Plastic pollution in coastal areas, particularly in subtropical and tropical regions, remains a pervasive environmental issue. Marine plastic debris provides an artificial surface that rapidly accumulates a dynamic microbial biofilm upon entering the marine ecosystem. Especially the early stages of colonization are critical in shaping the microbial community. This study investigates the early microbial colonization, in less than a week, on five different plastic polymers in Caribbean coastal waters through 16S rRNA gene amplicon sequencing. We discovered shared bacterial taxa among the various plastic polymers and sampling timepoints, with dominant orders being Flavobacteriales, Rhodobacterales, Rhizobiales, and Pseudomonadales. Statistical analysis confirmed significant differences in community composition between the two sampling points, with polystyrene exhibiting a distinct microbial community on day 6 compared to polyethylene, polypropylene, and nylon. We found the same for polyethylene compared to nylon and polyethylene-terephthalate. Further examination identified 47 genera responsible for these differences, primarily belonging to the phyla Proteobacteria and Bacteroidota. Our data indicate an influence of both environmentally related stochastic processes and plastic-related specific factors during early colonization. Interestingly, we noticed an increase in the relative abundance of hydrocarbon and potentially plastic-degrading bacteria (PDB) from 12.4 to 34.5% between the first and sixth day, suggesting their vital role in shaping the epiplastic community. Notably, some identified PDB have been reported to degrade the specific polymers studied, thus the monitored increase in relative abundance supports their role in plastic degradation. However, more research is required to fully understand their functioning and potential role in the epiplastic community. Our study provides insights into the prokaryotic colonization of marine plastics in the Caribbean basin, where to date studies have been limited despite high pollution rates.

With rapid climate change over the past decades, organisms living in seasonal environments are suggested to increasingly face trophic mismatches: the disruption of synchrony between different trophic levels due to a different phenological response to increasing temperatures. Strong effects of mismatches are especially expected in the Arctic region, where climatic changes are most pronounced. Nevertheless, relatively few studies have found strong evidence for trophic mismatches between the breeding period of Arctic-breeding shorebirds and the arthropod prey on which they rely. Here we argue that this is potentially caused by a generalization of trophic interactions. While many studies have measured the mismatch relative to the peak in abundance of all available arthropod species, we use metabarcoding of prey items in faeces to show that chicks of four different shorebird species (red knot, curlew sandpiper, little stint, and red phalarope) strongly differ in their arthropod diet. We found that two arthropod families, Tipulidae and Chironomidae, on average contributed >50% to the diet of chicks. While red knot chicks were relying mainly on Tipulidae (70% in 2018 and 39% in 2019), the chicks of the other three shorebird species were mainly preying on Chironomidae (43% for red phalarope, 37% for curlew sandpiper and 44% for little stint). We found that taking into account the species-specific diet changed our measure of trophic mismatch for two out of four shorebird species. We conclude that ignoring diet data may hamper our understanding of trophic mismatches.

Interactions between macroalgae and corals are omnipresent on eutrophied and overfished reefs worldwide. Contact with macroalgae can disrupt corals and their microbiomes through diverse mechanisms, including shading, abrasion, and the release of algal exudates. However, changes in the coral microbiome after algal contact ceases have not been studied. We investigated the recovery of the microbiome of massive reef-building Porites corals following experimental removal of the overgrowing green macroalga Halimeda macrophysa. We followed changes in the microbiome of macroalgal-removed and adjacent healthy-looking tissue of coral colonies over 40 days. Coral tissue was predominantly bleached underneath the macroalgae but regained almost its full pigmentation by day 40. Despite this recovery in pigmentation, the bacterial microbiome of macroalgal-removed coral tissue did not return to that of adjacent healthy-looking tissue (control). Overall, macroalgal contact led to the suppression of Gammaproteobacteria and increased diversity and dominance of Alphaproteobacteria, a shift that persevered for 40 days after algal removal. Causal effect analysis showed a positive effect of influential OTUs in healthy-looking tissue assigned to Gammaproteobacteria and Bacteroidia on the relative abundance of other OTUs within these classes. The effect of influential OTUs assigned to Alphaproteobacteria in macroalgal-removed tissue on the relative abundance of other OTUs was more diverse. Despite the high heterogeneity of coral microbiomes, differences in the relative abundance of main bacterial classes and orders between control/healthy and macroalgal-removed tissue showed temporal patterns. Differences in the Alpha-, Gamma-, Deltaproteobacteria and Bacteroidia between control/healthy and macroalgal-removed tissue increased after cessation of macroalga contact and stabilized or declined towards day 40. Acidimicrobiia, Deltaproteobacteria, Rhodospirillales and Rhodovibrionales returned to average relative abundances in the adjacent control/healthy tissue after 40 days. Nevertheless, Rhizobiales and Rhodobacterales (Alphaproteobacteria) still dominated the macroalgal-removed microbiome on day 40. We conclude that macroalgal overgrowth induces changes in the coral microbiome, and that algal removal did not lead to full recovery of the microbiome in 40 days. Return of pigmentation and distinct shifts in bacterial groups over time appear a possible pathway to the recovery of the coral microbiome after macroalgal removal.

Abstract. Interpreting lipid biomarkers in the sediment archive requires a good understanding of their application and limitations in modern systems. Recently it was discovered that marine bacteria performing anaerobic ammonium oxidation (anammox), belonging to the genus Ca. Scalindua, uniquely synthesize a stereoisomer of bacteriohopanetetrol (‘BHT-x’). The ratio of BHT-x over total bacteriohopanetetrol (BHT; ubiquitously synthesized by diverse bacteria) has been suggested as a proxy for water column anoxia. As BHT has been found in sediments over 50 Myr old, BHT-x has the potential to complement and extend the sedimentary biomarker record of marine anammox, conventionally constructed using ladderane lipids. Yet, little is known about the distribution of BHT-x in relation to the distribution of ladderanes and to the genetic evidence of Ca. Scalindua in modern marine systems. Here, we investigate the distribution of BHT-x and the application of the BHT-x ratio in relation to distributions of intact polar (IPL) ladderane lipids, ladderane fatty acids (FAs) and Ca. Scalindua 16S rRNA genes in suspended particulate matter (SPM) from the water column, sampled across a large oxygen gradient in the Benguela upwelling system (BUS). In BUS SPM, high BHT-x abundances were constrained to the oxygen deficient zone on the continental shelf (at [O2] < 45 µmol L−1, in all but one case). High BHT-x abundances co-occurred with high abundances of the Ca. Scalindua 16S rRNA gene (relative to the total number of bacterial 16S rRNA genes) and ladderane IPLs. At shelf stations with [O2] > 50 µmol L−1, the BHT-x ratio was < 0.04 (in all but one case). In apparent contradiction, ladderane FAs and low abundances of BHT and BHT-x (resulting in BHT-x ratio’s > 0.04) were also detected in oxygenated offshore waters ([O2] up to 180 µmol L−1), whereas ladderane IPLs were undetected. NL5-derived temperatures suggested that ladderane FAs in the offshore waters were not synthesized in situ but derived from warmer shelf waters. Thus, in sedimentary archives of systems with known lateral organic matter transport, such as the BUS, relative BHT and BHT-x abundances should be carefully considered. In such systems, a higher BHT-x ratio may act as a safer threshold for deoxygenation and/or Ca. Scalindua presence: in the BUS, at [O2] > 50 µmol L−1, the BHT-x ratio was < 0.18 at both off -and onshore sites (in all but one case) and a ratio > 0.18 corresponded in all cases (except one) with the presence of Ca. Scalindua 16S rRNA genes. Lastly, when investigating in situ anammox, we highlight the importance of using ladderane IPLs over BHT-x and/or ladderane FAs; these latter compounds are more recalcitrant and may derive from transported fossil anammox bacteria remnants.

Plastic particles in the ocean are typically covered with microbial biofilms, but it remains unclear whether distinct microbial communities colonize different polymer types. In this study, we analyzed microbial communities forming biofilms on floating microplastics in a bay of the island of Elba in the Mediterranean Sea. Raman spectroscopy revealed that the plastic particles mainly comprised polyethylene (PE), polypropylene (PP), and polystyrene (PS) of which polyethylene and polypropylene particles were typically brittle and featured cracks. Fluorescence in situ hybridization and imaging by high-resolution microscopy revealed dense microbial biofilms on the polymer surfaces. Amplicon sequencing of the 16S rRNA gene showed that the bacterial communities on all plastic types consisted mainly of the orders Flavobacteriales, Rhodobacterales, Cytophagales, Rickettsiales, Alteromonadales, Chitinophagales, and Oceanospirillales. We found significant differences in the biofilm community composition on PE compared with PP and PS (on OTU and order level), which shows that different microbial communities colonize specific polymer types. Furthermore, the sequencing data also revealed a higher relative abundance of archaeal sequences on PS in comparison with PE or PP. We furthermore found a high occurrence, up to 17% of all sequences, of different hydrocarbon-degrading bacteria on all investigated plastic types. However, their functioning in the plastic-associated biofilm and potential role in plastic degradation needs further assessment.

Marine plastic pollution has increased exponentially since the start of mass production in the 1950´s and the negative impacts of marine plastic debris (MPD) on marine life are general acknowledged. Typically, MPD is overgrown by diverse biofilms comprised of prokaryote and eukaryotes, but it is mostly unresolved whether polymers are colonized by opportunist that attach to any hard surface, or if different polymers attract specific communities. This question is further complicated by the fact that floating MPD is subjected to UV-induced photo-oxidation, which results in polymer degradation, i.e. the release of smaller and more bioavailable daughter products, and also causes changes in the polymer’s surface properties. If weathered surfaces are more prone to colonization than pristine ones and whether communities on these surfaces are different is unknown. In consonance, whether colonizers interact with the different polymers, e.g. degrade it, or are just ‘hitching a ride’ is ambiguous. To solve this complex problem we investigated the initial colonization of pristine plastics and the influence of photo-oxidation on community succession. We incubated five different polymer types (PE, PP, PET, PS and Nylon; one set UV pre-treated, one set pristine), in shallow coastal waters of the Caribbean island St. Eustatius at a depth of 5m. Multivariant-analyses to compare day 1 and day 6 revealed that the microbial community changed over time, which is a typical feature during colonisation. Communities of day 1 and day 6 were also analysed separately to assess the influences of UV pre-treatment and polymer type during separate stages of biofilm development. On day 1, UV pre-treated foils attracted a different community than non-pre-treated foils, while there was no statistical difference in community composition between the five polymer types. In contrast, on day 6, the influence of UV treatment on community composition was no longer significant, while different polymer types supported different communities. These results show that the community is dynamic in the initial stage of colonization of polymers. The effects of UV pre-treatment and polymer type indicate that colonizers are not purely opportunistic. With more in-depth analysis on OTU and/or order level, we aim to answer the following questions: 1) What is the main driver for community succession 2) Are there polymer-specific members of the community 3) Do the different polymer types select for communities that might utilise the polymer or its UV-degradation products for energy gain and/or growth.

Hydrothermal vent fields found at mid-ocean ridges emit hydrothermal fluids that disperse as neutrally buoyant plumes. From these fluids seafloor massive sulfides (SMS) deposits are formed, which are being explored as possible new mining sites for (trace) metals and rare earth elements (REEs). It has been suggested that during mining activities large amounts of suspended matter will appear in the water column due to excavation processes and discharge of mining waste from the surface vessel. Understanding how hydrothermal plumes can be characterised by means of geochemistry and microbiology as they spread away from their source and how they affect their surrounding environment may help in characterising the behaviour of the dilute distal part of chemically enriched mining plumes. This study on the extensive Rainbow hydrothermal plume, observed up to 25 km downstream from the vent site, enabled us to investigate how microbial communities and (trace) metal composition change in a natural plume with distance. The (trace) metal and REE content of suspended particulate matter (SPM) was determined using sector field inductively coupled plasma mass spectrometry (SF-ICP-MS) with high resolution (HR), and the microbial communities of the neutrally buoyant plume, above-plume, below-plume, and near-bottom water and sediment were characterised by using 16S rRNA amplicon sequencing methods. Both vertically in the water column and horizontally along the neutrally buoyant plume, geochemical and biological changes were evident, as the neutrally buoyant plume stood out by its enrichments in (trace) metals and REEs, as, for example, Fe, Cu, V, Mn and REEs were enriched by factors of up to ∼80, ∼90, ∼52, ∼2.5 and ∼40, respectively, compared to above-plume water samples taken at 1000 m water depth. The concentrations of these elements changed as the plume aged, shown by the decrease in element ∕ Fe molar ratios of chalcophile elements (Cu, Co, Zn), indicative of rapid removal from the hydrothermal plume or removal from the solid phase. Conversely, increasing REE ∕ Fe molar ratios imply uptake of REEs from the ambient seawater onto Fe-oxyhydroxides. This was also reflected in the background pelagic system, as Epsilonproteobacteria started to dominate and univariate microbial biodiversity declined with distance away from the Rainbow hydrothermal vent field. The Rainbow hydrothermal plume provides a geochemically enriched natural environment, which is a heterogeneous, dynamic habitat that is conducive to ecological changes in a short time span. This study of a hydrothermal plume provides a baseline study to characterise the natural plume before the interference of deep-sea mining.

Ocean plastic debris poses a large threat to the marine environment. Millions of tons of plastic end up in the ocean each year and the Mediterranean Sea is one of the most plastic polluted sea. Ocean plastic particles are typically covered with microbial biofilms, but it remains unclear if different polymer types are colonized by different communities. Knowledge in this aspect strengthens our understanding if microbes purely use plastic debris as attachment surface or if they may even contribute to the degradation of plastic. To gain a better understanding of the composition and structure of biofilms on micro plastic particles (MP) in the Mediterranean Sea, we analyzed microbial community covering floating MP in a bay/marina (Marina di Campo) on the island of Elba. MPs were collected with a plankton net (mesh size 50µm), fixed for fluorescence microscopy and stored for subsequent DNA extraction, and identification of the polymer with Raman spectroscopy. The particles were mainly comprised of polyethylene (PE), polypropylene (PP) and polystyrene (PS) and were often brittle and with cracks (PE, PP) and showed visual signs of biofouling (PE, PP, PS). Fluorescence in situ hybridization and imaging by high resolution confocal laser scanning microscopy of single MPs revealed high densities of colonization by microbes. 16S rRNA gene amplicon sequencing (Illumina Miseq) revealed higher abundance of archaeal sequences on PS (up to 29% of the reads) in comparison to PE or PP (up to 3% of the reads). The bacterial community in the biofilms on each of the three plastic types consisted mainly of the orders Flavobacteriales, Rickettsiales, Alteromonadales, Cytophagales, Rhodobacterales and Oceanospirillales. Furthermore, we found significant difference in the community composition of biofilms on PE compared to PP and PS but not between PP and PS. The indicator species on PE were Calditrichales, detected at 10 times higher sequence abundance on PE than on PP and PS, as well as several uncultured orders. This study sheds light on preferential microbial attachment and biofilm formation on microplastic particles, yet it remains to be revealed, whether and which of these may contribute to plastic degradation.

Marker gene sequencing of the rRNA operon (16S, 18S, ITS) or cytochrome c oxidase I (CO1) is a popular means to assess microbial communities of the environment, microbiomes associated with plants and animals, as well as communities of multicellular organisms via environmental DNA sequencing. Since this technique is based on sequencing a single gene, or even only parts of a single gene rather than the entire genome, the number of reads needed per sample to assess the microbial community structure is lower than that required for metagenome sequencing. This makes marker gene sequencing affordable to nearly any laboratory. Despite the relative ease and cost-efficiency of data generation, analyzing the resulting sequence data requires computational skills that may go beyond the standard repertoire of a current molecular biologist/ecologist. We have developed Cascabel, a scalable, flexible, and easy-to-use amplicon sequence data analysis pipeline, which uses Snakemake and a combination of existing and newly developed solutions for its computational steps. Cascabel takes the raw data as input and delivers a table of operational taxonomic units (OTUs) or Amplicon Sequence Variants (ASVs) in BIOM and text format and representative sequences. Cascabel is a highly versatile software that allows users to customize several steps of the pipeline, such as selecting from a set of OTU clustering methods or performing ASV analysis. In addition, we designed Cascabel to run in any linux/unix computing environment from desktop computers to computing servers making use of parallel processing if possible. The analyses and results are fully reproducible and documented in an HTML and optional pdf report. Cascabel is freely available at Github : https://github.com/AlejandroAb/CASCABEL.

AbstractAnalyses of environmental DNA have the potential to become an integrated tool in fish research and management. We performed a pilot study during the spring migration of fishes from the North Sea into the Wadden Sea and present comparative results from daily fyke catches (20 mm mesh) in the Marsdiep tidal inlet on two locations and results from weekly fish eDNA analyses on three locations, all within 2 km distance.Fish catches did not differ significantly between the two locations, whereas the eDNA composition showed a significant location effect. However, when eDNA analysis was restricted to species that were caught with the fykes, differences among locations became insignificant. Over ten weeks, from late April to early July, presence–absence calls of fishes based on weekly eDNA sampling significantly agreed with calls based on seven days of fyke fishing 1 km westwards. Fish eDNA compositions differed significantly among sample days and months but not between tides. Over the season, patterns in eDNA concentration (12S rRNA gene copies/L) inferred from quantitative PCR and Illumina HiSeq community composition corresponded to patterns in wet mass for the eight most abundant fish species in the fyke (>6 weeks present) despite changes in water temperature and changes in fish size class. Small sandeel and gobies, which are important prey for large fishes and birds, were typically missed with the fyke but contributed up to 25%–40% of the fish eDNA depending on the sample location.

ABSTRACTMarker gene sequencing of the rRNA operon (16S, 18S, ITS) or cytochrome c oxidase I (CO1) is a popular means to assess microbial communities of the environment, microbiomes associated with plants and animals, as well as communities of multicellular organisms via environmental DNA sequencing. Since this technique is based on sequencing a single gene rather than the entire genome, the number of reads needed per sample is lower than that required for metagenome sequencing, making marker gene sequencing affordable to nearly any laboratory. Despite the relative ease and cost-efficiency of data generation, analyzing the resulting sequence data requires computational skills that may go beyond the standard repertoire of a current molecular biologist/ecologist. We have developed Cascabel, a flexible and easy-to-use amplicon sequence data analysis pipeline, which uses Snakemake and a combination of existing and newly developed solutions for its computational steps. Cascabel takes the raw data as input and delivers a table of operational taxonomic units (OTUs) and a representative sequence tree. Our pipeline allows customizing the analyses by offering several choices for most of the steps, for example different OTU generating methods. The pipeline can make use of multiple computing nodes and scales from personal computers to computing servers. The analyses and results are fully reproducible and documented in an HTML and optional pdf report. Cascabel is freely available at Github: https://github.com/AlejandroAb/CASCABEL and licensed under GNU GPLv3.

The Dutch coastal zone is a region of the North Sea with a marked interannual and long-term abiotic and phytoplankton variability. To investigate the relationship between abiotic variability and phytoplankton composition, two routine water monitoring data sets (1991-2005) were examined. Multivariate statistics revealed two significant partitions in the data. The first consisted of interannual abiotic fluctuations that were correlated to Rhine discharge that affected the abundance of summer and autumn diatom species. The second partition was caused by a shift in the abiotic data from 1998 to 1999 that was followed by a shift in phytoplankton composition from 1999 to 2000. Important factors in the abiotic shift were decreases in suspended matter (SPM) and phosphate (DIP) concentrations, as well as in pH. The decrease in SPM was caused by a reduction in wind speed. The increase in water column daily irradiance from the decrease in SPM led to increases in the abundance of winter-spring species, notably the prymnesiophyte Phaeocystis globosa. Because wind speed is related to the North Atlantic Oscillation (NAO) index it was possible to correlate NAO index and P. globosa abundance. Only five abiotic variables representing interannual and long-term variability, including Rhine discharge and NAO index, were needed to model the observed partitions in phytoplankton composition. It was concluded that interannual variability in the coastal phytoplankton composition was related to year-to-year changes in river discharge while the long-term shift was caused by an alternating large-scale meteorological phenomenon.

AbstractKelp aquaculture is globally developing steadily as human food source, along with other applications. One of the newer crop species is Saccharina latissima, a northern hemisphere kelp inhabiting temperate to arctic rocky shores. To protect and document its natural genetic variation at the onset of this novel aquaculture, as well as increase knowledge on its taxonomy and phylogeography, we collected new genetic data, both nuclear and mitochondrial, and combined it with previous knowledge to estimate genetic connectivity and infer colonization history. Isolation‐with‐migration coalescent analyses demonstrate that gene flow among the sampled locations is virtually nonexistent. An updated scenario for the origin and colonization history of S. latissima is developed as follows: We propose that the species (or species complex) originated in the northwest Pacific, crossed to the northeast Pacific in the Miocene, and then crossed the Bering Strait after its opening ~5.5 Ma into the Arctic and northeast Atlantic. It subsequently crossed the Atlantic from east to west. During the Pleistocene, it was compressed in the south with evidence for northern refugia in Europe. Postglacial recolonization led to secondary contact in the Canadian Arctic. Saccharina cichorioides is shown to probably belong to the S. latissima species complex and to derive from ancestral populations in the Asian North Pacific. Our novel approach of comparing inferred gene flow based on coalescent analysis versus Wright's island model suggests that equilibrium levels of differentiation have not yet been reached in Europe and, hence, that genetic differentiation is expected to increase further if populations are left undisturbed.

Earlier studies show that the proliferation of phytoplankton viruses can be inhibited by depletion of soluble reactive phosphorus (SRP; orthophosphate). In natural marine waters, phytoplankton phosphorus (P) availability is, however, largely determined by the supply rate of SRP (e.g. through remineralization) and potentially by the source of P as well (i.e. the utilization of soluble non-reactive P; SNP). Here we show how a steady low supply of P (mimicking natural P recycling) to virally infected P-limited Micromonas pusilla stimulates virus proliferation. Independent of the degree of P limitation prior to infection (0.32 and 0.97μmax chemostat cultures), SRP supply resulted in 2-fold higher viral burst sizes (viruses lysed per host cell) as compared with no addition (P starvation). Delaying these spikes during the infection cycle showed that the added SRP was utilized for extra M. pusilla virus (MpV) production far into the lytic cycle (18 h post-infection). Moreover, P-limited M. pusilla utilized several SNP compounds with high efficiency and with the same extent of burst size stimulation as for SRP. Finally, addition of virus-free MpV lysate (representing a complex SNP mixture) to newly infected cells enhanced MpV production, implicating host-associated alkaline phosphatase activity, and highlighting its important role in oligotrophic environments.

An 8-year time-series in the Western Antarctic Peninsula (WAP) with an approximately weekly sampling frequency was used to elucidate changes in virioplankton abundance and their drivers in this climatically sensitive region. Virioplankton abundances at the coastal WAP show a pronounced seasonal cycle with interannual variability in the timing and magnitude of the summer maxima. Bacterioplankton abundance is the most influential driving factor of the virioplankton, and exhibit closely coupled dynamics. Sea ice cover and duration predetermine levels of phytoplankton stock and thus, influence virioplankton by dictating the substrates available to the bacterioplankton. However, variations in the composition of the phytoplankton community and particularly the prominence of Diatoms inferred from silicate drawdown, drive interannual differences in the magnitude of the virioplankton bloom; likely again mediated through changes in the bacterioplankton. Their findings suggest that future warming within the WAP will cause changes in sea ice that will influence viruses and their microbial hosts through changes in the timing, magnitude and composition of the phytoplankton bloom. Thus, the flow of matter and energy through the viral shunt may be decreased with consequences for the Antarctic food web and element cycling.

This study characterizes the microbial community composition over Haas Mound, one of the most prominent cold-water coral mounds of the Logachev Mound province (Rockall Bank, NE Atlantic). We outline patterns of distribution vertically – from the seafloor to the water column – and laterally – across the mound – and couple these to mound topography and hydrography. Samples of water, sediment and Lophelia pertusa were collected in 2012 and 2013 from locations that were chosen based on high definition video surveys. Temperature and current measurements were obtained at two sites at the summit and foot of Haas Mound to study near-bed hydrodynamic conditions. Overlaying water was collected from depths of 400 m as well as 5 and 10 m above the bottom using a CTD/Rosette system. Near-bottom water, sediment and L. pertusa mucus and skeleton samples were obtained with a box corer. Of all these biotopes, Roche GS-FLX amplicon sequencing targeting both Bacteria and Archaea was carried out, augmenting our understanding of deep sea microbial consortia. The pattern of similarities between samples, visualized by multi-dimensional scaling (MDS), indicates a strong link between the distribution of microbes and the specific biotopes. The microbial operational taxonomic unit (OTU) diversity was the highest in near-bottom water, which was sampled in the coral framework. For the first time, Thaumarchaeota marine group I (MGI) were found in L. pertusa mucus; Endozoicomonas was detected in skeleton, mucus and near-bottom water, whereas Mycoplasma was only detected in skeleton and near-bottom water, however not in mucus. Analysis of similarities (ANOSIM) indicates that overlaying water is well-mixed at 400 m depth but less so at 5 and 10 m above the bottom, where the composition of microbial communities differed significantly between summit, slope and off-mound. At all locations, the near-bottom water differed significantly from water at 5 m above the bottom, illustrating that the near-bottom water in between the coral framework represents a separate microbial habitat. Furthermore, the observed spatial heterogeneity in microbial communities is discussed in relation to environmental conditions.

DNA was analysed from external wounds on 3 dead harbour porpoises Phocoena phocoena that were stranded in the Netherlands. Puncture wounds as well as the edges of large open wounds were sampled with sterile cotton swabs. With specific primers that target the mtDNA control region of grey seal Halichoerus grypus, a 196 bp DNA fragment was amplified from 4 puncture wounds. Sequencing of the fragments confirmed the presence of grey seal DNA in the puncture wounds. DNA sequences differed between the cases, implying that 3 individual grey seals were involved. As 8 control swabs from intact skin and the transport bag as well as 6 swabs from open wounds on the same harbour porpoises were all negative, contamination with environmental DNA is considered unlikely. The results provide a link between strandings of mutilated harbour porpoises and recent observations of grey seals attacking harbour porpoises. Ours is the first study to use forensic techniques to identify DNA in bite marks from carcasses recovered from the marine environment. This approach can be extended to identify other marine aggressors, including cases involving persons mutilated at sea.

Harbour porpoises (Phocoena phocoena) stranding in large numbers around the southern North Sea with fatal, sharp-edged mutilations have spurred controversy among scientists, the fishing industry and conservationists, whose views about the likely cause differ. The recent detection of grey seal (Halichoerus grypus) DNA in bite marks on three mutilated harbour porpoises, as well as direct observations of grey seal attacks on porpoises, have identified this seal species as a probable cause. Bite mark characteristics were assessed in a retrospective analysis of photographs of dead harbour porpoises that stranded between 2003 and 2013 (n = 1081) on the Dutch coastline. There were 271 animals that were sufficiently fresh to allow macroscopic assessment of grey seal-associated wounds with certainty. In 25% of these, bite and claw marks were identified that were consistent with the marks found on animals that had tested positive for grey seal DNA. Affected animals were mostly healthy juveniles that had a thick blubber layer and had recently fed. We conclude that the majority of the mutilated harbour porpoises were victims of grey seal attacks and that predation by this species is one of the main causes of death in harbour porpoises in The Netherlands. We provide a decision tree that will help in the identification of future cases of grey seal predation on porpoises.

Viral infection of phytoplankton has major implications for biochemical and energy cycles, community dynamics, and microbial evolution in the marine environment. The non-bloom forming picoplankter Micromonas pusilla, a significant component of the plankton community worldwide, is known to be susceptible to infection by both dsDNA and dsRNA viruses. Logically, comprehensive knowledge of the ecology of M. pusilla requires a better understanding of the diversity and infection mechanisms of their viruses. Here, we investigated 19 new M. pusilla-specific viruses (MpVs) isolated from different locations and years. We performed partial characterization of those MpVs including structural characteristics, genome size, phylogenetic analysis based on partial DNA polymerase gene sequences, host range, and stability at different temperatures and upon exposure to chloroform. Combined, these characteristics allowed classification of the MpVs into 2 groups. Exposure to chloroform led to loss of infectivity by all MpVs in one group, which suggests the presence of an outer lipid envelope. In addition, all except one of the members in that group formed a monophylogenetic clade that was distinct from all other MpV isolates. The distinctive characteristics of the 2 MpV groups suggest different infection strategies, which may have important implications for the ecology of both host and virus populations in the environment. Knowledge gained from our study adds value to the MpV isolates as a scientific resource as it will aid in developing and testing in the laboratory new hypotheses about the ecological and biogeochemical implications of M. pusilla viral infection in the environment.