Adding Zooplankton to the OSMAC Toolkit: Effect of Grazing Stress on the Metabolic Profile and Bioactivity of a Diatom.

"One strain many compounds" (OSMAC) based approaches have been widely used in the search for bioactive compounds. Introducing stress factors like nutrient limitation, UV-light or cocultivation with competing organisms has successfully been used in prokaryote cultivation. It is known that diatom physiology is affected by changed cultivation conditions such as temperature, nutrient concentration and light conditions. Cocultivation, though, is less explored. Hence, we wanted to investigate whether grazing pressure can affect the metabolome of the marine diatom Porosira glacialis, and if the stress reaction could be detected as changes in bioactivity. P. glacialis cultures were mass cultivated in large volume bioreactor (6000 L), first as a monoculture and then as a coculture with live zooplankton. Extracts of the diatom biomass were screened in a selection of bioactivity assays: inhibition of biofilm formation, antibacterial and cell viability assay on human cells. Bioactivity was found in all bioassays performed. The viability assay towards normal lung fibroblasts revealed that P. glacialis had higher bioactivity when cocultivated with zooplankton than in monoculture. Cocultivation with diatoms had no noticeable effect on the activity against biofilm formation or bacterial growth. The metabolic profiles were analyzed showing the differences in diatom metabolomes between the two culture conditions. The experiment demonstrates that grazing stress affects the biochemistry of P. glacialis and thus represents a potential tool in the OSMAC toolkit.

Micro- and mesozooplankton successions in an Antarctic coastal environment during a warm year.

The rapid increase in atmospheric temperature detected in the last decades in the Western Antarctic Peninsula was accompanied by a strong glacier retreat and an increase in production of melting water, as well as changes in the sea-ice dynamic. The objective of this study was to analyze the succession of micro- and mesozooplankton during a warm annual cycle (December 2010-December 2011) in an Antarctic coastal environment (Potter Cove). The biomass of zooplankton body size classes was used to predict predator-prey size relationships (i.e., to test bottom-up/top-down control effects) using a Multiple Linear Regression Analysis. The micro- and mesozooplanktonic successions were graphically analyzed to detect the influence of environmental periods (defined by the degree of glacial melting, sea-ice freezing and sea-ice melting) on coupling/uncoupling planktonic biomass curves associated to possible predator-prey size relationship scenarios. At the beginning of the glacial melting, medium and large mesozooplankton (calanoid copepods, Euphausia superba, and Salpa thompsoni) exert a top-down control on Chl-a and microzooplankton. Stratification of the water column benefitted the availability of adequate food-size (Chl-a <20) for large microzooplankton (tintinnids) development observed during fall. High abundance of omnivores mesozooplankton (Oithona similis and furcilia of E. superba) during sea-ice freezing periods would be due to the presence of available heterotrophic food under or within the sea ice. Finally, the increase in microzooplankton abundance in the middle of spring, when sea-ice melting starts, corresponded to small and medium dinoflagellates and ciliates species, which were possibly part of the biota of sea ice. If glacier retreat continues and the duration and thickness of the sea ice layer fluctuates as predicted by climate models, our results predict a future scenario regarding the zooplankton succession in Antarctic coastal environments.

Global change drives modern plankton communities away from the pre-industrial state.

The ocean-the Earth's largest ecosystem-is increasingly affected by anthropogenic climate change1,2. Large and globally consistent shifts have been detected in species phenology, range extension and community composition in marine ecosystems3-5. However, despite evidence for ongoing change, it remains unknown whether marine ecosystems have entered an Anthropocene6 state beyond the natural decadal to centennial variability. This is because most observational time series lack a long-term baseline, and the few time series that extend back into the pre-industrial era have limited spatial coverage7,8. Here we use the unique potential of the sedimentary record of planktonic foraminifera-ubiquitous marine zooplankton-to provide a global pre-industrial baseline for the composition of modern species communities. We use a global compilation of 3,774 seafloor-derived planktonic foraminifera communities of pre-industrial age9 and compare these with communities from sediment-trap time series that have sampled plankton flux since AD 1978 (33 sites, 87 observation years). We find that the Anthropocene assemblages differ from their pre-industrial counterparts in proportion to the historical change in temperature. We observe community changes towards warmer or cooler compositions that are consistent with historical changes in temperature in 85% of the cases. These observations not only confirm the existing evidence for changes in marine zooplankton communities in historical times, but also demonstrate that Anthropocene communities of a globally distributed zooplankton group systematically differ from their unperturbed pre-industrial state.

The effects of environmental factors and geographic distance on species turnover in an agriculturally dominated river network.

Both environmental and geographic factors interact to structure the metacommunities in river networks, but the importance of these factors is difficult to distinguish. We used six aquatic taxonomic groups to test the relationship between environmental and geographic factors and their effect on species turnover patterns in an agriculturally dominated river (Chaohu Lake Basin, China). The relationships between three dissimilarity indices and geographic distance were assessed using the Mantel test while considering the differences in environmental factors between sites. Then, we employed a variation partitioning method to distinguish the isolated and combined effects of environmental and geographic distance on species turnover. There were significant relationships between environmental distance and species turnover in all groups. All organisms except periphytic diatoms were significantly correlated with two geographic (Euclidean and network) distances when the Chao dissimilarity index was considered. The results suggest that the strength of the correlations changed with environmental and geographic distances and with the aquatic community. The communities displayed more complex relationships with the distance measures when different dissimilarity (Jaccard, Chao, and Bray-Curtis dissimilarity) indices were considered. Nevertheless, aquatic communities are strongly influenced by both environmental and geographic distance, and the former has a stronger effect than the latter.

The Impact of Anthropogenic Disturbance on Bacterioplankton Communities During the Construction of Donghu Tunnel (Wuhan, China).

Bacterioplankton are both primary producers and primary consumers in aquatic ecosystems, which were commonly investigated to reflect environmental changes, evaluate primary productivity, and assess biogeochemical cycles. However, there is relatively less understanding of their responses to anthropogenic disturbances such as constructions of dams/tunnels/roads that may significantly affect the aquatic ecosystem. To fill such gap, this study focused on the bacterioplankton communities' diversity and turnover during a tunnel construction across an urban lake (Lake Donghu, Wuhan, China), and five batches of samples were collected within 2 months according to the tunnel construction progress. Results indicated that both resources and predator factors contributed significant to the variations of bacterioplankton communities, but the closed area and open areas showed different diversity patterns due to the impacts of tunnel construction. Briefly, the phytoplankton, TN, and TP in water were still significantly correlated with the bacterioplankton composition and diversity like that in normal conditions. Additionally, the organic matter, TN, and NH4-N in sediments also showed clear effects on the bacterioplankton. However, the predator effects on the bacterioplankton in the closed-off construction area mainly derived from large zooplankton (i.e., cladocerans), while small zooplankton such as protozoa and rotifers are only responsible for weak predator effects on the bacterioplankton in the open areas. Further analysis about the ecological driving forces indicated that the bacterioplankton communities' turnover during the tunnel construction was mainly governed by the homogeneous selection due to similar environments within the closed area or the open areas at two different stages. This finding suggests that bacterioplankton communities can quickly adapt to the environmental modifications resulting from tunnel construction activities. This study can also give references to enhance our understanding on bacterioplankton communities' response to ecological and environmental changes due to intensification of construction and urbanization in and around lake ecosystems.

Intra-monsoonal variation of zooplankton population in the Sundarbans Estuarine System, India.

The present study was conducted during July 2013 (early phase of monsoon or EM) and September 2013 (later phase of monsoon or LM) to ascertain the intra-monsoonal variation on zooplankton, by selecting 15 study stations in the river Saptamukhi, one of the main estuaries in the Sundarbans Estuarine System (SES). In 2013, SES experienced an unusually high monsoonal rainfall also exacerbated by cloud burst event at Himalayan region (upper stretches of SES) which tremendously increased the river runoff. The present work was aimed to decipher the effect of this unusual precipitation during the monsoon season on zooplankton assemblages along with different hydrological parameters. The abundance of zooplankton was recorded as lower during EM compared to LM. Altogether, 56 zooplankton taxa were identified with copepods forming the predominant population. Thirty-three copepod species were reported with 25 calanoid species forming the bulk of the biomass followed by 5 and 3 species of cyclopoids and harpacticoid, respectively. A combination of multivariate cluster analysis, biotic indices, and canonical correspondence analysis revealed noticeable alterations in the zooplankton community structure across the spatio-temporal scale. Furthermore, significant intra-monsoonal changes in zooplankton population correlated with several hydrological parameters were clearly noticed. Paracalanus parvus, Bestiolina similis and Oithona similis were observed to be the most dominant copepod species in both sampling periods. The result of the present study provides new insight on estuarine zooplankton community after unusual rainfall during monsoon season, and provides further evidence to support the conservation and management of the SES ecosystem.

Seasonal variability of diet and trophic level of the gelatinous predator Pelagia noctiluca (Scyphozoa).

Jellyfish populations apparently have increased in some places around the world and human problems with them also have increased. However, effects of jellyfish outbreaks in the ecosystems remain poorly understood and little or no information is available on their dietary preferences - in relation to the seasonal shifts of prey abundance - and on the potential variability of their impact on marine food webs. The mauve stinger Pelagia noctiluca (Forsskål, 1775) is by far the most common outbreak-forming scyphozoan jellyfish in the Western Mediterranean. By use of a combination of stomach contents, stable isotope (SI) and fatty acid (FA) analyses, we tested the hypothesis that changes in the seasonal dietary sources of P. noctiluca parallel changes in the FA and SI composition. Stomach content and biomarker analyses suggested that P. noctiluca is not a selective predator, cyclically shifting between carnivory and omnivory depending on the seasonality of accessible prey. The combination of SI and FA analyses highlighted the importance of microzooplankton as prey. Specific FA biomarkers showed that the diet of P. noctiluca changed seasonally depending on the availability of living plankton or suspended detritus. This study also revealed significant biochemical differences between jellyfish somatic and gonadal tissues, with total fatty acid concentration in the gonads up to ten times higher than in the somatic tissues.

Vertical gradients in species richness and community composition across the twilight zone in the North Pacific Subtropical Gyre.

Although metazoan animals in the mesopelagic zone play critical roles in deep pelagic food webs and in the attenuation of carbon in midwaters, the diversity of these assemblages is not fully known. A metabarcoding survey of mesozooplankton diversity across the epipelagic, mesopelagic and upper bathypelagic zones (0-1500 m) in the North Pacific Subtropical Gyre revealed far higher estimates of species richness than expected given prior morphology-based studies in the region (4,024 OTUs, 10-fold increase), despite conservative bioinformatic processing. Operational taxonomic unit (OTU) richness of the full assemblage peaked at lower epipelagic-upper mesopelagic depths (100-300 m), with slight shoaling of maximal richness at night due to diel vertical migration, in contrast to expectations of a deep mesopelagic diversity maximum as reported for several plankton groups in early systematic and zoogeographic studies. Four distinct depth-stratified species assemblages were identified, with faunal transitions occurring at 100 m, 300 m and 500 m. Highest diversity occurred in the smallest zooplankton size fractions (0.2-0.5 mm), which had significantly lower % OTUs classified due to poor representation in reference databases, suggesting a deep reservoir of poorly understood diversity in the smallest metazoan animals. A diverse meroplankton assemblage also was detected (350 OTUs), including larvae of both shallow and deep living benthic species. Our results provide some of the first insights into the hidden diversity present in zooplankton assemblages in midwaters, and a molecular reappraisal of vertical gradients in species richness, depth distributions and community composition for the full zooplankton assemblage across the epipelagic, mesopelagic and upper bathypelagic zones.

DNA metabarcoding of unfractionated water samples relates phyto-, zoo- and bacterioplankton dynamics and reveals a single-taxon bacterial bloom.

Most studies of aquatic plankton focus on either macroscopic or microbial communities, and on either eukaryotes or prokaryotes. This separation is primarily for methodological reasons, but can overlook potential interactions among groups. Here we tested whether DNA metabarcoding of unfractionated water samples with universal primers could be used to qualitatively and quantitatively study the temporal dynamics of the total plankton community in a shallow temperate lake. Significant changes in the relative proportions of normalized sequence reads of eukaryotic and prokaryotic plankton communities over a 3-month period in spring were found. Patterns followed the same trend as plankton estimates measured using traditional microscopic methods. The bloom of a conditionally rare bacterial taxon belonging to Arcicella was characterized, which rapidly came to dominate the whole lake ecosystem and would have remained unnoticed without metabarcoding. The data demonstrate the potential of universal DNA metabarcoding applied to unfractionated samples for providing a more holistic view of plankton communities.

Modified FlowCAM procedure for quantifying size distribution of zooplankton with sample recycling capacity.

We have developed a modified FlowCAM procedure for efficiently quantifying the size distribution of zooplankton. The modified method offers the following new features: 1) prevents animals from settling and clogging with constant bubbling in the sample container; 2) prevents damage to sample animals and facilitates recycling by replacing the built-in peristaltic pump with an external syringe pump, in order to generate negative pressure, creates a steady flow by drawing air from the receiving conical flask (i.e. vacuum pump), and transfers plankton from the sample container toward the main flowcell of the imaging system and finally into the receiving flask; 3) aligns samples in advance of imaging and prevents clogging with an additional flowcell placed ahead of the main flowcell. These modifications were designed to overcome the difficulties applying the standard FlowCAM procedure to studies where the number of individuals per sample is small, and since the FlowCAM can only image a subset of a sample. Our effective recycling procedure allows users to pass the same sample through the FlowCAM many times (i.e. bootstrapping the sample) in order to generate a good size distribution. Although more advanced FlowCAM models are equipped with syringe pump and Field of View (FOV) flowcells which can image all particles passing through the flow field; we note that these advanced setups are very expensive, offer limited syringe and flowcell sizes, and do not guarantee recycling. In contrast, our modifications are inexpensive and flexible. Finally, we compared the biovolumes estimated by automated FlowCAM image analysis versus conventional manual measurements, and found that the size of an individual zooplankter can be estimated by the FlowCAM image system after ground truthing.

Modeling sampling strategies for determination of zooplankton abundance in ballast water.

Ballast water has been a major source of non-indigenous species introductions. The International Maritime Organization has proposed performance standard that will establish an upper limit for viable organisms in discharged ballast. Here we test different sampling efforts for zooplankton in ballast water on a commercial vessel. We fit different probability density functions to find the most representative and evaluated sampling efforts necessary to achieve error rates (α, ß) of <0.05. Our tests encompassed four seasonal trials and five sample volumes. To estimate error rates, we performed simulations which drew from 1 to 30 replicates of each volume (0.10-3.00m3) for mean densities ranging between 1 and 20 organisms m-3. Fieldwork and simulations suggested that >0.5m3 samples had the best accuracy and precision, and that the Poisson distribution fit these communities best. This study provides the first field test of a sampling strategy to assess compliance with the future IMO standard for large vessels.

Indicator Properties of Baltic Zooplankton for Classification of Environmental Status within Marine Strategy Framework Directive.

The European Marine Strategy Framework Directive requires the EU Member States to estimate the level of anthropogenic impacts on their marine systems using 11 Descriptors. Assessing food web response to altered habitats is addressed by Descriptor 4 and its indicators, which are being developed for regional seas. However, the development of simple foodweb indicators able to assess the health of ecologically diverse, spatially variable and complex interactions is challenging. Zooplankton is a key element in marine foodwebs and thus comprise an important part of overall ecosystem health. Here, we review work on zooplankton indicator development using long-term data sets across the Baltic Sea and report the main findings. A suite of zooplankton community metrics were evaluated as putative ecological indicators that track community state in relation to Good Environmental Status (GES) criteria with regard to eutrophication and fish feeding conditions in the Baltic Sea. On the basis of an operational definition of GES, we propose mean body mass of zooplankton in the community in combination with zooplankton stock measured as either abundance or biomass to be applicable as an integrated indicator that could be used within the Descriptor 4 in the Baltic Sea. These metrics performed best in predicting zooplankton being in-GES when considering all datasets evaluated. However, some other metrics, such as copepod biomass, the contribution of copepods to the total zooplankton biomass or biomass-based Cladocera: Copepoda ratio, were equally reliable or even superior in certain basin-specific assessments. Our evaluation suggests that in several basins of the Baltic Sea, zooplankton communities currently appear to be out-of-GES, being comprised by smaller zooplankters and having lower total abundance or biomass compared to the communities during the reference conditions; however, the changes in the taxonomic structure underlying these trends vary widely across the sea basins due to the estuarine character of the Baltic Sea.

In situ imaging reveals the biomass of giant protists in the global ocean.

Planktonic organisms play crucial roles in oceanic food webs and global biogeochemical cycles. Most of our knowledge about the ecological impact of large zooplankton stems from research on abundant and robust crustaceans, and in particular copepods. A number of the other organisms that comprise planktonic communities are fragile, and therefore hard to sample and quantify, meaning that their abundances and effects on oceanic ecosystems are poorly understood. Here, using data from a worldwide in situ imaging survey of plankton larger than 600 µm, we show that a substantial part of the biomass of this size fraction consists of giant protists belonging to the Rhizaria, a super-group of mostly fragile unicellular marine organisms that includes the taxa Phaeodaria and Radiolaria (for example, orders Collodaria and Acantharia). Globally, we estimate that rhizarians in the top 200 m of world oceans represent a standing stock of 0.089 Pg carbon, equivalent to 5.2% of the total oceanic biota carbon reservoir. In the vast oligotrophic intertropical open oceans, rhizarian biomass is estimated to be equivalent to that of all other mesozooplankton (plankton in the size range 0.2-20 mm). The photosymbiotic association of many rhizarians with microalgae may be an important factor in explaining their distribution. The previously overlooked importance of these giant protists across the widest ecosystem on the planet changes our understanding of marine planktonic ecosystems.

Warming accelerates termination of a phytoplankton spring bloom by fungal parasites.

Climate change is expected to favour infectious diseases across ecosystems worldwide. In freshwater and marine environments, parasites play a crucial role in controlling plankton population dynamics. Infection of phytoplankton populations will cause a transfer of carbon and nutrients into parasites, which may change the type of food available for higher trophic levels. Some phytoplankton species are inedible to zooplankton, and the termination of their population by parasites may liberate otherwise unavailable carbon and nutrients. Phytoplankton spring blooms often consist of large diatoms inedible for zooplankton, but the zoospores of their fungal parasites may serve as a food source for this higher trophic level. Here, we investigated the impact of warming on the fungal infection of a natural phytoplankton spring bloom and followed the response of a zooplankton community. Experiments were performed in ca. 1000 L indoor mesocosms exposed to a controlled seasonal temperature cycle and a warm (+4 °C) treatment in the period from March to June 2014. The spring bloom was dominated by the diatom Synedra. At the peak of infection over 40% of the Synedra population was infected by a fungal parasite (i.e. a chytrid) in both treatments. Warming did not affect the onset of the Synedra bloom, but accelerated its termination. Peak population density of Synedra tended to be lower in the warm treatments. Furthermore, Synedra carbon: phosphorus stoichiometry increased during the bloom, particularly in the control treatments. This indicates enhanced phosphorus limitation in the control treatments, which may have constrained chytrid development. Timing of the rotifer Keratella advanced in the warm treatments and closely followed chytrid infections. The chytrids' zoospores may thus have served as an alternative food source to Keratella. Our study thus emphasizes the importance of incorporating not only nutrient limitation and grazing, but also parasitism in understanding the response of plankton communities towards global warming.

A New Phaeodarian Species Discovered from the Japan Sea Proper Water, Auloscena pleuroclada sp. nov. (Aulosphaeridae, Phaeosphaerida, Phaeodaria).

A new phaeodarian species, characterized by the presence of long developed side branches recurved proximally and distally on the surface of its radial tube, was described as Auloscena pleuroclada. This new species was only collected from the layers below the 250 m depth in the Sea of Japan. They have never been found in the shallower layers (above 250 m) of this sea or in other investigated areas. The distribution of the present new species is presumably restricted within the deep water of this area, and this species could be a specific phaeodarian adapted to the deep-sea environment.

Five Years of Experimental Warming Increases the Biodiversity and Productivity of Phytoplankton.

Phytoplankton are key components of aquatic ecosystems, fixing CO2 from the atmosphere through photosynthesis and supporting secondary production, yet relatively little is known about how future global warming might alter their biodiversity and associated ecosystem functioning. Here, we explore how the structure, function, and biodiversity of a planktonic metacommunity was altered after five years of experimental warming. Our outdoor mesocosm experiment was open to natural dispersal from the regional species pool, allowing us to explore the effects of experimental warming in the context of metacommunity dynamics. Warming of 4°C led to a 67% increase in the species richness of the phytoplankton, more evenly-distributed abundance, and higher rates of gross primary productivity. Warming elevated productivity indirectly, by increasing the biodiversity and biomass of the local phytoplankton communities. Warming also systematically shifted the taxonomic and functional trait composition of the phytoplankton, favoring large, colonial, inedible phytoplankton taxa, suggesting stronger top-down control, mediated by zooplankton grazing played an important role. Overall, our findings suggest that temperature can modulate species coexistence, and through such mechanisms, global warming could, in some cases, increase the species richness and productivity of phytoplankton communities.

Water stratification affects the microeukaryotic community in a subtropical deep reservoir.

Producers, consumers, and decomposers are the three key functional groups that form the basis of all ecosystems. But, little is known about how these functional groups coexist with each other in aquatic environments, particularly in subtropical reservoirs. In this study, we describe the nature of microeukaryotic communities in a subtropical deep reservoir during the strongly stratified period. Denaturing gradient gel electrophoresis gel band sequencing, pyrosequencing, and light microscopy were used together to facilitate an in-depth investigation of the community structure of phytoplankton, zooplankton, and fungi. Our results showed that thermal and oxygen stratification shaped the composition of the phytoplankton, zooplankton, and fungi populations in the reservoir. Stratification was evident among ecological functional groups in autumn: producers and consumers were overwhelmingly dominant in the epilimnion characterized by high temperatures and oxygen levels, whereas decomposers were inclined to inhabit the hypolimnion. These results contribute to our understanding of the relationship of ecosystem functional groups in the man-made aquatic systems and have important practical implications for reservoir management. Results suggest that the strategies for the control of eutrophication and harmful algal bloom prevention should focus on a fuller understanding of the consequences of both thermal stratification and vertical distribution of microplankton.

Distribution of hydro-biological parameters in coastal waters off Rushikulya Estuary, East Coast of India: a premonsoon case study.

The hydro-biological parameters of coastal waters off Rushikulya estuary was investigated during premonsoon 2011. Important hydro-biological parameters such as water temperature, salinity, pH, DO, NO2, NO3, NH4, PO4, SiO4, TSM, Chl-a, phytoplankton and zooplankton were measured during the present study. Temperature established a strong positive correlation with salinity and pH during the present study. Chl-a found in positive relation with NO3, SiO, and TSM. Analysis of variance revealed significant monthly variation in pH, salinity and TSM. Significant station wise variation was observed in DO and most of the nutrients i.e., NO3, NH4, PO4, SiO4. A total of 119 species of phytoplankton were identified of which 84 species are of diatoms, 22 species of dinoflagellates, 7 species of green algae, 5 species of cyanobacteria (blue green algae) and 1 species of cocolithophore. Phytoplankton abundance varied between 25543 (Nos. L(-1)) and 36309 (Nos. L(-1)). Diatoms dominated the phytoplankton community followed by dinoflagellates in all the months. Diatoms contributed to 82-89% of the total phytoplankton population density whereas dinoflagellates contributed to 6-12%. The regression between Chl-a and phytoplankton abundance resulted with weak relation (R(2) = 0.042). Zooplankton fauna composed of 134 species of holoplankton and 20 types of meroplankton were encountered during the study period. Zooplankton population dominated by copepod during all months and accounted for 74 to 85% to the total zooplankton. The population density ranged from 6959 to 35869 Nos./10 m(3). Analysis of variance explained no significant variation in total zooplankton abundance and also for different groups of zooplankton.

Early detection of protozoan grazers in algal biofuel cultures.

Future micro-algal biofuels will most likely be derived from open-pond production systems. These are by definition open to "invasion" by grazers, which could devastate micro-algal mass-cultures. There is an urgent requirement for methodologies capable of early detection and control of grazers in dense algal cultures. In this study a model system employing the marine alga Nannochloropsis oculata was challenged by grazers including ciliates, amoebae and a heterotrophic dinoflagellate. A FlowCAM flow-cytometer was used to detect all grazers investigated (size range <20->80 µm in length) in the presence of algae. Detection limits were <10 cells ml(-1) for both "large" and "small" model grazers, Euplotes vannus (80 × 45 µm) and an unidentified holotrichous ciliate (~18 × 8 µm) respectively. Furthermore, the system can distinguish the presence of ciliates in N. oculata cultures with biotechnologically relevant cell densities; i.e. >1.4 × 10(8) cells ml(-1) (>0.5 g l(-1) dry wt.).