Effects of phthalates on marine organisms: cytotoxicity and genotoxicity of mono-(2-ethylhexyl)-phthalate (MEHP) on European sea bass (Dicentrarchus labrax) embryonic cell line.

INTRODUCTION: Mono-(2-ethylhexyl) phthalate (MEHP) represents a toxicological risk for marine organisms due to its widespread presence in aquatic environments. METHODS: MEHP effects on cell viability, cell death and genotoxicity were investigated on the DLEC cell line, derived from early embryos of the European sea bass Dicentrarchus labrax L. RESULTS: A dose-dependent cytotoxic effect, with no induction of necrotic process, except at its highest concentration, was observed. Moreover, chromosomal instability was detected, both in binucleated and mononucleated cells, coupled with a minor inhibition of cell proliferation, whereas genomic instability was not revealed. To our knowledge, the overall results suggest the first evidence of a possible aneugenic effect of this compound in the DLEC cell line, that is the induction of chromosomal loss events without the induction of primary DNA damage. CONCLUSIONS: MEHP should be considered more harmful than its parent compound DEHP, because it induces genomic instability in the DLEC cell line without triggering cell death.

Comparative study of peripheral blood cells in two marine fishes (Synechogobius hasta and Sebastes schlegelii): Morphological and cytochemical characterization.

The morphology, cell size and relative number of peripheral blood cells in two teleosts, Synechogobius hasta and Sebastes schlegelii, were compared using different staining methods. The results showed significant differences in cell size. The percentage of monocytes in S. hasta was greater than that in S. schlegelii (P < 0.01); however, the opposite results were obtained in the percentage of lymphocytes and thrombocytes. The two fishes shared common cytochemical-staining results, which showed that both erythrocytes were negative for all cytochemical staining; monocytes were strongly positive for PAS and positive for SBB and NAE; lymphocytes were negative for SBB, POX and NAE; neutrophils were positive for SBB and NAE; and thrombocytes were negative for SBB, ALP, POX and NAE. However, species specificity existed in the cytochemical properties. For S. hasta, monocytes were strongly positive for ALP and ACP; lymphocytes were strongly positive for ALP and weakly positive for ACP; neutrophils were strongly positive for ACP and POX; and thrombocytes were weakly positive for PAS and positive for ACP. Unlike S. hasta, monocytes were strongly positive for ACP and positive for ALP in S. schlegelii; lymphocytes were positive for ALP and partially positive for PAS; neutrophils were positive for ACP and POX; and thrombocytes were negative for PAS and ACP. The POX activity of monocytes in S. hasta was greater than that in S. schlegelii (P < 0.05), while the POX activity of neutrophils and the NAE activity of monocytes in S. hasta were significantly greater than those in S. schlegelii (P < 0.01). The results of this study can be used as a reference for the construction of haematological parameters in S. hasta and S. schlegelii for the assessment of fish health and can provide a research basis for fish diseases and immunity.

Stem Cells and Innate Immunity in Aquatic Invertebrates: Bridging Two Seemingly Disparate Disciplines for New Discoveries in Biology.

The scopes related to the interplay between stem cells and the immune system are broad and range from the basic understanding of organism's physiology and ecology to translational studies, further contributing to (eco)toxicology, biotechnology, and medicine as well as regulatory and ethical aspects. Stem cells originate immune cells through hematopoiesis, and the interplay between the two cell types is required in processes like regeneration. In addition, stem and immune cell anomalies directly affect the organism's functions, its ability to cope with environmental changes and, indirectly, its role in ecosystem services. However, stem cells and immune cells continue to be considered parts of two branches of biological research with few interconnections between them. This review aims to bridge these two seemingly disparate disciplines towards much more integrative and transformative approaches with examples deriving mainly from aquatic invertebrates. We discuss the current understanding of cross-disciplinary collaborative and emerging issues, raising novel hypotheses and comments. We also discuss the problems and perspectives of the two disciplines and how to integrate their conceptual frameworks to address basic equations in biology in a new, innovative way.

Diversity and phylogeny of percolomonads based on newly discovered species from hypersaline and marine waters.

Percolomonads are common freshwater, marine and hypersaline tetraflagellated organisms. Current phylogenetic analyses of eukaryotes comprise only two species of this underinvestigated family. Here, we studied the morphology, salinity tolerance and 18S rDNA gene-based phylogeny of seven percolomonad cultures. We describe three new genera and five novel species of Percolomonadida based on phylogenetic distances and morphological characteristics: Barbelia atlantica, B. abyssalis, Lula jakobsenorum, Nakurumonas serrata and Percolomonas doradorae. The new species show features typical for percolomonads, one long flagellum for skidding, three shorter flagella of equal length and a ventral feeding groove. The new species comprise organisms living in marine and athalassic hypersaline waters with salinity ranging from 10 to 150 PSU. Based on these novel taxa, the taxonomy and phylogeny of Percolatea was extended and further resolved.

Additions to the genus Gimesia: description of Gimesia alba sp. nov., Gimesia algae sp. nov., Gimesia aquarii sp. nov., Gimesia aquatilis sp. nov., Gimesia fumaroli sp. nov. and Gimesia panareensis sp. nov., isolated from aquatic habitats of the Northern Hemisphere.

Thirteen novel planctomycetal strains were isolated from five different aquatic sampling locations. These comprise the hydrothermal vent system close to Panarea Island (Italy), a biofilm on the surface of kelp at Monterey Bay (CA, USA), sediment and algae on Mallorca Island (Spain) and Helgoland Island (Germany), as well as a seawater aquarium in Braunschweig, Germany. All strains were shown to belong to the genus Gimesia. Their genomes cover a size range from 7.22 to 8.29 Mb and have a G+C content between 45.1 and 53.7%. All strains are mesophilic (Topt 26-33 °C) with generation times between 12 and 32 h. Analysis of fatty acids yielded palmitic acid (16:0) and a fatty acid with the equivalent chain length of 15.817 as major compounds. While five of the novel strains belong to the already described species Gimesia maris and Gimesia chilikensis, the other strains belong to novel species, for which we propose the names Gimesia alba (type strain Pan241wT = DSM 100744T = LMG 31345T = CECT 9841T = VKM B-3430T), Gimesia algae (type strain Pan161T = CECT 30192T = STH00943T = LMG 29130T), Gimesia aquarii (type strain V144T = DSM 101710T = VKM B-3433T), Gimesia fumaroli (type strain Enr17T = DSM 100710T = VKM B-3429T) and Gimesia panareensis (type strain Enr10T = DSM 100416T = LMG 29082T). STH numbers refer to the Jena Microbial Resource Collection (JMRC).

Periplasma, gen. nov., a new oomycete lineage with isogamous sexual reproduction.

This paper describes the taxonomy, developmental morphology, and phylogeny of Periplasma isogametum, a new monotypic member of the Leptomitales (Oomycota). In phylogenetic trees inferred from concatenated and separate nuc 18S rDNA (18S) and nuc 28S rDNA (28S) sequences, P. isogametum forms a well-supported clade related to but distinct from Apodachlya, a member of the Leptomitales. The organism is a holocarpic facultative saprotroph of moribund aquatic insects but grows well on a variety of mycological media in which it produces large eucarpic hyphae with a peripheral layer of protoplasm surrounding a central vacuole. In zoosporogenesis, a peripheral network of zoospore initials collapses to the center of the zoosporangium and is partitioned into individual heterokont zoospores. Sexual reproduction involves morphological isogamy in which male gametes produce elongate fertilization tubes, which fuse with female gametes, which are subsequently converted into thick-walled oospores. Developmental morphology is detailed in photomicrographs and pointillism drawings.

Fourfold daily growth rate in multicellular marine alga Ulva meridionalis.

Microalgae with high growth rates have been considered as promising organisms to replace fossil resources with contemporary primary production as a renewable source. However, their microscopic size makes it hard to be harvested for industrial applications. In this regard, multicellular macroalgae are more suitable for harvesting. Here, we show that Ulva meridionalis has the highest growth rate ever reported for a multicellular autotrophic plant. Contrasted to the known bloom-forming species U. prolifera growing at an approximately two-fold growth rate per day in optimum conditions, U. meridionalis grows at a daily rate of over fourfold. The high growth ability of this multicellular alga would provide the most effective method for CO2 fixation and biomass production.

Microfluidic accumulation assay to quantify the attachment of the marine bacterium Cobetia marina on fouling-release coatings.

Testing the adhesion of marine biofilm formers on bioresistant coatings is important to determine their fouling-release and antifouling properties. A dynamic attachment assay for the marine bacterium Cobetia marina (C. marina) was developed to test the adhesion on coatings and bioresistant surfaces. With well-defined culture conditions, the reproducibility of the microfluidic accumulation experiments with C. marina was verified using self-assembling monolayers as model surfaces. The assay discriminated the attachment of C. marina on four different surfaces with different wettability and protein resistances. In addition to these benchmark experiments on self-assembled monolayers, the adhesion of C. marina on polyglycerol coatings with different thicknesses was investigated.

Analysis of 13,312 benthic invertebrate samples from German streams reveals minor deviations in ecological status class between abundance and presence/absence data.

Benthic invertebrates are the most commonly used organisms used to assess ecological status as required by the EU Water Framework Directive (WFD). For WFD-compliant assessments, benthic invertebrate communities are sampled, identified and counted. Taxa × abundance matrices are used to calculate indices and the resulting scores are compared to reference values to determine the ecological status class. DNA-based tools, such as DNA metabarcoding, provide a new and precise method for species identification but cannot deliver robust abundance data. To evaluate the applicability of DNA-based tools to ecological status assessment, we evaluated whether the results derived from presence/absence data are comparable to those derived from abundance data. We analysed benthic invertebrate community data obtained from 13,312 WFD assessments of German streams. Broken down to 30 official stream types, we compared assessment results based on abundance and presence/absence data for the assessment modules "organic pollution" (i.e., the saprobic index) and "general degradation" (a multimetric index) as well as their underlying metrics. In 76.6% of cases, the ecological status class did not change after transforming abundance data to presence/absence data. In 12% of cases, the status class was reduced by one (e.g., from good to moderate), and in 11.2% of cases, the class increased by one. In only 0.2% of cases, the status shifted by two classes. Systematic stream type-specific deviations were found and differences between abundance and presence/absence data were most prominent for stream types where abundance information contributed directly to one or several metrics of the general degradation module. For a single stream type, these deviations led to a systematic shift in status from 'good' to 'moderate' (n = 201; with only n = 3 increasing). The systematic decrease in scores was observed, even when considering simulated confidence intervals for abundance data. Our analysis suggests that presence/absence data can yield similar assessment results to those for abundance-based data, despite type-specific deviations. For most metrics, it should be possible to intercalibrate the two data types without substantial efforts. Thus, benthic invertebrate taxon lists generated by standardised DNA-based methods should be further considered as a complementary approach.

Breakthrough in Marine Invertebrate Cell Culture: Sponge Cells Divide Rapidly in Improved Nutrient Medium.

Sponges (Phylum Porifera) are among the oldest Metazoa and considered critical to understanding animal evolution and development. They are also the most prolific source of marine-derived chemicals with pharmaceutical relevance. Cell lines are important tools for research in many disciplines, and have been established for many organisms, including freshwater and terrestrial invertebrates. Despite many efforts over multiple decades, there are still no cell lines for marine invertebrates. In this study, we report a breakthrough: we demonstrate that an amino acid-optimized nutrient medium stimulates rapid cell division in 9 sponge species. The fastest dividing cells doubled in less than 1 hour. Cultures of 3 species were subcultured from 3 to 5 times, with an average of 5.99 population doublings after subculturing, and a lifespan from 21 to 35 days. Our results form the basis for developing marine invertebrate cell models to better understand early animal evolution, determine the role of secondary metabolites, and predict the impact of climate change to coral reef community ecology. Furthermore, sponge cell lines can be used to scale-up production of sponge-derived chemicals for clinical trials and develop new drugs to combat cancer and other diseases.

Organically-preserved multicellular eukaryote from the early Ediacaran Nyborg Formation, Arctic Norway.

Eukaryotic multicellularity originated in the Mesoproterozoic Era and evolved multiple times since, yet early multicellular fossils are scarce until the terminal Neoproterozoic and often restricted to cases of exceptional preservation. Here we describe unusual organically-preserved fossils from mudrocks, that provide support for the presence of organisms with differentiated cells (potentially an epithelial layer) in the late Neoproterozoic. Cyathinema digermulense gen. et sp. nov. from the Nyborg Formation, Vestertana Group, Digermulen Peninsula in Arctic Norway, is a new carbonaceous organ-taxon which consists of stacked tubes with cup-shaped ends. It represents parts of a larger organism (multicellular eukaryote or a colony), likely with greater preservation potential than its other elements. Arrangement of open-ended tubes invites comparison with cells of an epithelial layer present in a variety of eukaryotic clades. This tissue may have benefitted the organism in: avoiding overgrowth, limiting fouling, reproduction, or water filtration. C. digermulense shares characteristics with extant and fossil groups including red algae and their fossils, demosponge larvae and putative sponge fossils, colonial protists, and nematophytes. Regardless of its precise affinity, C. digermulense was a complex and likely benthic marine eukaryote exhibiting cellular differentiation, and a rare occurrence of early multicellularity outside of Konservat-Lagerstätten.

Gertia stigmatica gen. et sp. nov. (Kareniaceae, Dinophyceae), a New Marine Unarmored Dinoflagellate Possessing the Peridinin-type Chloroplast with an Eyespot.

Marine unarmored dinoflagellates in the family Kareniaceae are known to possess chloroplasts of haptophyte origin, which contain fucoxanthin and its derivatives as major carotenoids, and lack peridinin. In the present study, the first species with the peridinin-type chloroplast in this family, Gertia stigmatica gen. et sp. nov., is described on the basis of ultrastructure, photosynthetic pigment composition, and molecular phylogeny inferred from nucleus- and chloroplast-encoded genes. Cells of G. stigmatica were small and harboring a chloroplast with an eyespot and two pyrenoids. The apical structure complex was straight, similar to Karenia and Karlodinium. Under transmission electron microscopy, the chloroplast was surrounded by two membranes, and the eyespot was composed of a single layer of osmiophilic globules (eyespot type A); this was never previously reported from the Kareniaceae. High performance liquid chromatography demonstrated the chloroplast contains peridinin, and neither fucoxanthin nor 19'-acyloxyfucoxanthins was identified. A phylogeny based on nucleus-encoded rDNAs suggested a position of G. stigmatica in the Kareniaceae, but not clustered within the previously described genera, i.e., Karenia, Karlodinium and Takayama. A phylogeny of chloroplast-encoded psbA, psbC and psbD indicated the chloroplast is of peridinin-type typical of dinoflagellates, but the most related species remains unclear.

Collective intercellular communication through ultra-fast hydrodynamic trigger waves.

The biophysical relationships between sensors and actuators1-5 have been fundamental to the development of complex life forms. Swimming organisms generate abundant flows that persist in aquatic environments6-13, and responding promptly to external stimuli is key to survival14-19. Here we present the discovery of 'hydrodynamic trigger waves' in cellular communities of the protist Spirostomum ambiguum that propagate-in a manner similar to a chain reaction20-22-hundreds of times faster than their swimming speed. By coiling its cytoskeleton, Spirostomum can contract its long body by 60% within milliseconds23, experiencing accelerations that can reach forces of 14g. We show that a single cellular contraction (the transmitter) generates long-ranged vortex flows at intermediate Reynolds numbers that can, in turn, trigger neighbouring cells (the receivers). To measure the sensitivity to hydrodynamic signals in these receiver cells, we present a high-throughput suction-flow device for probing mechanosensitive ion channels24 by back-calculating the microscopic forces on the cell membrane. We analyse and quantitatively model the ultra-fast hydrodynamic trigger waves in a universal framework of antenna and percolation theory25,26, and reveal a phase transition that requires a critical colony density to sustain collective communication. Our results suggest that this signalling could help to organize cohabiting communities over large distances and influence long-term behaviour through gene expression (comparable to quorum sensing16). In more immediate terms, because contractions release toxins27, synchronized discharges could facilitate the repulsion of large predators or immobilize large prey. We postulate that numerous aquatic organisms other than protists could coordinate their behaviour using variations of hydrodynamic trigger waves.

Taxonomy and phylogeny of two poorly studied genera of marine oligotrich ciliates including descriptions of two new species: Cyrtostrombidium paraboreale sp. n. and Apostrombidium orientale sp. n. (Ciliophora: Spirotrichea).

Although it is widely recognized that oligotrich ciliates are the dominant constituent of microzooplankton communities and perform key functions in energy flow and material cycling in marine microbial food webs, knowledge of their diversity is scant. In the present study, we investigate the oligotrich genera, Cyrtostrombidium and Apostrombidium, with emphasis on their morphology and evolutionary relationships. Three isolates were collected from coastal waters of northern and southern China including two new species, viz., Cyrtostrombidium paraboreale sp. n., Apostrombidium orientale sp. n., and Apostrombidium pseudokielum Xu et al., 2009. Cyrtostrombidium paraboreale sp. n. is characterized by possessing 64-98 cytopharyngeal rods and two macronuclear nodules. Apostrombidium orientale sp. n. is characterized by its somatic kinety consisting of five fragments including a horizontally orientated subterminal fragment and possessing conspicuously long dorsal cilia. Apostrombidium pseudokielum is redescribed based on the new population and a re-examination of the type material. Phylogenetic analyses were performed for the subclass Oligotrichia, incorporating SSU rRNA gene sequences of the three species investigated here. The results indicate that the genus Cyrtostrombidium is monophyletic with C. paraboreale sp. n. occupying the basal position. The genus Apostrombidium is not monophyletic as Varistrombidium kielum is nested within it.

Workshop report: Cryopreservation of aquatic biomedical models.

The genetic resources of aquatic biomedical model organisms are the products of millions of years of evolution, decades of scientific development, and hundreds of millions of dollars of research funding investment. Genetic resources (e.g., specific alleles, transgenes, or combinations) of each model organism can be considered a form of scientific wealth that can be accumulated and exchanged, typically in the form of live animals or germplasm. Large-scale maintenance of live aquatic organisms that carry these genetic resources is inefficient, costly, and risky. In situ maintenance may be substantially enhanced and backed up by combining cryopreserved germplasm repositories and genetic information systems with live animal culture. Unfortunately, cryopreservation has not advanced much beyond the status of an exploratory research for most aquatic species, lacks widespread application, and methods for successful cryopreservation remain poorly defined. For most aquatic species biological materials other than sperm or somatic cells are not comprehensively banked to represent and preserve a broad range of genetic diversity for each species. Therefore, new approaches and standardization are needed for repository-level application to ensure reproducible recovery of cryopreserved materials. Additionally, development of new technologies is needed to address preservation of novel biological materials, such as eggs and embryos of aquatic species. To address these goals, the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health (NIH) hosted the Cryopreservation of Aquatic Biomedical Models Workshop on January 7 to 8, 2017, in conjunction with the 8th Aquatic Animal Models of Human Disease Conference in Birmingham, Alabama. The goals of the workshop were to assess the status of germplasm cryopreservation in various biomedical aquatic models and allow representatives of the scientific community to develop and prioritize a consensus of specific actionable recommendations that will move the field of cryopreservation of aquatic resources forward. This workshop included sessions devoted to new approaches for cryopreservation of aquatic species, discussion of current efforts and approaches in preservation of aquatic model germplasm, consideration of needs for standardization of methods to support reproducibility, and enhancement of repository development by establishment of scalable high-throughput technologies. The following three broad recommendations were forwarded from workshop attendees: 1: Establish a comprehensive, centralized unit ("hub") to programmatically develop training for and documentation of cryopreservation methods for aquatic model systems. This would include development of species-specific protocols and approaches, outreach programs, community development and standardization, freezing services and training of the next generation of experts in aquatic cryopreservation. 2: Provide mechanisms to support innovative technical advancements that will increase the reliability, reproducibility, simplicity, throughput, and efficiency of the cryopreservation process, including vitrification and pipelines for sperm, oocytes, eggs, embryos, larvae, stem cells, and somatic cells of all aquatic species. This recommendation encompasses basic cryopreservation knowledge and engineering technology, such as microfluidics and automated processing technologies. 3: Implement mechanisms that allow the various aquatic model stock centers to increase their planning, personnel, ability to secure genetic resources and to promote interaction within an integrated, comprehensive repository network for aquatic model species repositories.

Loricate choanoflagellates (Acanthoecida) from warm water seas. I. Conioeca gen. nov. and Nannoeca Thomsen.

The main outcome of this and subsequent papers is to provide a baseline survey of heterotrophic protist diversity from warm water marine ecosystems, exemplified by loricate choanoflagellates (Acanthoecida). Loricate choanoflagellates are heterotrophic, nano-sized protists that are ubiquitous in marine and brackish water habitats. They dwell in a lorica formed by silicified costal strips organized in species specific patterns. The single anteriorly directed flagellum is surrounded by a collar formed by microvilli which together constitute the feeding apparatus. Keystone benefits from this warm water survey, which covers all three major oceans, is an improved understanding of global biogeographical patterns, and a further consolidation of the morphospecies matrix, that constitutes a highly essential reference framework for the current efforts to provide barcodes for as many species of loricate choanoflagellates as possible, based on e.g. single cell pipetting techniques. We describe here Conioeca gen. et sp. nov., which is so far distributionally confined to warm water habitats, and elaborate on the morphological variability encountered within the N. minuta complex. This leads to both the circumscription of a new N. minuta form A as well as the description of N. mexicana sp. nov.

Micro-CT 3D imaging reveals the internal structure of three abyssal xenophyophore species (Protista, Foraminifera) from the eastern equatorial Pacific Ocean.

Xenophyophores, giant foraminifera, are distinctive members of the deep-sea megafauna that accumulate large masses of waste material ('stercomare') within their agglutinated tests, and organise their cells as branching strands enclosed within an organic tube (the 'granellare' system). Using non-destructive, three-dimensional micro-CT imaging we explored these structures in three species from the abyssal eastern Pacific Clarion-Clipperton Zone (CCZ). In Psammina spp., the low-density stercomare occupied much of the test interior, while high-density granellare strands branched throughout the structure. In Galatheammina sp. the test comprised a mixture of stercomare and test particles, with the granellare forming a web-like system of filaments. The granellare occupied 2.8-5.1%, the stercomare 72.4-82.4%, and test particles 14.7-22.5%, of the 'body' volume in the two Psammina species. The corresponding proportions in Galatheammina sp. were 1.7% (granellare), 39.5% (stercomare) and 58.8% (test particles). These data provide a potential basis for estimating the contribution of xenophyophores to seafloor biomass in areas like the CCZ where they dominate the megafauna. As in most xenophyophore species, the granellare hosted huge numbers of tiny barite crystals. We speculate that these help to support the extensive granellare system, as well as reducing the cell volume and lightening the metabolic burden required to maintain it.

Cross-shelf investigation of coral reef cryptic benthic organisms reveals diversity patterns of the hidden majority.

Coral reefs harbor diverse assemblages of organisms yet the majority of this diversity is hidden within the three dimensional structure of the reef and neglected using standard visual surveys. This study uses Autonomous Reef Monitoring Structures (ARMS) and amplicon sequencing methodologies, targeting mitochondrial cytochrome oxidase I and 18S rRNA genes, to investigate changes in the cryptic reef biodiversity. ARMS, deployed at 11 sites across a near- to off-shore gradient in the Red Sea were dominated by Porifera (sessile fraction), Arthropoda and Annelida (mobile fractions). The two primer sets detected different taxa lists, but patterns in community composition and structure were similar. While the microhabitat of the ARMS deployment affected the community structure, a clear cross-shelf gradient was observed for all fractions investigated. The partitioning of beta-diversity revealed that replacement (i.e. the substitution of species) made the highest contribution with richness playing a smaller role. Hence, different reef habitats across the shelf are relevant to regional diversity, as they harbor different communities, a result with clear implications for the design of Marine Protected Areas. ARMS can be vital tools to assess biodiversity patterns in the generally neglected but species-rich cryptic benthos, providing invaluable information for the management and conservation of hard-bottomed habitats over local and global scales.

Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact.

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity.

Sheldon spectrum and the plankton paradox: two sides of the same coin-a trait-based plankton size-spectrum model.

The Sheldon spectrum describes a remarkable regularity in aquatic ecosystems: the biomass density as a function of logarithmic body mass is approximately constant over many orders of magnitude. While size-spectrum models have explained this phenomenon for assemblages of multicellular organisms, this paper introduces a species-resolved size-spectrum model to explain the phenomenon in unicellular plankton. A Sheldon spectrum spanning the cell-size range of unicellular plankton necessarily consists of a large number of coexisting species covering a wide range of characteristic sizes. The coexistence of many phytoplankton species feeding on a small number of resources is known as the Paradox of the Plankton. Our model resolves the paradox by showing that coexistence is facilitated by the allometric scaling of four physiological rates. Two of the allometries have empirical support, the remaining two emerge from predator-prey interactions exactly when the abundances follow a Sheldon spectrum. Our plankton model is a scale-invariant trait-based size-spectrum model: it describes the abundance of phyto- and zooplankton cells as a function of both size and species trait (the maximal size before cell division). It incorporates growth due to resource consumption and predation on smaller cells, death due to predation, and a flexible cell division process. We give analytic solutions at steady state for both the within-species size distributions and the relative abundances across species.