Impact of hypoxia conditions on the Mnemiopsis leidyi A. Agassiz, 1865 bioluminescence.

The findings of the study on the impact of hypoxia on the glow of the Black Sea ctenophore Mnemiopsis leidyi A. Agassiz, 1865 of three size groups (20-30, 30-45, and 45-60 mm) were obtained under experimental conditions. Peculiarities of ctenophore bioluminescence were studied during mechanical and chemical stimulation under the conditions of normoxia (at an oxygen concentration of 5.6-6.7 mg O2  L-1 ), moderate hypoxia (2.5-2.8 mg O2  L-1 ), and acute hypoxia (1.2-1.5 mg O2  L-1 ). An increase in the amplitude and energy of luminescence of the ctenophores mechanically and chemically stimulated was observed at an oxygen concentration of 1.2-1.5 mg O2  L-1 (acute hypoxia) in two size groups in the lobate form (30-45 and 45-60 mm). The inhibition of amplitude, energy, and duration of the signal was registered in M. leidyi ctenophores at the transitional stage from larva to the lobate form under conditions of acute hypoxia. It was noted that in normoxia, the values of the amplitude and energy of the bioluminescent signal of M. leidyi increase along with a size growth of an individual. This phenomenon was observed both during mechanical and chemical stimulations. Under conditions of acute hypoxia, this trend was mainly preserved. The universality of the relation between the bioluminescence of the organisms and their bioenergetics is obvious. The bioluminescent system of ctenophores has the role of an antioxidant system and is engaged in the neutralization of reactive oxygen species (ROS), that is the process during which photons are emitted. The response of the bioluminescent system to a decrease in oxygen concentration can be associated with an increase in the production of ROS that provides high values of the ctenophore luminescence under hypoxic conditions.

Recombinant light-sensitive photoprotein berovin from ctenophore Beroe abyssicola: Bioluminescence and absorbance characteristics.

The bright bioluminescence of ctenophores inhabiting the oceans worldwide is caused by light-sensitive Ca2+-regulated photoproteins. By now, the cDNAs encoding photoproteins from the four different ctenophore species have been cloned and the recombinant proteins have been characterized to some extent. In this work, we report on the specific activity and the quantum yield of bioluminescence reaction as well as the absorbance characteristics of high-purity recombinant berovin. To determine those, we applied the amino acid composition analysis to accurately measure berovin concentration and the recombinant aequorin as a light standard to convert relative light units to quanta. The extinction coefficient of 1% berovin solution at 435 nm was found to be 1.82. The one can be employed to precisely determine the protein concentration of active photoproteins from other ctenophore species. The specific activity and the bioluminescence quantum yield were respectively found to be 1.98 × 1015 quanta/mg and 0.083. These values appeared to be several times lower than those of the cnidarian photoproteins, which is obviously due to differences in amino acid environments of the substrate in active sites of these photoproteins.

The IS630/Tc1/mariner transposons in three ctenophore genomes.

Transposable elements (TEs) exert a significant effect on the structure and functioning of the genomes and also serve as a source of the new genes. The study of the TE diversity and evolution in different taxa is indispensable for the fundamental understanding of their roles in the genomes. IS630/Tc1/mariner (ITm) transposable elements represent the most prevalent and diverse group of DNA transposons. In this work, we studied the diversity, evolutionary dynamics and the phylogenetic relationships of the ITm transposons found in three ctenophore species: Mnemiopsis leidyi, Pleurobrachia bachei, Beroe ovata. We identified 29 ITm transposons, seven of which possess the terminal inverted repeats (TIRs) and an intact transposase, and, thus, are, presumably, active. Four other ITm transposons have the features of domesticated TEs. According to the results of the phylogenetic analysis, the ITm transposons of the ctenophores represent five groups - MLE/DD34D, TLE/DD34-38E, mosquito/DD37E, Visiror/DD41D and pogo/DDxD. Pogo/DDxD superfamily turnes out to be the most diverse and prevalent, since it accounts for more than 40% of the TEs identified. The data obtained in this research will fill the gap of knowledge of the diversity and evolution of the ITm transposons in the multicellular genomes and will lay the ground for the study of the TE effects on the evolution of the ctenophores.

Depth- and temperature-specific fatty acid adaptations in ctenophores from extreme habitats.

Animals are known to regulate the composition of their cell membranes to maintain key biophysical properties in response to changes in temperature. For deep-sea marine organisms, high hydrostatic pressure represents an additional, yet much more poorly understood, perturbant of cell membrane structure. Previous studies in fish and marine microbes have reported correlations with temperature and depth of membrane-fluidizing lipid components, such as polyunsaturated fatty acids. Because little has been done to isolate the separate effects of temperature and pressure on the lipid pool, it is still not understood whether these two environmental factors elicit independent or overlapping biochemical adaptive responses. Here, we use the taxonomic and habitat diversity of the phylum Ctenophora to test whether distinct low-temperature and high-pressure signatures can be detected in fatty acid profiles. We measured the fatty acid composition of 105 individual ctenophores, representing 21 species, from deep and shallow Arctic, temperate, and tropical sampling locales (sea surface temperature, -2° to 28°C). In tropical and temperate regions, remotely operated submersibles (ROVs) enabled sampling down to 4000 m. We found that among specimens with body temperatures 7.5°C or colder, depth predicted fatty acid unsaturation levels. In contrast, in the upper 200 m of the water column, temperature predicted fatty acid chain lengths. Taken together, our findings suggest that lipid metabolism may be specialized with respect to multiple physical variables in diverse marine environments. Largely distinct modes of adaptation to depth and cold imply that polar marine invertebrates may not find a ready refugium from climate change in the deep.

Bioluminescence of ctenophores near the boundary of oxygen-depleted waters at the redoxcline of the Black Sea.

Vertical distribution of ctenophores near the boundary of oxygen-depleted waters of the Black Sea redoxcline was studied by use of video observations with real-time water sampling, horizontal MultiNet towing, and soundings using bathyphotometers with simultaneous vertical plankton net sampling. The results of the study showed for the first time that the daytime accumulation of ctenophores above the upper boundary of the suboxic zone changes the biophysical properties of the medium, causing an increase in the daytime intensity of bioluminescence near the redoxcline. The dynamics of this glow is in antiphase to that in the surface layers, where it is associated with the bioluminescence of phytoplankton. Therefore, in the deep-sea areas, two types of bioluminescence peaks differ in the light generation sources: the nighttime glow of phytoplankton in surface layers and the daytime glow of zooplankton in layers of oxygen-depleted waters at the redoxcline. The discovery of this new phenomenon allows the use of bioluminescent methods for the rapid assessment of the depth of the daytime zooplankton layers for the subsequent hauls of plankton nets. This significantly expands the possibilities of studying the structure and functioning of the pelagic ecosystem of the Black Sea and other marine basins with a redoxcline.

Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes.

Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.

Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes.

Septate junctions (SJs) insure barrier properties and control paracellular diffusion of solutes across epithelia in invertebrates. However, the origin and evolution of their molecular constituents in Metazoa have not been firmly established. Here, we investigated the genomes of early branching metazoan representatives to reconstruct the phylogeny of the molecular components of SJs. Although Claudins and SJ cytoplasmic adaptor components appeared successively throughout metazoan evolution, the structural components of SJs arose at the time of Placozoa/Cnidaria/Bilateria radiation. We also show that in the scleractinian coral Stylophora pistillata, the structural SJ component Neurexin IV colocalizes with the cortical actin network at the apical border of the cells, at the place of SJs. We propose a model for SJ components in Cnidaria. Moreover, our study reveals an unanticipated diversity of SJ structural component variants in cnidarians. This diversity correlates with gene-specific expression in calcifying and noncalcifying tissues, suggesting specific paracellular pathways across the cell layers of these diploblastic animals.

The search for ancestral nervous systems: an integrative and comparative approach.

Even the most basal multicellular nervous systems are capable of producing complex behavioral acts that involve the integration and combination of simple responses, and decision-making when presented with conflicting stimuli. This requires an understanding beyond that available from genomic investigations, and calls for a integrative and comparative approach, where the power of genomic/transcriptomic techniques is coupled with morphological, physiological and developmental experimentation to identify common and species-specific nervous system properties for the development and elaboration of phylogenomic reconstructions. With careful selection of genes and gene products, we can continue to make significant progress in our search for ancestral nervous system organizations.

Electrogenesis in the lower Metazoa and implications for neuronal integration.

Electrogenic communication appears to have evolved independently in a variety of animal and plant lineages. Considered here are metazoan cells as disparate as the loose three-dimensional parenchyma of glass sponges, the two-dimensional epithelial sheets of hydrozoan jellyfish and the egg cell membranes of the ctenophore Beroe ovata, all of which are capable of generating electrical impulses. Neuronal electrogenesis may have evolved independently in ctenophores and cnidarians but the dearth of electrophysiological data relating to ctenophore nerves means that our attention is focused on the Cnidaria, whose nervous systems have been the subject of extensive study. The aim here is to show how their active and passive neuronal properties interact to give integrated behaviour. Neuronal electrogenesis, goes beyond simply relaying 'states of excitement' and utilizes the equivalent of a set of basic electrical 'apps' to integrate incoming sensory information with internally generated pacemaker activity. A small number of membrane-based processes make up these analogue applications. Passive components include the decremental spread of current determined by cellular anatomy; active components include ion channels specified by their selectivity and voltage dependence. A recurring theme is the role of inactivating potassium channels in regulating performance. Although different aspects of cnidarian behaviour are controlled by separate neuronal systems, integrated responses and coordinated movements depend on interactions between them. Integrative interactions discussed here include those between feeding and swimming, between tentacle contraction and swimming and between slow and fast swimming in the hydrozoan jellyfish Aglantha digitale.

Ctenophora: Illustrated Guide and Taxonomy.

Ctenophores or comb jellies represent the first diverging lineage of extant animals - sister to all other Metazoa. As a result, they occupy a unique place in the biological sciences. Despite their importance, this diverse group of marine predators has remained relatively poorly known, with both the species and higher-level taxonomy of the phylum in need of attention. We present a checklist of the phylum based on a review of the current taxonomic literature and illustrate their diversity with images. The current classification presented remains substantially in conflict with recent phylogenetic results, and many of the taxa are not monophyletic or untested. This chapter summarizes the existing classification focusing on recognized families and genera with 185 currently accepted, extant species listed. We provide illustrative examples of ctenophore diversity covering all but one of the 33 families and 47 of the 48 genera, as well as about 25-30 undescribed species. We also list the 14 recognized ctenophore fossil species and note others that have been controversially attributed to the phylum. Analyses of unique ctenophore adaptations are critical to understanding early animal evolution and adaptive radiation of this clade of basal metazoans.

Expression, Purification, and Determination of Sensitivity to Calcium Ions of Ctenophore Photoproteins.

Light-sensitive Ca2+-regulated photoproteins of ctenophores are single-chain polypeptide proteins of 206-208 amino acids in length comprising three canonical EF-hand Ca2+-binding sites, each of 12 contiguous residues. These photoproteins are a stable complex of apoprotein and 2-hydroperoxy adduct of coelenterazine. Addition of calcium ions to photoprotein is only required to trigger bright bioluminescence. However, in contrast to the related Ca2+-regulated photoproteins of jellyfish their capacity to bioluminescence disappears on exposure to light over the entire absorption spectral range of ctenophore photoproteins. Here, we describe protocols for expression of gene encoding ctenophore photoprotein in Escherichia coli cells, obtaining of the recombinant apoprotein of high purity and its conversion into active photoprotein with synthetic coelenterazine as well as determination of its sensitivity to calcium ions using light-sensitive Ca2+-regulated photoprotein berovin from ctenophore Beroe abyssicola as an illustrative case.

Functional Screening of cDNA Expression Library for Novel Ctenophore Photoproteins.

The functional screening of cDNA libraries (or functional cloning) enables isolation of cDNA genes encoding novel proteins with unknown amino acid sequences. This approach is the only way to identify a protein sequence in the event of shortage of biological material for obtaining pure target protein in amounts sufficient to determine its primary structure, since sensitive functional test for a target protein is only required to successfully perform functional cloning. Commonly, bioluminescent proteins from representatives belonging to different taxa significantly differ in sequences due to independent origin of bioluminescent systems during evolution. Nonetheless, these proteins are frequently similar in functions and can use even the same substrate of bioluminescence reaction, allowing the use of the same functional test for screening. The cDNA genes encoding unknown light-emitting proteins can be identified during functional screening with high sensitivity, which is provided by modern light recording equipment making possible the detection of a very small amount of a target protein. Here, we present the protocols for isolation of full-size cDNA genes for the novel bioluminescent protein family of light-sensitive Ca2+-regulated photoproteins in the absence of any sequence information by functional screening of plasmid cDNA expression library. The protocols describe all the steps from gathering animals to isolation of individual E. coli colonies carrying full-size cDNA genes using photoprotein berovin from ctenophore Beroe abyssicola as an illustrative example.

Gene Expression Patterns in the Ctenophore Pleurobrachia bachei: In Situ Hybridization.

In situ hybridization is a powerful and precise tool for revealing cell- and tissue-specific gene expression and a critical approach to validating single-cell RNA-seq (scRNA-seq). However, applying it to highly fragile animals such as ctenophores is challenging. Here, we present an in situ hybridization protocol for adult Pleurobrachia bachei (Cydippida)-a notable reference species representing the earliest-branching metazoan lineage, Ctenophora, sister to the rest of Metazoa. We provided expression patterns for several markers of cell phenotypes, as illustrated examples. The list includes predicted small secretory molecules/neuropeptides, WntX, genes encoding RNA-binding proteins (Musashi, Elav, Dicer, Argonaut), Neuroglobin, and selected transcription factors such as BarX. Both cell- and organ-specific expression of these genes further support the convergent evolution of many ctenophore innovations, which are remarkably distinct from tissue and organ specification in other basal metazoan lineages.

From gelatinous to muscle food chain: rock cod Patagonotothen ramsayi recycles coelenterate and tunicate resources on the Patagonian Shelf.

Stomach contents of 4808 fishes of 20 species caught in the eastern part of the Patagonian Shelf between 1999 and 2012 were analysed to assess dietary contributions of gelatinous plankton resources. Gelatinous plankton occurred in diets of seven species with two species, Patagonotothen ramsayi and Squalus acanthias, having >10% ctenophores in their diet. Consumption of gelatinous plankton was important in P. ramsayi and was strikingly seasonal, with maximum occurrence (up to 46% of non-empty stomachs) in late summer to autumn. Ctenophores were most abundant in P. ramsayi of 25-34 cm total length, L(T) whereas salps were more frequent in larger >35 cm L(T) individuals. In winter to spring, occurrence of gelatinous plankton in diets was minimal, reflecting their overall seasonal abundance in the ocean. The recent increase in abundance of P. ramsayi has enabled the species to recycle a significant proportion of the ecosystem production from gelatinous dead end to the main muscular food chain via seasonal reliance on ctenophores, jellyfish and tunicates. This additional influx of production that has been diverted from the gelatinous food chain favours the increase in abundance of several piscivorous top predators and affects the trophic web structure of the Patagonian Shelf ecosystem.

Ecological role of bioluminescence of Black sea ctenophores.

Bioluminescence, which is a manifestation of the vital activity of an organism in the form of electromagnetic radiation in the visible area of the spectrum, is a highly important ecological and optical factor of the marine environment. Until recently, it was believed that microplankton - bacteria and dinoflagellates - exceptionally contribute to the formation of the bioluminescence field in the Black Sea, as well as in other regions of the World Ocean. However, the ctenophores Mnemiopsis leidyi A. Agassiz, 1865, and Beroe ovata Mayer, 1912, which invaded the Black Sea in the 1980s-1990s, are also luminous organisms whose bioluminescence intensity is millions of times greater than that of most microplankton representatives. It is known that the characteristics of bioluminescence can reveal the state of the organism and, consequently, the state of the environment. At present, there is a fairly large number of works devoted to the physiology and ecology of the Black Sea ctenophores. In recent studies, the variability of light emission parameters of ctenophores following their functional state was revealed. Intensity and duration of light emission as parameters of the ctenophore bioluminescent signal as well as the influence of various abiotic and anthropogenic environmental factors on the ctenophore luminescence have been studied. However, the significance of bioluminescence for the living activity of ctenophores remains unclear. In connection with the above, it is extremely important to assess the ecological role of the bioluminescence of the Black Sea ctenophores.

The premetazoan ancestry of the synaptic toolkit and appearance of first neurons.

Neurons, especially when coupled with muscles, allow animals to interact with and navigate through their environment in ways unique to life on earth. Found in all major animal lineages except sponges and placozoans, nervous systems range widely in organization and complexity, with neurons possibly representing the most diverse cell-type. This diversity has led to much debate over the evolutionary origin of neurons as well as synapses, which allow for the directed transmission of information. The broad phylogenetic distribution of neurons and presence of many of the defining components outside of animals suggests an early origin of this cell type, potentially in the time between the first animal and the last common ancestor of extant animals. Here, we highlight the occurrence and function of key aspects of neurons outside of animals as well as recent findings from non-bilaterian animals in order to make predictions about when and how the first neuron(s) arose during animal evolution and their relationship to those found in extant lineages. With advancing technologies in single cell transcriptomics and proteomics as well as expanding functional techniques in non-bilaterian animals and the close relatives of animals, it is an exciting time to begin unraveling the complex evolutionary history of this fascinating animal cell type.

Towards robust and repeatable methods for studying interactions between jellyfish and microplastics.

Studies of microplastics are increasing exponentially and standard protocols are only beginning to be established. Jellyfish are considered susceptible to ingesting microplastics because they feed on small, suspended particles. Inconsistent approaches used to study interactions between jellyfish and microplastics, however, make comparisons among studies difficult. Here we review aspects of the methods used to sample jellyfish in the field and experimental approaches used in the laboratory to study interactions between jellyfish and microplastics, recommend some standard protocols and identify areas for further research. We highlight the need for experiments to be environmentally relevant, to study a greater diversity of species and to study different life history stages.

Emerging models: The "development" of the ctenophore Mnemiopsis leidyi and the cnidarian Nematostella vectensis as useful experimental models.

The goal of this chapter is to explain the reasoning for developing two understudied invertebrate animal species for asking specific biological questions. The first is the ctenophore (comb jelly) Mnemiopsis leidyi and the second is the anthozoan cnidarian (starlet sea anemone) Nematostella vectensis. Although these two taxa belong to some of the earliest branching extant metazoan clades, their developmental features could hardly be more different from one another. This should serve as a general warning to be careful when extrapolating comparisons of one species to another. Two-taxon comparisons are especially flawed; and to interpret features in a phylogenetic context one must sample carefully within a given taxon to determine how representative certain features are before comparing with other clades. The other benefit of this comparison is to identify key practical factors when attempting to develop new species for experimental investigation.

Evolution of glutamatergic signaling and synapses.

Glutamate (Glu) is the primary excitatory transmitter in the mammalian brain. But, we know little about the evolutionary history of this adaptation, including the selection of l-glutamate as a signaling molecule in the first place. Here, we used comparative metabolomics and genomic data to reconstruct the genealogy of glutamatergic signaling. The origin of Glu-mediated communications might be traced to primordial nitrogen and carbon metabolic pathways. The versatile chemistry of L-Glu placed this molecule at the crossroad of cellular biochemistry as one of the most abundant metabolites. From there, innovations multiplied. Many stress factors or injuries could increase extracellular glutamate concentration, which led to the development of modular molecular systems for its rapid sensing in bacteria and archaea. More than 20 evolutionarily distinct families of ionotropic glutamate receptors (iGluRs) have been identified in eukaryotes. The domain compositions of iGluRs correlate with the origins of multicellularity in eukaryotes. Although L-Glu was recruited as a neuro-muscular transmitter in the early-branching metazoans, it was predominantly a non-neuronal messenger, with a possibility that glutamatergic synapses evolved more than once. Furthermore, the molecular secretory complexity of glutamatergic synapses in invertebrates (e.g., Aplysia) can exceed their vertebrate counterparts. Comparative genomics also revealed 15+ subfamilies of iGluRs across Metazoa. However, most of this ancestral diversity had been lost in the vertebrate lineage, preserving AMPA, Kainate, Delta, and NMDA receptors. The widespread expansion of glutamate synapses in the cortical areas might be associated with the enhanced metabolic demands of the complex brain and compartmentalization of Glu signaling within modular neuronal ensembles.

Effect of invasive ctenophores Mnemiopsis leidyi and Beroe ovata on low trophic webs of the Black Sea ecosystem.

The study focuses on the impact of life excretion and mucus released by the "biological pollutants" invasive ctenophore Mnemiopsis leidyi and its predator Beroe ovata on the marine environment and lower trophic levels of the Black Sea ecosystem (bacteria, pico-phytoplankton, nano-autotrophic/heterotrophic flagellates, micro-phytoplankton, chlorophyll a, primary production (PP), micro-zooplankton). The chemical and biological variables were analysed in two sets of lab experiments with natural communities from mesotrophic (Gelendzhik) and eutrophic (Varna) coastal waters. While both species altered the chemical properties of experimental media, exerting structural and functional changes in the low food-web biological compartments, the results showed a stronger effect of B. ovata, most likely related to the measured higher rate of excretion and amount of released mucus. In addition the alterations in the Gelendzhik experiment were more pronounced, indicating that environmental implications on lower food-web are more conspicuous in mesotrophic than in eutrophic coastal waters.