Metabarcoding as a tool to enhance marine surveillance of nonindigenous species in tropical harbors: A case study in Tahiti

Globalization has increased connectivity between countries enhancing the spread of marine nonindigenous species (NIS). The establishment of marine NIS shows substantial negative effects on the structure and functioning of the natural ecosystems by competing for habitats and resources. Ports are often hubs for the spread of NIS via commercial and recreational vessels. Prevention, detection, and mitigation efforts are required to avoid and manage the establishment of NIS in new ecosystems. In this study, metabarcoding approaches targeting the nuclear small-subunit ribosomal RNA (18S rRNA) gene and mitochondrial cytochrome c oxidase I (COI) gene were used to investigate planktonic and sessile (i.e., biofouling) communities and NIS at four locations in Tahiti, including two marinas and one port with varying anthropogenic impacts, and a relatively pristine site (Manava) used as a control. ASV richness values showed significant differences (18S rRNA gene: p = .023; COI: p < .001) between locations in the plankton samples, with the control site (low impact) having the highest diversity for both genes. ASV richness was also significantly different among locations for the biofouling samples in the COI dataset (p = .002). Community composition differed between locations with spatial patterns appearing stronger for the plankton samples compared with the biofouling samples. Detection of NIS based on selected lists of globally invasive species revealed a wide diversity of potentially invasive taxa especially in the more anthropogenically impacted regions. The use of a multigene approach improved the detection of NIS. This study demonstrates the utility of using a metabarcoding approach to routinely monitor areas most at risk from NIS establishment in Tahiti and other coastal nations. These coastal nations are vulnerable to shipping-mediated incursions, and baseline information is required for both native diversity and nonindigenous diversity.publishedVersio

A step towards the validation of bacteria biotic indices using DNA metabarcoding for benthic monitoring

Environmental genomics is a promising field for monitoring biodiversity in a timely fashion. Efforts have increasingly been dedicated to the use of bacteria DNA derived data to develop biotic indices for benthic monitoring. However, a substantial debate exists about whether bacteria‐derived data using DNA metabarcoding should follow, for example, a taxonomy‐based or a taxonomy‐free approach to marine bioassessments. Here, we showcase the value of DNA‐based monitoring using the impact of fish farming as an example of anthropogenic disturbances in coastal areas and compare the performance of taxonomy‐based and taxonomy‐free approaches in detecting environmental alterations. We analysed samples collected near to the farm cages and along distance gradients from two aquaculture installations, and at control sites, to evaluate the effect of this activity on bacterial assemblages. Using the putative response of bacterial taxa to stress we calculated the taxonomy‐based biotic index microgAMBI. The distribution of individual amplicon sequence variants (ASVs), as a function of a gradient in sediment acid volatile sulphides, was then used to derive a taxonomy‐free bacterial biotic index specific for this data set using a de novo approach based on quantile regression splines. Our results show that microgAMBI revealed a organically enriched environment along the gradient. However, the de novo biotic index outperformed microgAMBI by providing a higher discriminatory power in detecting changes in abiotic factors directly related to fish production, whilst allowing the identification of new ASVs bioindicators. The de novo strategy applied here represents a robust method to define new bioindicators in regions or habitats where no previous information about the response of bacteria to environmental stressors exists.This work was partially funded by the project CGL2015-70136-R from the Spanish Ministry of Economy and Competitiveness (MINECO) and the EU ERDF funding program. E.A. and S.C. are supported by funding from a collaboration between KAUST and Saudi Aramco within the framework of the Saudi Aramco –KAUST Center for Marine Environmental Observations. K.T.-G. is supported by Ministerio de Ciencia, Innnovación y Universidades through the Juan de la Cierva Incorporación program (IJCI-2017-34174)

Comparing sediment DNA extraction methods for assessing organic enrichment associated with marine aquaculture

Marine sediments contain a high diversity of micro- and macro-organisms which are important in the functioning of biogeochemical cycles. Traditionally, anthropogenic perturbation has been investigated by identifying macro-organism responses along gradients. Environmental DNA (eDNA) analyses have recently been advocated as a rapid and cost-effective approach to measuring ecological impacts and efforts are underway to incorporate eDNA tools into monitoring. Before these methods can replace or complement existing methods, robustness and repeatability of each analytical step has to be demonstrated. One area that requires further investigation is the selection of sediment DNA extraction method. Environmental DNA sediment samples were obtained along a disturbance gradient adjacent to a Chinook (Oncorhynchus tshawytscha) salmon farm in Otanerau Bay, New Zealand. DNA was extracted using four extraction kits (Qiagen DNeasy PowerSoil, Qiagen DNeasy PowerSoil Pro, Qiagen RNeasy PowerSoil Total RNA/DNA extraction/elution and Favorgen FavorPrep Soil DNA Isolation Midi Kit) and three sediment volumes (0.25, 2, and 5 g). Prokaryotic and eukaryotic communities were amplified using primers targeting the 16S and 18S ribosomal RNA genes, respectively, and were sequenced on an Illumina MiSeq. Diversity and community composition estimates were obtained from each extraction kit, as well as their relative performance in established metabarcoding biotic indices. Differences were observed in the quality and quantity of the extracted DNA amongst kits with the two Qiagen DNeasy PowerSoil kits performing best. Significant differences were observed in both prokaryotes and eukaryotes (p < 0.001) richness among kits. A small proportion of amplicon sequence variants (ASVs) were shared amongst the kits (~3%) although these shared ASVs accounted for the majority of sequence reads (prokaryotes: 59.9%, eukaryotes: 67.2%). Differences were observed in the richness and relative abundance of taxonomic classes revealed with each kit. Multivariate analysis showed that there was a significant interaction between “distance” from the farm and “kit” in explaining the composition of the communities, with the distance from the farm being a stronger determinant of community composition. Comparison of the kits against the bacterial and eukaryotic metabarcoding biotic index suggested that all kits showed similar patterns along the environmental gradient. Overall, we advocate for the use of Qiagen DNeasy PowerSoil kits for use when characterizing prokaryotic and eukaryotic eDNA from marine farm sediments. We base this conclusion on the higher DNA quality values and richness achieved with these kits compared to the other kits/amounts investigated in this study. The additional advantage of the PowerSoil Kits is that DNA extractions can be performed using an extractor robot, offering additional standardization and reproducibility of results.publishedVersio

Polarization spectroscopy and magnetically-induced dichroism of the potassium D2 lines

We study modulation-free methods for producing sub-Doppler, dispersive line shapes for laser stabilization near the potassium D2 transitions at 767 nm. Polarization spectroscopy is performed and a comparison is made between the use of a mirror or beam splitter for aligning the counter-propagating pump and probe beams. Conventional magnetically-induced dichroism is found to suffer from a small dispersion and large background offset. We therefore introduce a modified scheme, using two spatially separated pump-probe beam pairs. Finally we compare our results to methods using phase modulation and heterodyne detection.Comment: 11 pages, 8 figures; published versio

Seasonal and spatial variations in bacterial communities from tetrodotoxin-bearing and non-tetrodotoxin-bearing clams

Tetrodotoxin (TTX) is one of the most potent naturally occurring compounds and is responsible for many human intoxications worldwide. Paphies australis are endemic clams to New Zealand which contain varying concentrations of TTX. Research suggests that P. australis accumulate the toxin exogenously, but the source remains uncertain. The aim of this study was to identify potential bacterial TTX-producers by exploring differences in bacterial communities in two organs of P. australis: the siphon and digestive gland. Samples from the digestive glands of a non-toxic bivalve Austrovenus stutchburyi that lives amongst toxic P. australis populations were also analyzed. Bacterial communities were characterized using 16S ribosomal RNA gene metabarcoding in P. australis sourced monthly from the Hokianga Harbor, a site known to have TTX-bearing clams, for 1 year, from ten sites with varying TTX concentrations around New Zealand, and in A. stutchburyi from the Hokianga Harbor. Tetrodotoxin was detected in P. australis from sites all around New Zealand and in all P. australis collected monthly from the Hokianga Harbor. The toxin averaged 150 μg kg–1 over the year of sampling in the Hokianga Harbor but no TTX was detected in the A. stutchburyi samples from the same site. Bacterial species diversity differed amongst sites (p < 0.001, F = 5.9) and the diversity in siphon samples was significantly higher than in digestive glands (p < 0.001, F = 65.8). Spirochaetaceae (4–60%) and Mycoplasmataceae (16–78%) were the most abundant families in the siphons and the digestive glands, respectively. The bacterial communities were compared between sites with the lowest TTX concentrations and the Hokianga Harbor (site with the highest TTX concentrations), and the core bacterial communities from TTX-bearing individuals were analyzed. The results from both spatial and temporal studies corroborate with previous hypotheses that Vibrio and Bacillus could be responsible for the source of TTX in bivalves. The results from this study also indicate that marine cyanobacteria, in particular picocyanobacteria (e.g., Cyanobium, Synechococcus, Pleurocapsa, and Prochlorococcus), should be investigated further as potential TTX producers

Sequencing effort dictates gene discovery in marine microbial metagenomes

© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd. Massive metagenomic sequencing combined with gene prediction methods were previously used to compile the gene catalogue of the ocean and host-associated microbes. Global expeditions conducted over the past 15 years have sampled the ocean to build a catalogue of genes from pelagic microbes. Here we undertook a large sequencing effort of a perturbed Red Sea plankton community to uncover that the rate of gene discovery increases continuously with sequencing effort, with no indication that the retrieved 2.83 million non-redundant (complete) genes predicted from the experiment represented a nearly complete inventory of the genes present in the sampled community (i.e., no evidence of saturation). The underlying reason is the Pareto-like distribution of the abundance of genes in the plankton community, resulting in a very long tail of millions of genes present at remarkably low abundances, which can only be retrieved through massive sequencing. Microbial metagenomic projects retrieve a variable number of unique genes per Tera base-pair (Tbp), with a median value of 14.7 million unique genes per Tbp sequenced across projects. The increase in the rate of gene discovery in microbial metagenomes with sequencing effort implies that there is ample room for new gene discovery in further ocean and holobiont sequencing studies

The role of seagrass vegetation and local environmental conditions in shaping benthic bacterial and macroinvertebrate communities in a tropical coastal lagoon

We investigated the influence of seagrass canopies on the benthic biodiversity of bacteria and macroinvertebrates in a Red Sea tropical lagoon. Changes in abundance, number of taxa and assemblage structure were analyzed in response to seagrass densities (low, SLD; high, SHD; seagrasses with algae, SA), and compared with unvegetated sediments. Biological and environmental variables were examined in these four habitats (hereafter called treatments), both in the underlaying sediments and overlaying waters, at three randomly picked locations in March 2017. Differences between treatments were more apparent in the benthic habitat than in the overlaying waters. The presence of vegetation (more than its cover) and changes in sedimentary features (grain size and metals) at local scales influenced the observed biological patterns, particularly for macroinvertebrates. Of note, the highest percentage of exclusive macroinvertebrate taxa (18% of the gamma diversity) was observed in the SHD treatment peaking in the SA for bacteria. Benthic macroinvertebrates and bacteria shared a generally low number of taxa across treatments and locations; approximately, 25% of the gamma diversity was shared among all treatments and locations for macrofauna, dropping to 11% for bacteria. Given the low overlap in the species distribution across the lagoon, sustaining the connectivity among heterogeneous soft sediment habitats appears to be essential for maintaining regional biodiversity. This study addresses a current scientific gap related to the relative contributions of vegetated and unvegetated habitats to biodiversity in tropical regions.Peer reviewe

WISE/NEOWISE Preliminary Analysis and Highlights of the 67P/Churyumov-Gerasimenko Near Nucleus Environs

On January 18-19 and June 28-29 of 2010, the Wide-field Infrared Survey Explorer (WISE) spacecraft imaged the Rosetta mission target, comet 67P/Churyumov-Gerasimenko. We present a preliminary analysis of the images, which provide a characterization of the dust environment at heliocentric distances similar to those planned for the initial spacecraft encounter, but on the outbound leg of its orbit rather than the inbound. Broad-band photometry yields low levels of CO2 production at a comet heliocentric distance of 3.32 AU and no detectable production at 4.18 AU. We find that at these heliocentric distances, large dust grains with mean grain diameters on the order of a millimeter or greater dominate the coma and evolve to populate the tail. This is further supported by broad-band photometry centered on the nucleus, which yield an estimated differential dust particle size distribution with a power law relation that is considerably shallower than average. We set a 3-sigma upper limit constraint on the albedo of the large-grain dust at <= 0.12. Our best estimate of the nucleus radius (1.82 +/- 0.20 km) and albedo (0.04 +/- 0.01) are in agreement with measurements previously reported in the literature

WISE/NEOWISE observations of Active Bodies in the Main Belt

We report results based on mid-infrared photometry of 5 active main belt objects (AMBOs) detected by the Wide-field Infrared Survey Explorer (WISE) spacecraft. Four of these bodies, P/2010 R2 (La Sagra), 133P/Elst-Pizarro, (596) Scheila, and 176P/LINEAR, showed no signs of activity at the time of the observations, allowing the WISE detections to place firm constraints on their diameters and albedos. Geometric albedos were in the range of a few percent, and on the order of other measured comet nuclei. P/2010 A2 was observed on April 2-3, 2010, three months after its peak activity. Photometry of the coma at 12 and 22 {\mu}m combined with ground-based visible-wavelength measurements provides constraints on the dust particle mass distribution (PMD), dlogn/dlogm, yielding power-law slope values of {\alpha} = -0.5 +/- 0.1. This PMD is considerably more shallow than that found for other comets, in particular inbound particle fluence during the Stardust encounter of comet 81P/Wild 2. It is similar to the PMD seen for 9P/Tempel 1 in the immediate aftermath of the Deep Impact experiment. Upper limits for CO2 & CO production are also provided for each AMBO and compared with revised production numbers for WISE observations of 103P/Hartley 2.Comment: 32 Pages, including 5 Figure