Unlocking the secret life of blue mussels: Exploring connectivity in the Skagerrak through biophysical modeling and population genomics.

Knowledge of functional dispersal barriers in the marine environment can be used to inform a wide variety of management actions, such as marine spatial planning, restoration efforts, fisheries regulations, and invasive species management. Locations and causes of dispersal barriers can be studied through various methods, including movement tracking, biophysical modeling, demographic models, and genetics. Combining methods illustrating potential dispersal, such as biophysical modeling, with realized dispersal through, e.g., genetic connectivity estimates, provides particularly useful information for teasing apart potential causes of observed barriers. In this study, we focus on blue mussels (Mytilus edulis) in the Skagerrak-a marginal sea connected to the North Sea in Northern Europe-and combine biophysical models of larval dispersal with genomic data to infer locations and causes of dispersal barriers in the area. Results from both methods agree; patterns of ocean currents are a major structuring factor in the area. We find a complex pattern of source-sink dynamics with several dispersal barriers and show that some areas can be isolated despite an overall high dispersal capability. Finally, we translate our finding into management advice that can be used to sustainably manage this ecologically and economically important species in the future.

Are silicone foul-release coatings a viable and environmentally sustainable alternative to biocidal antifouling coatings in the Baltic Sea region?

To combat unwanted fouling on immersed hulls, biocidal antifouling coatings are commonly applied to vessels trafficking the Baltic Sea. Here, the efficacy, environmental sustainability and market barriers of silicone foul-release coatings (FRCs) was assessed for this region to evaluate their viability as replacements for biocidal coatings. Coated panels were exposed statically over a 1 year period at three locations in the Baltic Sea region to assess the long-term performance of a biocide-free FRC and two copper coatings. The FRC was found to perform equally well or significantly better than the copper coatings. Even though most silicone FRCs on the market are biocide-free, a review of the literature regarding toxic effects and the identity and environmental fate of leachables shows that they may not be completely environmentally benign, simply for the lack of biocides. Nonetheless, FRCs are substantially less toxic compared to biocidal antifouling coatings and their use should be promoted.

Monitoring biofouling as a management tool for reducing toxic antifouling practices in the Baltic Sea.

Over two million leisure boats use the coastal areas of the Baltic Sea for recreational purposes. The majority of these boats are painted with toxic antifouling paints that release biocides into the coastal ecosystems and negatively impact non-targeted species. Regulations concerning the use of antifouling paints differ dramatically between countries bordering the Baltic Sea and most of them lack the support of biological data. In the present study, we collected data on biofouling in 17 marinas along the Baltic Sea coast during three consecutive boating seasons (May-October 2014, 2015 and 2016). In this context, we compared different monitoring strategies and developed a fouling index (FI) to characterise marinas according to the recorded biofouling abundance and type (defined according to the hardness and strength of attachment to the substrate). Lower FI values, i.e. softer and/or less abundant biofouling, were consistently observed in marinas in the northern Baltic Sea. The decrease in FI from the south-western to the northern Baltic Sea was partially explained by the concomitant decrease in salinity. Nevertheless, most of the observed changes in biofouling seemed to be determined by local factors and inter-annual variability, which emphasizes the necessity for systematic monitoring of biofouling by end-users and/or authorities for the effective implementation of non-toxic antifouling alternatives in marinas. Based on the obtained results, we discuss how monitoring programs and other related measures can be used to support adaptive management strategies towards more sustainable antifouling practices in the Baltic Sea.

Osmoregulation in Barnacles: An Evolutionary Perspective of Potential Mechanisms and Future Research Directions.

Barnacles form a globally ubiquitous group of sessile crustaceans that are particularly common in the coastal intertidal. Several barnacle species are described as highly euryhaline and a few species even have the ability to colonize estuarine and brackish habitats below 5 PSU. However, the physiological and/or morphological adaptations that allow barnacles to live at low salinities are poorly understood and current knowledge is largely based on classical eco-physiological studies offering limited insight into the molecular mechanisms. This review provides an overview of available knowledge of salinity tolerance in barnacles and what is currently known about their osmoregulatory strategies. To stimulate future studies on barnacle euryhalinity, we briefly review and compare barnacles to other marine invertebrates with known mechanisms of osmoregulation with focus on crustaceans. Different mechanisms are described based on the current understanding of molecular biology and integrative physiology of osmoregulation. We focus on ion and water transport across epithelial cell layers, including transport mechanisms across cell membranes and paracellular transfer across tight junctions as well as on the use of intra- and extracellular osmolytes. Based on this current knowledge, we discuss the osmoregulatory mechanisms possibly present in barnacles. We further discuss evolutionary consequences of barnacle osmoregulation including invasion-success in new habitats and life-history evolution. Tolerance to low salinities may play a crucial role in determining future distributions of barnacles since forthcoming climate-change scenarios predict decreased salinity in shallow coastal areas. Finally, we outline future research directions to identify osmoregulatory tissues, characterize physiological and molecular mechanisms, and explore ecological and evolutionary implications of osmoregulation in barnacles.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression.

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

Copper release rate needed to inhibit fouling on the west coast of Sweden and control of copper release using zinc oxide.

How zinc oxide influences copper release has been tested and the lowest release rate of copper from various combinations of copper and zinc in a paint matrix evaluated, whilst still deterring macrofouling, including barnacles and bryozoans. Copper (I) oxide was added to a generic AF paint in 0, 8.5, 11.7 or 16.3 wt% copper oxide in combination with 0, 10 or 20 wt% zinc oxide and applied on PMMA panels. The results show that zinc influences the release rate of copper. When 10 and 20 wt% zinc was added, the total amount of copper released significantly increased by on average 32 and 47% respectively. All treatments that included copper were successful in deterring macrofouling, including the treatment with the lowest average Cu release rate, ie 4.68 µg cm-2 day-1.

A new flow-through bioassay for testing low-emission antifouling coatings.

Current antifouling (AF) technologies are based on the continuous release of biocides into the water, and consequently discharge into the environment. Major efforts to develop more environmentally friendly coatings require efficient testing in laboratory assays, followed by field studies. Barnacles are important fouling organisms worldwide, increasing hydrodynamic drag on ships and damaging coatings on underwater surfaces, and thus are extensively used as models in AF research, mostly in static, laboratory-based systems. Reliable flow-through test assays for the screening of biocide-containing AF paints, however, are rare. Herein, a flow-through bioassay was developed to screen for diverse low-release biocide paints, and to evaluate their effects on pre- and post-settlement traits in barnacles. The assay distinguishes between the effects from direct surface contact and bulk-water effects, which are crucial when developing low-emission AF coatings. This flow-through bioassay adds a new tool for rapid laboratory-based first-stage screening of candidate compounds and novel AF formulations.

Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity.

Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle B. improvisus, based on genome and transcriptome sequencing. Our analyses reveal that B. improvisus contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the B. improvisus aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU) compared to high salinity (33 PSU). Our study provides a base for future mechanistic studies on the role of aquaporins in osmoregulation.

Low sensitivity of reproductive life-stages in the Pacific oyster (Crassostrea gigas) to abamectin.

Hard surfaces submerged in the marine environment often become colonised by macro-organisms unless the surfaces have some form of biofouling protection. While protective paints that contain tributyltin or copper work well to prevent biofouling, release of these materials into the environment has been shown to have wider negative impacts. Consequently, new low-release antifouling paints are being developed with alternative active ingredients, such as avermectins, yet little is known about their potential effects on non-target organisms in marine environments. Here we investigated the toxicity of a key avermectin, specifically abamectin, on several aspects of reproduction (sperm motility, fertilisation success, early larval development) in the Pacific oyster, Crassostrea gigas. Oyster reproduction was generally insensitive to the low concentrations of abamectin, although greater concentrations of abamectin did negatively affect all three endpoints - LOECs were 1000 µg l-1, 500 µg l-1, and 100 µg l-1 abamectin for sperm motility, fertilisation success, and larval development, respectively. A similar pattern was found in the EC50s of the three endpoints (mean ± SE) 934 ± 59 µg l-1, 1076.26 ± 725.61 µg l-1, and 140 ± 78 µg l-1 abamectin (sperm motility, fertilisation success, and larval development, respectively). Together, these results clearly indicate that of the three endpoints considered, larval development was more sensitive to abamectin (lower LOEC, EC50) than fertilisation success and sperm motility. Although more data are needed from a wider range of marine species and environments to fully assess potential toxicity effects on non-target organisms, our results highlight the potential utility of abamectin in low-release antifouling paints.

Population and life-stage specific sensitivities to temperature and salinity stress in barnacles.

Temperature and salinity shape the distribution and genetic structure of marine communities. Future warming and freshening will exert an additional stress to coastal marine systems. The extent to which organisms respond to these shifts will, however, be mediated by the tolerances of all life-stages and populations of species and their potential to adapt. We investigated nauplius and cypris larvae of the barnacle Balanus (Amphibalanus) improvisus from the Swedish west coast with respect to temperature (12, 20, and 28 °C) and salinity (5, 15, and 30) tolerances. Warming accelerated larval development and increased overall survival and subsequent settlement success. Nauplii developed and metamorphosed best at intermediate salinity. This was also observed in cypris larvae when the preceding nauplii stages had been reared at a salinity of 30. Direct comparisons of the present findings with those on a population from the more brackish Baltic Sea demonstrate contrasting patterns. We conclude that i) B. improvisus larvae within the Baltic region will be favoured by near-future seawater warming and freshening, that ii) salinity tolerances of larvae from the two different populations reflect salinities in their native habitats, but are nonetheless suboptimal and that iii) this species is generally highly plastic with regard to salinity.

DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

The Story of a Hitchhiker: Population Genetic Patterns in the Invasive Barnacle Balanus(Amphibalanus) improvisus Darwin 1854.

Understanding the ecological and evolutionary forces that determine the genetic structure and spread of invasive species is a key component of invasion biology. The bay barnacle, Balanus improvisus (= Amphibalanus improvisus), is one of the most successful aquatic invaders worldwide, and is characterised by broad environmental tolerance. Although the species can spread through natural larval dispersal, human-mediated transport through (primarily) shipping has almost certainly contributed to the current global distribution of this species. Despite its worldwide distribution, little is known about the phylogeography of this species. Here, we characterize the population genetic structure and model dispersal dynamics of the barnacle B. improvisus, and describe how human-mediated spreading via shipping as well as natural larval dispersal may have contributed to observed genetic variation. We used both mitochondrial DNA (cytochrome c oxidase subunit I: COI) and nuclear microsatellites to characterize the genetic structure in 14 populations of B. improvisus on a global and regional scale (Baltic Sea). Genetic diversity was high in most populations, and many haplotypes were shared among populations on a global scale, indicating that long-distance dispersal (presumably through shipping and other anthropogenic activities) has played an important role in shaping the population genetic structure of this cosmopolitan species. We could not clearly confirm prior claims that B. improvisus originates from the western margins of the Atlantic coasts; although there were indications that Argentina could be part of a native region. In addition to dispersal via shipping, we show that natural larval dispersal may play an important role for further colonisation following initial introduction.

Importance of plasticity and local adaptation for coping with changing salinity in coastal areas: a test case with barnacles in the Baltic Sea.

BACKGROUND: Salinity plays an important role in shaping coastal marine communities. Near-future climate predictions indicate that salinity will decrease in many shallow coastal areas due to increased precipitation; however, few studies have addressed this issue. The ability of ecosystems to cope with future changes will depend on species' capacities to acclimatise or adapt to new environmental conditions. Here, we investigated the effects of a strong salinity gradient (the Baltic Sea system--Baltic, Kattegat, Skagerrak) on plasticity and adaptations in the euryhaline barnacle Balanus improvisus. We used a common-garden approach, where multiple batches of newly settled barnacles from each of three different geographical areas along the Skagerrak-Baltic salinity gradient were exposed to corresponding native salinities (6, 15 and 30 PSU), and phenotypic traits including mortality, growth, shell strength, condition index and reproductive maturity were recorded. RESULTS: We found that B. improvisus was highly euryhaline, but had highest growth and reproductive maturity at intermediate salinities. We also found that low salinity had negative effects on other fitness-related traits including initial growth and shell strength, although mortality was also lowest in low salinity. Overall, differences between populations in most measured traits were weak, indicating little local adaptation to salinity. Nonetheless, we observed some population-specific responses--notably that populations from high salinity grew stronger shells in their native salinity compared to the other populations, possibly indicating adaptation to differences in local predation pressure. CONCLUSIONS: Our study shows that B. improvisus is an example of a true brackish-water species, and that plastic responses are more likely than evolutionary tracking in coping with future changes in coastal salinity.

Molecular characterization of the α-subunit of Na⁺/K⁺ ATPase from the euryhaline barnacle Balanus improvisus reveals multiple genes and differential expression of alternative splice variants.

The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na⁺/K⁺ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO2 levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions.