Feeding selectivity of bivalve larvae on natural plankton assemblages in the Western English Channel

Meroplankton, including bivalve larvae, are an important and yet understudied component of coastal marine food webs. Understanding the baseline of meroplankton ecology is imperative to establish and predict their sensitivity to local and global marine stressors. Over an annual cycle (October 2009–September 2010), bivalve larvae were collected from the Western Channel Observatory time series station L4 (50°15.00′N, 4°13.02′W). The morphologically similar larvae were identified by analysis of the 18S nuclear small subunit ribosomal RNA gene, and a series of incubation experiments were conducted to determine larval ingestion rates on natural plankton assemblages. Complementary gut content analysis was performed using a PCR-based method for detecting prey DNA both from field-collected larvae and those from the feeding experiments. Molecular identification of bivalve larvae showed the community composition to change over the course of the sampling period with domination by Phaxas in winter and higher diversity in autumn. The larvae selected for nanoeukaryotes (2–20 µm) including coccolithophores (<20 µm) which together comprised >75 % of the bivalve larvae diet. Additionally, a small percentage of carbon ingested originated from heterotrophic ciliates (<30 µm). The molecular analysis of bivalve larvae gut content provided increased resolution of identification of prey consumed and demonstrated that the composition of prey consumed established through bottle incubations conferred with that established from in situ larvae. Despite changes in bivalve larvae community structure, clearance rates of each prey type did not change significantly over the course of the experiment, suggesting different bivalve larvae species may consume similar prey.

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.

Validation of the detection of Alexandrium species using specific RNA probes tested in a microarray format: Calibration of signal using variability of RNA content with environmental conditions.

The dinoflagellate genus Alexandrium contains several toxin producing species and strains, which can cause major economic losses to the shell fish industry. It is therefore important to be able to detect these toxin producers and also distinguish toxic strains from some of the morphologically identical non-toxic strains. To facilitate this DNA probes to be used in a microarray format were designed in silico or developed from existing published probes. These probes targeted either the 18S or 28S ribosomal ribonucleic acid (rRNA) gene in Alexandrium tamarense Group I, Group III and Group IV, Alexandrium ostenfeldii and Alexandrium minutum. Three strains of A. tamarense Group I, A. tamarense Group III, A. minutum and two strains of A. ostenfeldii were grown at optimal conditions and transferred into new environmental conditions changing either the light intensity, salinity, temperature or nutrient concentrations, to check if any of these environmental conditions induced changes in the cellular ribonucleic acid (RNA) concentration or growth rate. The aim of this experiment was the calibration of several species-specific probes for the quantification of the toxic Alexandrium strains. Growth rates were highly variable but only elevated or lowered salinity significantly lowered growth rate for A. tamarense Group I and Group III; differences in RNA content were not significant for the majority of the treatments. Only light intensity seemed to affect significantly the RNA content in A. tamarense Group I and Group III, but this was still within the same range as for the other treatments meaning that a back calibration from RNA to cell numbers was possible. The designed probes allow the production of quantitative information for Alexandrium species for the microarray chip.

Ocean acidification shows negligible impacts on high-latitude bacterial community structure in coastal pelagic mesocosms

The impact of ocean acidification and carbonation on microbial community structure was assessed during a large-scale in situ costal pelagic mesocosm study, included as part of the EPOCA 2010 Arctic campaign. The mesocosm experiment included ambient conditions (fjord) and nine mesocosms with pCO(2) levels ranging from similar to 145 to similar to 1420 mu atm. Samples for the present study were collected at ten time points (t-1, t1, t5, t7, t12, t14, t18, t22, t26 to t28) in seven treatments (ambient fjord (similar to 145), 2x similar to 185, similar to 270, similar to 685, similar to 820, similar to 1050 mu atm) and were analysed for "small" and "large" size fraction microbial community composition using 16S rRNA (ribosomal ribonucleic acid) amplicon sequencing. This high-throughput sequencing analysis produced similar to 20 000 000 16S rRNA V4 reads, which comprised 7000OTUs. The main variables structuring these communities were sample origins (fjord or mesocosms) and the community size fraction (small or large size fraction). The community was significantly different between the unenclosed fjord water and enclosed mesocosms (both control and elevated CO2 treatments) after nutrients were added to the mesocosms, suggesting that the addition of nutrients is the primary driver of the change in mesocosm community structure. The relative importance of each structuring variable depended greatly on the time at which the community was sampled in relation to the phytoplankton bloom. The sampling strategy of separating the small and large size fraction was the second most important factor for community structure. When the small and large size fraction bacteria were analysed separately at different time points, the only taxon pCO(2) was found to significantly affect were the Gammaproteobacteria after nutrient addition. Finally, pCO(2) treatment was found to be significantly correlated (non-linear) with 15 rare taxa, most of which increased in abundance with higher CO2.

Minor impact of ocean acidification to the composition of the active microbial community in an Arctic sediment

Effects of ocean acidification on the composition of the active bacterial and archaeal community within Arctic surface sediment was analysed in detail using 16S rRNA 454 pyrosequencing. Intact sediment cores were collected and exposed to one of five different pCO(2) concentrations [380 (present day), 540, 750, 1120 and 3000 atm] and RNA extracted after a period of 14 days exposure. Measurements of diversity and multivariate similarity indicated very little difference between pCO(2) treatments. Only when the highest and lowest pCO(2) treatments were compared were significant differences evident, namely increases in the abundance of operational taxonomic units most closely related to the Halobacteria and differences to the presence/absence structure of the Planctomycetes. The relative abundance of members of the classes Planctomycetacia and Nitrospira increased with increasing pCO(2) concentration, indicating that these groups may be able to take advantage of changing pH or pCO(2) conditions. The modest response of the active microbial communities associated with these sediments may be due to the low and fluctuating pore-water pH already experienced by sediment microbes, a result of the pH buffering capacity of marine sediments, or due to currently unknown factors. Further research is required to fully understand the impact of elevated CO2 on sediment physicochemical parameters, biogeochemistry and microbial community dynamics.

Evaluating The Ribosomal Internal Transcribed Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker

DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.

Response of an Arctic Sediment Nitrogen Cycling Community to Increased CO2

Ocean acidification influences sediment/water nitrogen fluxes, possibly by impacting on the microbial process of ammonia oxidation. To investigate this further, undisturbed sediment cores collected from Ny Alesund harbour (Svalbard) were incubated with seawater adjusted to CO2 concentrations of 380, 540, 760, 1,120 and 3,000 μatm. DNA and RNA were extracted from the sediment surface after 14 days' exposure and the abundance of bacterial and archaeal ammonia oxidising (amoA) genes and transcripts quantified using quantitative polymerase chain reaction. While there was no change to the abundance of bacterial amoA genes, an increase to 760 μatm pCO2 reduced the abundance of bacterial amoA transcripts by 65 %, and this was accompanied by a shift in the composition of the active community. In contrast, archaeal amoA gene and transcript abundance both doubled at 3,000 μatm, with an increase in species richness also apparent. This suggests that ammonia oxidising bacteria and archaea in marine sediments have different pH optima, and the impact of elevated CO2 on N cycling may be dependent on the relative abundances of these two major microbial groups. Further evidence of a shift in the balance of key N cycling groups was also evident: the abundance of nirS-type denitrifier transcripts decreased alongside bacterial amoA transcripts, indicating that NO3 − produced by bacterial nitrification fuelled denitrification. An increase in the abundance of Planctomycete-specific 16S rRNA, the vastmajority of which grouped with known anammox bacteria, was also apparent at 3,000 μatm pCO2. This could indicate a possible shift from coupled nitrification–denitrification to anammox activity at elevated CO2.

Validation of the detection of Pseudo-nitzschia spp. using specific RNA probes tested in a microarray format: Calibration of signal based on variability of RNA content with environmental conditions.

Harmful algal blooms (HAB) occur worldwide and cause health problems and economic damage to fisheries and tourism. Monitoring for toxic algae is therefore essential but is based primarily on light microscopy, which is time consuming and can be limited by insufficient morphological characters such that more time is needed to examine critical features with electron microscopy. Monitoring with molecular tools is done in only a few places world-wide. EU FP7 MIDTAL (Microarray Detection of Toxic Algae) used SSU and LSU rRNA genes as targets on microarrays to identify toxic species. In order to comply with current monitoring requirements to report cell numbers as the relevant threshold measurement to trigger closure of fisheries, it was necessary to calibrate our microarray to convert the hybridisation signal obtained to cell numbers. Calibration curves for two species of Pseudo-nitzschia for use with the MIDTAL microarray are presented to obtain cell numbers following hybridisation. It complements work presented by Barra et al. (2012b. Environ. Sci. Pollut. Res. doi: 10.1007/s11356-012-1330-1v) for two other Pseudo-nitzschia spp., Dittami and Edvardsen (2012a. J. Phycol. 48, 1050) for Pseudochatonella, Blanco et al. (2013. Harmful Algae 24, 80) for Heterosigma, McCoy et al. (2013. FEMS. doi: 10.1111/1574-6941.12277) for Prymnesium spp., Karlodinium veneficum, and cf. Chatonella spp. and Taylor et al. (2014. Harmful Algae, in press) for Alexandrium.

High-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study

Despite the ecological importance of copepods, few Next Generation Sequencing studies (NGS) have been performed on small crustaceans, and a standard method for RNA extraction is lacking. In this study, we compared three commonly-used methods: TRIzol®, Aurum Total RNA Mini Kit and Qiagen RNeasy Micro Kit, in combination with preservation reagents TRIzol® or RNAlater®, to obtain high-quality and quantity of RNA from copepods for NGS. Total RNA was extracted from the copepods Calanus helgolandicus, Centropages typicus and Temora stylifera and its quantity and quality were evaluated using NanoDrop, agarose gel electrophoresis and Agilent Bioanalyzer. Our results demonstrate that preservation of copepods in RNAlater® and extraction with Qiagen RNeasy Micro Kit were the optimal isolation method for high-quality and quantity of RNA for NGS studies of C. helgolandicus. Intriguingly, C. helgolandicus 28S rRNA is formed by two subunits that separate after heat-denaturation and migrate along with 18S rRNA. This unique property of protostome RNA has never been reported in copepods. Overall, our comparative study on RNA extraction protocols will help increase gene expression studies on copepods using high-throughput applications, such as RNA-Seq and microarrays.