Next Generation Sequencing Reveals the Hidden Diversity of Zooplankton Assemblages

Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel next generation sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify richness and diversity of a mixed zooplankton assemblage from a productive time series site in the Western English Channel. Methodology/Principle Findings Plankton net hauls (200 µm) were taken at the Western Channel Observatory station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,041 sequences were obtained for all samples. The sequences clustered into 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 135 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 58 taxonomic groups. Conclusions Metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and hard-to-identify meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for elucidating the true diversity and species richness of zooplankton communities. While this approach allows for broad diversity assessments of plankton it may become increasingly attractive in future if sequence reference libraries of accurately identified individuals are better populated.

Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph

A variety of culture-independent techniques have been developed that can be used in conjunction with culture-dependent physiological and metabolic studies of key microbial organisms, in order to better understand how the activity of natural populations influences and regulates all major biogeochemical cycles. In this study, we combined DNA-stable isotope probing with metagenomics and metaproteomics to characterize an as yet uncultivated marine methylotroph that actively incorporated carbon from 13C-labeled methanol into biomass. By metagenomic sequencing of the heavy DNA, we retrieved virtually the whole genome of this bacterium and determined its metabolic potential. Through protein-stable isotope probing, the RuMP cycle was established as the main carbon assimilation pathway, and the classical methanol dehydrogenase-encoding gene mxaF, as well as three out of four identified xoxF homologues were found to be expressed. This proof-of-concept study is the first in which theculture-independent techniques of DNA- and protein-stable isotope probing have been used to characterize the metabolism of a naturally-ocurring Methylophaga-like bacterium in the marine environment (i.e. M. thiooxydans L4) and thus provides a powerful approach to access the genome and proteome of uncultivated microbes involved in key processes in the environment

A long-term chlorophyll dataset reveals regime shift in North Sea phytoplankton biomass unconnected to nutrient levels

During the 1980s, a rapid increase in the Phytoplankton Colour Index (PCI), a semiquantitative visual estimate of algal biomass, was observed in the North Sea as part of a regionwide regime shift. Two new data sets created from the relationship between the PCI and SeaWiFS chlorophyll a (Chl a) quantify differences in the previous and current regimes for both the anthropogenically affected coastal North Sea and the comparatively unaffected open North Sea. The new regime maintains a 13% higher Chl a concentration in the open North Sea and a 21% higher concentration in coastal North Sea waters. However, the current regime has lower total nitrogen and total phosphorus concentrations than the previous regime, although the molar N: P ratio in coastal waters is now well above the Redfield ratio and continually increasing. Besides becoming warmer, North Sea waters are also becoming clearer (i.e., less turbid), thereby allowing the normally light-limited coastal phytoplankton to more effectively utilize lower concentrations of nutrients. Linear regression analyses indicate that winter Secchi depth and sea surface temperature are the most important predictors of coastal Chl a, while Atlantic inflow is the best predictor of open Chl a; nutrient concentrations are not a significant predictor in either model. Thus, despite decreasing nutrient concentrations, Chl a continues to increase, suggesting that climatic variability and water transparency may be more important than nutrient concentrations to phytoplankton production at the scale of this study.

1H NMR metabolomics reveals contrasting response by male and female mussels exposed to reduced seawater pH, increased temperature and a pathogen

Human activities are fundamentally altering the chemistry of the world's oceans. Ocean acidification (OA) is occurring against a background of warming and an increasing occurrence of disease outbreaks, posing a significant threat to marine organisms, communities, and ecosystems. In the current study, (1)H NMR spectroscopy was used to investigate the response of the blue mussel, Mytilus edulis, to a 90-day exposure to reduced seawater pH and increased temperature, followed by a subsequent pathogenic challenge. Analysis of the metabolome revealed significant differences between male and female organisms. Furthermore, males and females are shown to respond differently to environmental stress. While males were significantly affected by reduced seawater pH, increased temperature, and a bacterial challenge, it was only a reduction in seawater pH that impacted females. Despite impacting males and females differently, stressors seem to act via a generalized stress response impacting both energy metabolism and osmotic balance in both sexes. This study therefore has important implications for the interpretation of metabolomic data in mussels, as well as the impact of environmental stress in marine invertebrates in general.

Satellite-detected fluorescence reveals global physiology of ocean phytoplankton

Phytoplankton photosynthesis links global ocean biology and climate-driven fluctuations in the physical environment. These interactions are largely expressed through changes in phytoplankton physiology, but physiological status has proven extremely challenging to characterize globally. Phytoplankton fluorescence does provide a rich source of physiological information long exploited in laboratory and field studies, and is now observed from space. Here we evaluate the physiological underpinnings of global variations in satellite-based phytoplankton chlorophyll fluorescence. The three dominant factors influencing fluorescence distributions are chlorophyll concentration, pigment packaging effects on light absorption, and light-dependent energy-quenching processes. After accounting for these three factors, resultant global distributions of quenching-corrected fluorescence quantum yields reveal a striking consistency with anticipated patterns of iron availability. High fluorescence quantum yields are typically found in low iron waters, while low quantum yields dominate regions where other environmental factors are most limiting to phytoplankton growth. Specific properties of photosynthetic membranes are discussed that provide a mechanistic view linking iron stress to satellite-detected fluorescence. Our results present satellite-based fluorescence as a valuable tool for evaluating nutrient stress predictions in ocean ecosystem models and give the first synoptic observational evidence that iron plays an important role in seasonal phytoplankton dynamics of the Indian Ocean. Satellite fluorescence may also provide a path for monitoring climate-phytoplankton physiology interactions and improving descriptions of phytoplankton light use efficiencies in ocean productivity models.

Genotyping an Emiliania huxleyi (Prymnesiophyceae) bloom event in the North Sea reveals evidence of asexual reproduction

Due to the unprecedented rate at which our climate is changing, the ultimate consequence for many species is likely to be either extinction or migration to an alternate habitat. Certain species might, however, evolve at a rate that could make them resilient to the effects of a rapidly changing environment. This scenario is most likely to apply to species that have large population sizes and rapid generation times, such that the genetic variation required for adaptive evolution can be readily supplied. Emiliania huxleyi (Lohm.) Hay and Mohler (Prymnesiophyceae) is likely to be such a species as it is the most conspicuous extant calcareous phytoplankton species in our oceans with generation times of 1 day−1. Here we report on a validated set of microsatellites, in conjunction with the coccolithophore morphology motif genetic marker, to genotype 93 clonal isolates collected from across the world. Of these, 52 came from a single bloom event in the North Sea collected on the D366 UK Ocean Acidification cruise in June-July 2011. There were 26 multilocus genotypes (MLGs) encountered only once in the North Sea bloom and 8 MLGs encountered twice or up to six times. Each of these repeated MLGs exhibited Psex values of less than 0.05 indicating each repeated MLG was the product of asexual reproduction and not separate meiotic events. In addition, we show that the two most polymorphic microsatellite loci, EHMS37 and P01E05, are reporting on regions likely undergoing rapid genetic drift during asexual reproduction. Despite the small sample size, there were many more repeated genotypes than previously reported for other bloom-forming phytoplankton species, including a previously genotyped E. huxleyi bloom event. This study challenges our current assumption that sex is the predominant mode of reproduction during bloom events. Whilst genetic diversity is high amongst extant populations of E. huxleyi, the root cause for this diversity and ultimate fate of these populations still requires further examination. Nonetheless, we show that certain CMM genotypes are found everywhere; while others appear to have a regional bias.

Mesoscale fronts as foraging habitats: composite front mapping reveals oceanographic drivers of habitat use for a pelagic seabird

The oceanographic drivers of marine vertebrate habitat use are poorly understood yet fundamental to our knowledge of marine ecosystem functioning. Here, we use composite front mapping and high-resolution GPS tracking to determine the significance of mesoscale oceanographic fronts as physical drivers of foraging habitat selection in northern gannets Morus bassanus. We tracked 66 breeding gannets from a Celtic Sea colony over 2 years and used residence time to identify area-restricted search (ARS) behaviour. Composite front maps identified thermal and chlorophyll-a mesoscale fronts at two different temporal scales—(i) contemporaneous fronts and (ii) seasonally persistent frontal zones. Using generalized additive models (GAMs), with generalized estimating equations (GEE-GAMs) to account for serial autocorrelation in tracking data, we found that gannets do not adjust their behaviour in response to contemporaneous fronts. However, ARS was more likely to occur within spatially predictable, seasonally persistent frontal zones (GAMs). Our results provide proof of concept that composite front mapping is a useful tool for studying the influence of oceanographic features on animal movements. Moreover, we highlight that frontal persistence is a crucial element of the formation of pelagic foraging hotspots for mobile marine vertebrates.

Pathogenic challenge reveals immune trade-off in mussels exposed to reduced seawater pH and increased temperature

Mussels tolerant to seawater pH's that are projected to occur by 2300 due to ocean acidification.•Exposure to pH 6.50 reduced mussel immune response, yet in the absence of a pathogen.•Subsequent pathogenic challenge led to a reversal of immune suppression at pH 6.50.•Study highlights the importance of undertaking multiple stressor exposures.•Shows a need to consider physiological trade-offs and measure responses functionally