concentrations ranged from 7.47 ng/g in the proboscis to 72.32 ng/g in the posterior body region. The differences observed between the two nemerteans in the distribution of the TTXs were consistent with the differences in the localization of TTXs-positive cells. In addition, TTXs-positive glandular cell types were found in the intestine and characterized for the first time. TTXs in the new cell types were assumed to play a unique physiological role for nemerteans.

Tetrodotoxin and Its Analogues (TTXs) in the Food-Capture and Defense Organs of the Palaeonemertean Cephalothrix cf. simula.

Tetrodotoxin (TTX), an extremely potent low-molecular-weight neurotoxin, is widespread among marine animals including ribbon worms (Nemertea). Previously, studies on the highly toxic palaeonemertean Cephalothrix cf. simula showed that toxin-positive structures are present all over its body and are mainly associated with glandular cells and epithelial tissues. The highest TTXs concentrations were detected in a total extract from the intestine of the anterior part of the body and also in a total extract from the proboscis. However, many questions as to the TTXs distribution in the organs of the anterior part of the worm's body and the functions of the toxins in these organs are still unanswered. In the present report, we provide additional results of a detailed and comprehensive analysis of TTXs distribution in the nemertean's proboscis, buccal cavity, and cephalic gland using an integrated approach including high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), confocal laser scanning microscopy with anti-TTX antibodies, light and electron microscopies, and observations of feeding behavior. For the proboscis, we have found a TTXs profile different from that characteristic of other organs and tissues. We have also shown for the first time that the major amount of TTXs is localized in the anterior part of the proboscis that is mainly involved in hunting. TTX-containing glandular cells, which can be involved in the prey immobilization, have been found in the buccal cavities of the nemerteans. A significant contribution of the cephalic gland to the toxicity of this animal has been shown for the first time, and the role of the gland is hypothesized to be involved not only in protection against potential enemies but also in immobilizing prey. The data obtained have made it possible to extend the understanding of the role and features of the use of TTXs in the organs of the anterior part of nemertean's body.

Tetrodotoxins in Larval Development of Ribbon Worm Cephalothrix cf. simula (Palaeonemertea, Nemertea).

The toxic ribbon worm, Cephalothrix cf. simula (Palaeonemertea, Nemertea), possesses extremely high concentrations of tetrodotoxin (TTX). Although TTX has been found in the eggs of this species, the fate of the toxin in the ontogeny of the animal has not been explored. Here, using high performance liquid chromatography with tandem mass spectrometry and immunohistochemistry with anti-TTX antibodies, we examined levels, profile, and localization of TTX and its analogues (TTXs) in larvae of C. cf. simula throughout 41 days post-fertilization. A detailed investigation of cells in sites of TTX-accumulation was performed with light and electron microscopy. Newly hatched larvae possessed weak TTX-like immunoreactivity in all cells. With subsequent development, intensity of TTX-labeling in the ectodermal structures, mesodermal cells and apical cylinder of the apical gland increased. In the ectodermal structures, an intense TTX-labeling was observed in the multiciliated, type II granular, type I mucoid, and basal cells of the epidermis, and in the type III granular cells of the mouth gland. In the mesoderm, TTX was localized in the muscle and unigranular parenchyma-like cells. Eggs and larvae of C. cf. simula contained five TTXs, with two major toxins - TTX and 5,6,11-trideoxyTTX. Level and relative proportion of TTXs did not differ significantly among developmental stages, confirming that larvae obtained toxins from maternal eggs and were able to retain it. The results of this study provide insights into the formation of TTX-bearing apparatus of C. cf. simula through the larval development.

Lipidome of the Brown Macroalga Undaria pinnatifida: Influence of Season and Endophytic Infection.

An analysis of the lipidome of the brown alga Undaria pinnatifida (Laminariales) was performed' more than 900 molecular species were identified in 12 polar lipids and 1 neutral lipid using HPLC/MS-MS. The seasonal changes of U. pinnatifida lipidome were determined. It was shown that acclimatization to winter and spring was accompanied by an increase in the unsaturation of both polar and neutral lipids. In autumn and summer, on the contrary, the contents of more saturated molecular species of all lipid classes increased. Based on the data obtained, a scheme for the polar and neutral lipid synthesis in brown algae was proposed. In addition, the influence of infection with the brown filamentous endophyte Laminariocolax aecidioides (Ectocarpales) on U. pinnatifida lipidome was studied. It was found that infection has the most noticeable effect on the molecular species composition of triacylglycerides, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylhydroxyethylglycine of the host macrophyte. In infected samples of algae, changes in the composition of triacylglycerides were revealed both in areas with the presence of an endophyte and in adjacent intact tissues, which may indicate the occurrence of a secondary infection.

Description and genome-wide analysis of Profundicola chukchiensis gen. nov., sp. nov., marine bacteria isolated from bottom sediments of the Chukchi Sea.

Two Gram-negative, aerobic halophilic non-motile strains designated KMM 9713 and KMM 9724T were isolated from the bottom sediments sampled from the Chukchi Sea in the Arctic Ocean, Russia. The novel strains grew in 0.5-5% NaCl, at 7-42°C, and pH 5.5-10.5. Phylogenetic analyses based on 16S rRNA gene and whole genome sequences revealed that strains KMM 9713 and KMM 9724T were close to each other and shared the highest 16S rRNA gene sequence similarity of 91.28% with the type strain Ornithobacterium rhinotracheale DSM 15997T and 90.15-90.92% with the members of the genus Empedobacter in the family Weeksellaceae. Phylogenetic trees indicated that strains KMM 9713 and KMM 9724T formed a distinct line adjacent to their relative O. rhinotracheale DSM 15997T. The average nucleotide identity values between strain KMM 9724T and O. rhinotracheale DSM 15997T, Empedobacter brevis NBRC 14943T, and Moheibacter sediminis CGMCC 1.12708T were 76.73%, 75.78%, and 74.65%, respectively. The novel strains contained the predominant menaquinone MK-6 and the major fatty acids of iso-C17:0 3-OH, iso-C15:0 followed by iso-C17:1ω6. Polar lipids consisted of phosphatidylethanolamine, one an unidentified aminophospholipid, two unidentified aminolipids, and two or three unidentified lipids. The DNA G+C contents of 34.5% and 34.7% were calculated from genome sequence of the strains KMM 9713 and KMM 9724T, respectively. Based on the phylogenetic evidence and distinctive phenotypic characteristics, strains KMM 9713 and KMM 9724T are proposed to be classified as a novel genus and species Profundicola chukchiensis gen. nov., sp. nov. The type strain of Profundicola chukchiensis gen. nov., sp. nov. is strain KMM 9724T (= KACC 22806T).

Microbulbifer okhotskensis sp. nov., isolated from a deep bottom sediment of the Okhotsk Sea.

A Gram-negative, aerobic, non-motile bacterium КMM 9862T was isolated from a deep bottom sediment sample obtained from the Okhotsk Sea, Russia. Based on the 16S rRNA gene and whole genome sequences analyses the novel strain КMM 9862T fell into the genus Microbulbifer (class Gammaproteobacteria) sharing the highest 16S rRNA gene sequence similarities of 97.4% to Microbulbifer echini AM134T and Microbulbifer epialgicus F-104T, 97.3% to Microbulbifer pacificus SPO729T, 97.1% to Microbulbifer variabilis ATCC 700307T, and similarity values of < 97.1% to other recognized Microbulbifer species. The average nucleotide identity and digital DNA-DNA hybridization values between strain КMM 9862T and M. variabilis ATCC 700307T and M. thermotolerans DSM 19189T were 80.34 and 77.72%, and 20.2 and 19.0%, respectively. Strain КMM 9862T contained Q-8 as the predominant ubiquinone and C16:0, C16:1 ω7c, C12:0, and C10:0 3-OH as the major fatty acids. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, an unidentified aminophospholipid, an unidentified aminolipid, two unidentified phospholipids, phosphatidic acid, and an unidentified lipid. The DNA G+C content of 49.8% was calculated from the genome sequence. On the basis of the phylogenetic evidence and distinctive phenotypic characteristics, the marine bacterium KMM 9862T is proposed to be classified as a novel species Microbulbifer okhotskensis sp. nov. The type strain of the species is strain KMM 9862T (= KACC 22804T).

Effect of Temperature and Light Intensity on the Polar Lipidome of Endophytic Brown Algae Streblonema corymbiferum and Streblonema sp. In Vitro.

The effect of temperature and light intensity on the polar lipidome of endophytic brown algae Streblonema corymbiferum and Streblonema sp. in vitro was investigated. More than 460 molecular species have been identified in four glycoglycerolipids classes, five phosphoglycerolipids classes and one betaine lipid class. The lipids glucuronosyldiacylglycerol and diacylglyceryl-N,N,N-trimethyl-homoserine were found in the algae of the order Ectocarpales for the first time. A decrease in cultivation temperature led to an increase in the unsaturation level in all classes of polar lipids. Thus, at low temperatures, the content of 18:4/18:4 monogalactosyldiacylglycerol (MGDG), 20:5/18:4 digalactosyldiacylglycerol (DGDG), 18:3/16:0 sulfoquinovosyldiacylglycerol (SQDG), 18:3/18:3 and 18:3/18:4 phosphatidylglycerol (PG), 20:4/20:5 and 20:5/20:5 phosphatidylethanolamine (PE), 14:0/20:5, 16:0/20:5 and 20:5/20:5 phosphatidylcholine (PC), 20:5/20:4 phosphatidylhydroxyethylglycine and 18:1/18:2 DGTS increased. At high temperatures, an increase in the content of chloroplast-derived MGDG, DGDG and PG was observed. Both low and high light intensities caused an increase in 20:5/18:3 MGDG and 18:3/16:1 PG. At low light intensity, the content of DGDG with fatty acid (FA) 18:3 increased, and at high light intensity, it was with FA 20:5. The molecular species composition of extraplastid lipids also showed a dependence on light intensity. Thus, the content of PC and PE species with C20-polyunsaturated FA at both sn-positions, 18:1/18:1 DGTS and 16:0/18:1 phosphatidylinositol increased. Low light intensity induced a significant increase in the content of chloroplast-derived 18:1/16:1 phosphatidylethanolamine.

Current Progress in Lipidomics of Marine Invertebrates.

Marine invertebrates are a paraphyletic group that comprises more than 90% of all marine animal species. Lipids form the structural basis of cell membranes, are utilized as an energy reserve by all marine invertebrates, and are, therefore, considered important indicators of their ecology and biochemistry. The nutritional value of commercial invertebrates directly depends on their lipid composition. The lipid classes and fatty acids of marine invertebrates have been studied in detail, but data on their lipidomes (the profiles of all lipid molecules) remain very limited. To date, lipidomes or their parts are known only for a few species of mollusks, coral polyps, ascidians, jellyfish, sea anemones, sponges, sea stars, sea urchins, sea cucumbers, crabs, copepods, shrimp, and squid. This paper reviews various features of the lipid molecular species of these animals. The results of the application of the lipidomic approach in ecology, embryology, physiology, lipid biosynthesis, and in studies on the nutritional value of marine invertebrates are also discussed. The possible applications of lipidomics in the study of marine invertebrates are considered.

Lipidomic profiling reveals biosynthetic relationships between phospholipids and diacylglycerol ethers in the deep-sea soft coral Paragorgia arborea.

The cold-water gorgonian coral Paragorgia arborea is considered as a foundation species of deep-sea ecosystems in the northern Atlantic and Pacific oceans. To advance lipidomic studies of deep-sea corals, molecular species compositions of diacylglycerol ethers (DAGE), which are specific storage lipids of corals, and structural glycerophospholipids (GPL) including ethanolamine, choline, inositol and serine GPL (PE, PC, PI, and PS, respectively) were analyzed in P. arborea by HPLC and tandem mass spectrometry. In DAGE molecules, alkyl groups (16:0, 14:0, and 18:1), polyunsaturated fatty acids (PUFA), and monounsaturated FA are mainly substituted the glycerol moiety at position sn-1, sn-2, and sn-3, respectively. The ether form (1-O-alkyl-2-acyl) predominates in PE and PC, while PI is comprised of the 1,2-diacyl form. Both ether and diacyl forms were observed in PS. At position sn-2, C20 PUFA are mainly attached to PC, but C24 PUFA, soft coral chemotaxonomic markers, concentrate in PS, PI, and PE. A comparison of non-polar parts of molecules has shown that DAGE, ether PE, and ether PC can originate from one set of 1-O-alkyl-2-acyl-sn-glycerols. Ether PE may be converted to ether PS by the base-exchange reaction. A diacylglycerol unit generated from phosphatidic acid can be a precursor for diacyl PS, PC, and PI. Thus, a lipidomic approach has confirmed the difference in biosynthetic origins between ether and diacyl lipids of deep-sea gorgonians.

Tetrodotoxins Secretion and Voltage-Gated Sodium Channel Adaptation in the Ribbon Worm Kulikovia alborostrata (Takakura, 1898) (Nemertea).

Nemertea is a phylum of marine worms whose members bear various toxins, including tetrodotoxin (TTX) and its analogues. Despite the more than 30 years of studying TTXs in nemerteans, many questions regarding their functions and the mechanisms ensuring their accumulation and usage remain unclear. In the nemertean Kulikovia alborostrata, we studied TTX and 5,6,11-trideoxyTTX concentrations in body extracts and in released mucus, as well as various aspects of the TTX-positive-cell excretion system and voltage-gated sodium (Nav1) channel subtype 1 mutations contributing to the toxins' accumulation. For TTX detection, an immunohistological study with an anti-TTX antibody and HPLC-MS/MS were conducted. For Nav1 mutation searching, PCR amplification with specific primers, followed by Sanger sequencing, was used. The investigation revealed that, in response to an external stimulus, subepidermal TTX-positive cells released secretions actively to the body surface. The post-release toxin recovery in these cells was low for TTX and high for 5,6,11-trideoxyTTX in captivity. According to the data obtained, there is low probability of the targeted usage of TTX as a repellent, and targeted 5,6,11-trideoxyTTX secretion by TTX-bearing nemerteans was suggested as a possibility. The Sanger sequencing revealed identical sequences of the P-loop regions of Nav1 domains I-IV in all 17 studied individuals. Mutations comprising amino acid substitutions, probably contributing to nemertean channel resistance to TTX, were shown.

Harenicola maris gen. nov., sp. nov. isolated from the Sea of Japan shallow sediments.

A Gram-negative, non-motile bacterium КMM 3653T was isolated from a sediment sample from the Sea of Japan seashore, Russia. On the basis of the 16S rRNA gene sequence analysis the strain КMM 3653T was positioned within the family Rhodobacteraceae (class Alphaproteobacteria) forming a distinct lineage with the highest gene sequence similarities to the members of the genera Pacificibacter (95.2-94.7%) and Nioella (95.1-94.5%), respectively. According to the phylogenomic tree based on 400 conserved protein sequences, strain КMM 3653T was placed in the cluster comprising Vannielia litorea, Nioella nitratireducens, Litoreibacter albidus and Pseudoruegeria aquimaris as a separate lineage adjacent to V. litorea KCTC 32083T. The average nucleotide identity values between strain КMM 3653T and V. litorea KCTC 32083T, N. nitratireducens KCTC 32417T, L. albidus KMM 3851T, and P. aquimaris CECT 7680T were 71.1, 70.3, 69.6, and 71.0%, respectively. Strain КMM 3653T contained Q-10 as the predominant ubiquinone and C18:1ω7c as the major fatty acid followed by C16:0. The polar lipids were phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified phospholipid, two unidentified aminolipids, and five unidentified lipids. The DNA G+C content of 61.8% was calculated from the genome sequence. Based on the phylogenetic evidence and distinctive phenotypic characteristics, we proposed strain KMM 3653T (= KCTC 82575T) to be classified as a novel genus and species Harenicola maris gen. nov., sp. nov.

Thalassobius aquimarinus sp. nov., isolated from the Sea of Japan seashore.

An aerobic, Gram-negative, non-pigmented non-motile bacterium designed КMM 8518T was isolated from a seawater sampled from the Sea of Japan seashore. Strain КMM 8518T grew at 7-42 °C and in the presence of 1-7% NaCl. The phylogenetic analyses based on 16S rRNA gene and whole-genome sequences placed the novel strain КMM 8518T into the genus Thalassobius as a separate lineage. Strain КMM 8518T shared the highest 16S rRNA gene sequence similarity of 98% to Thalassobius gelatinovorus KCTC 22092T and similarity values of ≤ 97% to other recognized Thalassobius species. The average nucleotide identity and digital DNA-DNA hybridization values between strain КMM 8518T and T. gelatinovorus KCTC 22092T were 79.6% and 23.5%, respectively. The major respiratory quinone was ubiquinone-10. The major fatty acid was C18:1ω7c followed by 11-methyl C18:1ω7c. Polar lipids comprised phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminolipid, an unidentified phospholipid, and three unidentified lipids. The DNA G+C content of 62.7% was calculated from genome sequence analysis. Based on the phylogenetic analyses and distinctive phenotypic characteristics, the marine bacterium КMM 8518T is concluded to represent a novel species of the genus Thalassobius for which the name Thalassobius aquimarinus sp. nov. is proposed. The type strain of the species is strain KMM 8518T (= KCTC 82576T).

Fatty Acid Composition and Thermotropic Behavior of Glycolipids and Other Membrane Lipids of Ulva lactuca (Chlorophyta) Inhabiting Different Climatic Zones.

Increasing global temperatures are expected to increase the risk of extinction of various species due to acceleration in the pace of shifting climate zones. Nevertheless, there is no information on the physicochemical properties of membrane lipids that enable the adaptation of the algae to different climatic zones. The present work aimed to compare fatty acid composition and thermal transitions of membrane lipids from green macroalgae Ulva lactuca harvested in the Sea of Japan and the Adriatic Sea in summer. U. lactuca inhabiting the Adriatic Sea had bleached parts of thalli which were completely devoid of chloroplast glycolipids. The adaptation to a warmer climatic zone was also accompanied by a significant decrease in the ratio between unsaturated and saturated fatty acids (UFA/SFA) of membrane lipids, especially in bleached thalli. Hence, bleaching of algae is probably associated with the significant decrease of the UFA/SFA ratio in glycolipids. The decreasing ratio of n-3/n-6 polyunsaturated fatty acids (PUFAs) was observed in extra-plastidial lipids and only in the major glycolipid, non-lamellar monogalactosyldiacylglycerol. The opposite thermotropic behavior of non-lamellar and lamellar glycolipids can contribute to maintenance of the highly dynamic structure of thylakoid membranes of algae in response to the increasing temperatures of climatic zones.

Effects of elevated growth temperature and heat shock on the lipid composition of the inner and outer membranes of Yersinia pseudotuberculosis.

Differences in the distribution of individual phospholipids between the inner (IM) and outer membranes (OM) of gram-negative bacteria have been detected in mesophilic Escherichia, Erwinia and Salmonella species but have never been investigated in the psychrotrophic Yersinia genus. Therefore, the influence of an elevated growth temperature and heat shock on the phospholipid and fatty acid (FA) compositions of the fractionated Yersinia pseudotuberculosis envelope was investigated. The shift of the growth temperature from 8 °C to 37 °C to mimic the switch from saprophytic to parasitic growth of this bacteria and the exposure of the cells to heat shock, which was induced by a sharp increase in the temperature from 8 °C to 45 °C, increased the lysophosphatidylethanolamine content from zero and 1% to 6% and 10% in the IM and OM, respectively. These changes were accompanied by a decrease in the phosphatidylethanolamine (PE) content and a drastic increase (up to 3-fold higher) in the phosphatidylglycerol (PG) level in the OM of the bacteria, which increases the net negative charge of the cell envelope. The levels of the predominant saturated palmitic (16:0) and cyclopropane FAs were approximately 1.5- and 7.5-fold higher, respectively, but the content of the predominant unsaturated palmitoleic (16:1n-7) and cis-vaccenic (18:1n-7) FAs was approximately 10-30-fold lower in both membranes that were isolated from the cells grown at elevated temperatures. Due to these changes, reflecting the process of "homeoviscous adaptation", the ratio between the unsaturated and saturated FAs decreased but remained higher in the IM than that in the OM. Simultaneously, no significant changes were observed in the FA composition of cells subjected to heat shock, demonstrating a difference between the responses of the heat-shocked and heat-adapted Y. pseudotuberculosis. The unique ability of Y. pseudotuberculosis to reciprocally regulate the ratio of anionic PG and net neutral PE and therefore adjust the negative charge of the OM may be a common strategy used by pathogenic bacteria to promote the barrier function of the OM.

Opposite Effects of Lysophosphatidylethanolamines on Conformation of OmpF-like Porin from Yersinia pseudotuberculosis.

Lysophosphatidyletnolamine (LPE) is one of enigmatic lipids of bacteria. It is generated from major membrane lipid - phosphatidylethanolamine at severe changes of the bacterial growth conditions. Accumulation of this phospholipid in cells of Gram-negative enterobacterium Yersinia pseudotuberculosis results in the enhanced thermostability of OmpF-like porin (YOmpF) from the same bacteria. The respective integral conformational rearrangements may disturb the channel permeability of protein under stress conditions. However, role of fatty acid composition of LPE in this effect remained unclear. Present work demonstrated that the level of unsaturated LPE is 3.5 times higher than saturated one in total LPE of bacterial cells exposed to stress (phenol treatment). Unsaturated 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (MOPE) and saturated LPE 1-palmitoyl-2- hydroxy-sn-glycero-3-phosphoethanolamine (MPPE) oppositely affect the conformation of YOmpF. MOPE increases the protein thermal stability due to more dense packing of monomers in porin and preserves its trimeric form at elevated temperature, while MPPE weakens the contact between monomers and promotes dissociation of the protein.