A Substrate-Independent Benthic Sampler (SIBS) for Hard and Mixed-Bottom Marine Habitats: A Proof-of-Concept Study

Sea cage fish farms are increasingly situated over hard and mixed substrate habitats for production and waste-dispersion reasons; yet in many cases, these installations are not being effectively managed with respect to benthic impacts due to the lack of a practical sampling method. This study presents the first set of results from a newly developed Substrate Independent Benthic Sampler (SIBS) device that captures the unconsolidated organic and inorganic matter that overlies almost all substrates. The contents of the samples were analyzed using extracted environmental DNA (eDNA) followed by metabarcoding of the bacterial 16S rRNA gene. SIBS microbial assemblages reliably changed with proximity to farm and concurred with visual assessments of impact. Moreover, the approach appeared to be very sensitive with respect to the enrichment gradient, being able to discern influences at distances of 500–1500 m from the impact source. Other spatial differences, due to region and farm, were small in comparison, and the effect of the underlying substrate type was minor. The samples contained sufficient previously described bacterial bioindicator taxa from enriched sediments, such that a meaningful biotic index could be calculated, thereby placing them on a well-established benthic enrichment spectrum with established environmental thresholds. SIBS-derived bacterial data provide a powerful new approach for mapping spatial boundaries of farm effects irrespective of substrate type and topography. More importantly, the tool should also permit quantitative assessment of benthic enrichment levels irrespective of substrate type from depths of at least 100 m. It therefore has the potential to solve the hard-bottom problem that has until now prohibited effective environmental monitoring at mixed and hard-bottom locations.publishedVersio

Epifaunal habitat associations on mixed and hard bottom substrates in coastal waters of Northern Norway

Hard and mixed seafloor substrates are an important benthic habitat in coastal northern Norway and they are known to be colonized by relatively diverse communities of sessile epifauna. These assemblages are highly susceptible to physical damage and stresses imposed by organic material from industrial and municipal sources. However, despite increasing prevalence of stressors, the diversity and distribution of benthic substrates and biological communities in coastal Arctic and sub-Arctic regions remain poorly documented. In response, this study has characterized the composition of mixed and hard bottom substrates and associated sessile epifauna in fjords in Finnmark, northern Norway, using remote sensing and an innovation low-cost towed camera method. The study fjords supported a dense covering (0.1 to 0.68 individuals m–2) of sponge taxa common to deep-water ostur sponge habitats (Geodia sp., Mycale lingua, Polymastia sp., Phakellia ventilabrum, and Axinella infundibuliformis). In addition, aggregations of the soft coral (Duva florida), the tunicate (Ascidia sp.), the seastar (Ceramaster granularis) and anemone (Hormathia digitata) were prominent fauna. The small-scale spatial patterns of the epifaunal communities in this study were primarily influenced by the local hydrodynamic regime, depth, the topographical slope and the presence of hard bedrock substrates. This description of the composition, distribution and the identification of environmental drivers of epibenthic communities is valuable for the development of predictive habitat models to manage the benthic impact of multiple stressor on these ecological valuable and vulnerable Arctic habitats.publishedVersio

Beyond taxonomy: Validating functional inference approaches in the context of fish-farm impact assessments

Characterization of microbial assemblages via environmental DNA metabarcoding is increasingly being used in routine monitoring programs due to its sensitivity and cost-effectiveness. Several programs have recently been developed which infer functional profiles from 16S rRNA gene data using hidden-state prediction (HSP) algorithms. These might offer an economic and scalable alternative to shotgun metagenomics. To date, HSP-based methods have seen limited use for benthic marine surveys and their performance in these environments remains unevaluated. In this study, 16S rRNA metabarcoding was applied to sediment samples collected at 0 and ≥1,200 m from Norwegian salmon farms, and three metabolic inference approaches (Paprica, Picrust2 and Tax4Fun2) evaluated against metagenomics and environmental data. While metabarcoding and metagenomics recovered a comparable functional diversity, the taxonomic composition differed between approaches, with genera richness up to 20× higher for metabarcoding. Comparisons between the sensitivity (highest true positive rates) and specificity (lowest true negative rates) of HSP-based programs in detecting functions found in metagenomic data ranged from 0.52 and 0.60 to 0.76 and 0.79, respectively. However, little correlation was observed between the relative abundance of their specific functions. Functional beta-diversity of HSP-based data was strongly associated with that of metagenomics (r ≥ 0.86 for Paprica and Tax4Fun2) and responded similarly to the impact of fish farm activities. Our results demonstrate that although HSP-based metabarcoding approaches provide a slightly different functional profile than metagenomics, partly due to recovering a distinct community, they represent a cost-effective and valuable tool for characterizing and assessing the effects of fish farming on benthic ecosystems.publishedVersio

SuspensionFeeding Benthic Species’ Physiological and Microbiome Response to Salmon Farming and Associated Environmental Changes

Caged salmon farming is increasingly undertaken in water bodies with strong hydrodynamics where hard and mixed substrate habitats are more prevalent. Yet, these structurally complex and heterogeneous habitats support diverse benthic communities including several cnidarians and sponges that remain poorly characterized. This study used a combination of respirometry measurements, gas chromatography and 16S rRNA metabarcoding to define the respiration rate, stable carbon (δ13C) and nitrogen isotopes (δ15N), fatty acid (FA) and microbial profiles, and assess the impact of salmon farming on four important epibenthic suspension-feeders along the western Norwegian coast: the sponges Craniella and Weberella, the soft coral Duva florida and the anemone Hormathia digitata. Our results showed striking differences in fatty acid profiles and host microbiome communities in terms of identity, functional capabilities and genetic properties across the suspension-feeders. We found evidence of increased mortality rate in specimens located near fish farm activities and of a species-specific effect on respiration rate, with D. florida showing increased activity under the farm. Effects of fish farming on the suspension feeders were also species-specific and particularly evidenced by functional microbial turnover and by alteration of overall FA profiles in the soft coral and sea anemone. In particular, D. florida showed reduced level of FAs close to the farm (0-350 m), with significant difference in composition along a distance gradient. Only H. digitata showed evidence of incorporation of organic material from the fish farm waste via fatty acids trophic markers (FATM) and stable isotope analysis. Overall, our study demonstrates that suspension feeders have taxon-specific sensitivity towards the effect of salmon farming, and identified several potential molecular indicators that could be used as surrogate of impact gradient upon further research and validation. It also provides a wealth of ecological and physiological information on some of the most common sessile epibenthic organisms within Arctic and sub-Arctic regions, enabling us to better understand their response and evaluate their resilience to environmental changes.publishedVersio

Effects of fish farm activities on the sponge Weberella bursa, and its associated microbiota

Sustained growth of world-wide sea farming and the search of optimal growing conditions have driven several countries, including Norway, to establish new finfish sites in more exposed, high current locations. Characterized by a range of gravel, broken rock and/or bedrock, these complex environments and the associated diverse range of epifauna species are not easily monitored via traditional methodologies (e.g. morpho-taxonomic identification and enumeration, and compound analyses of sediment grabs). Consequently, little is known about many of the benthic inhabitants, or how they may respond to fish farming. In this study, we aimed to initiate addressing this knowledge gap by assessing the response of the sponge Weberella bursa (Polymastidea) to salmon aquaculture. Fourteen specimens were translocated along a distance gradient from a salmon farm located along the mid-west coast of Norway. Following 7 months of exposure, their epithelial tissue were analysed for gene expression analysis (mRNA), fatty acid (FA), stable isotope and taxonomic and functional microbiome characterization. Among all datasets, only fatty acid profiles showed significant changes associated with fish farm activities, with higher proportion of terrestrial FAs and long saturated and monounsaturated FAs near the farm. These results suggest that W. bursa sponges may be more resistant to organic enrichment than previously thought. Nonetheless, several putative indicators of non-lethal response could be identified. Specifically, W. bursa specimens located underneath the farm tended to have reduced ribosomal activity while having increased expression of genes controlling cell apoptosis (e.g. caspase-3, cytochrome c oxidase and death domain proteins). Based on predictive functional analysis, specimens near to the farm were also found to be particularly enriched in sulfur and nitrogen cycling bacteria, and in microbial taxa with anti-toxin and xenobiotic biodegradation capability, notably of benzyl benzoate compounds used in sea lice treatments. These results indicate that potentially harmful elements such as sulfite, nitrite and pesticides may be neutralized and degraded by a particularly enriched set of bacteria in W. bursa microbiome. While additional research is needed to validate these putative indicators, our study provides a first glimpse as to how sessile organisms may respond and adapt to environmental changes induced by fin fish farming, and pave the way to the development of novel monitoring tools adapted to mix and hard bottom habitats.publishedVersio

Effect of substrate type and pellet age on the resuspension of Atlantic salmon faecal material

publishedVersio

Global Trends of Benthic Bacterial Diversity and Community Composition Along Organic Enrichment Gradients of Salmon Farms

The analysis of benthic bacterial community structure has emerged as a powerful alternative to traditional microscopy-based taxonomic approaches to monitor aquaculture disturbance in coastal environments. However, local bacterial diversity and community composition vary with season, biogeographic region, hydrology, sediment texture, and aquafarm-specific parameters. Therefore, without an understanding of the inherent variation contained within community complexes, bacterial diversity surveys conducted at individual farms, countries, or specific seasons may not be able to infer global universal pictures of bacterial community diversity and composition at different degrees of aquaculture disturbance. We have analyzed environmental DNA (eDNA) metabarcodes (V3–V4 region of the hypervariable SSU rRNA gene) of 138 samples of different farms located in different major salmon-producing countries. For these samples, we identified universal bacterial core taxa that indicate high, moderate, and low aquaculture impact, regardless of sampling season, sampled country, seafloor substrate type, or local farming and environmental conditions. We also discuss bacterial taxon groups that are specific for individual local conditions. We then link the metabolic properties of the identified bacterial taxon groups to benthic processes, which provides a better understanding of universal benthic ecosystem function(ing) of coastal aquaculture sites. Our results may further guide the continuing development of a practical and generic bacterial eDNA-based environmental monitoring approach.publishedVersio

Habitat associations of juvenile Atlantic cod (Gadus morhua L.) and sympatric demersal fish communities within shallow inshore nursery grounds

publishedVersionPaid open acces

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

Attraction of cod Gadus morhua from coastal spawning grounds to salmon farms

Wild fish aggregate at aquaculture net-pens, but the underlying mechanisms are not fully understood. This study examined how salmon farms attract coastal Atlantic cod Gadus morhua from their inshore spawning grounds. Acoustic receivers were deployed at 5 known cod spawning grounds and 6 salmon Salmo salar farms located at varying distances from these grounds in a mid-Norway study site. Cod were caught at each spawning ground annually from 2017-2019, fitted with acoustic transmitters and released (n = 535). A total of 289 tagged cod (54%) were detected at the salmon farms, with more cod detected at farms closest to the focal spawning grounds and at operational farms. The latter result is likely linked to the availability of feeding opportunities at farm locations. Those cod that were detected by the receivers spent less time at farms farther from their release locations. For the farm-associated cod, 70% were detected for 1 mo close to the farms, with 1 individual staying 720 d underneath the farm. A total of 135 cod visited 2 or more farms, with farms in proximity more connected in terms of inter-farm movement. Some of the cod utilizing these local spawning grounds likely have considerable dietary input from salmon feed.publishedVersionpublishedVersio