Comparative analysis of bottom trawl and nanopore sequencing in fish biodiversity assessment: The sylt outer reef example.

The assessment of fish diversity is crucial for effective conservation and management strategies, especially in ecologically sensitive regions such as marine protected areas. This study contrasts the effectiveness of environmental DNA (eDNA) metabarcoding analysis employing Nanopore technology with compare beam trawl surveys at the Sylt Outer Reef, a Natura 2000 site in the North Sea, Germany. Out of the 17 fish species caught in a bottom trawl (using a 3m beam trawl), 14 were also identified through eDNA extracted from water samples. The three species not detected in the eDNA results were absent because they lacked representation in public DNA databases. The eDNA method detected twice as many fish species as the beam trawl, totalling 36 species, of which 14 were also detected by the trawl. Additionally, the selection of primers (Mifish) facilitated the identification of one marine mammal species, the harbour porpoise. In conclusion, the findings underscore the potential of eDNA coupled with MinION sequencing (Long read technology) as a robust tool for biodiversity assessment, surpassing traditional methods in detecting species richness.

Occurrence of the pugnose pipefish Bryx dunckeri in the Sargasso Sea.

Juvenile specimens of the pugnose pipefish, Bryx dunckeri, were collected during a multipurpose research survey conducted within the Sargasso Sea Subtropical Convergence Zone, extending the known distribution range of this species to include open ocean areas of the Western North Atlantic. Novel spatial data are of scientific interest as information on the distribution, population structure, and population size of this species is limited. Additionally, we present detailed photographs and morphological data on the collected specimens. The results are discussed in relation to the dispersal abilities and population structure in syngnathids.

Temperature and pressure dependency of oxygen consumption during long-term sustained swimming of European eels.

Many aspects of the typically 5000-10,000 km spawning migration of the European eel (Anguilla anguilla) remain unknown. As part of this migration, eels undertake extensive diurnal vertical migrations to depths below 1000 m, being exposed to a wide range of temperatures and hydrostatic pressures. In this experimental study, we exposed eels to different combinations of temperature (12-20°C) and pressure (100--800 kPa) during long-term sustained swimming (32-47 days). Both temperature and pressure affected oxygen consumption rate, such that there was a significant increase of metabolic rate with temperature, whereas pressure reduced oxygen consumption, albeit only at higher temperatures. Average oxygen consumption rates ranged between 15 mg kg-1 h-1 (12°C, 100 kPa) and 30.2 mg kg-1 h-1 (20°C, 100 kPa), highlighting the remarkably high swimming efficiency of this species and, more importantly, indicating that past evaluations of the cost of transport are potentially overestimates as they are often based on experiments conducted at atmospheric pressure at higher temperatures.

Evidence of hybridization between genetically distinct Baltic cod stocks during peak population abundance(s).

Range expansions can lead to increased contact of divergent populations, thus increasing the potential of hybridization events. Whether viable hybrids are produced will most likely depend on the level of genomic divergence and associated genomic incompatibilities between the different entities as well as environmental conditions. By taking advantage of historical Baltic cod (Gadus morhua) otolith samples combined with genotyping and whole genome sequencing, we here investigate the genetic impact of the increased spawning stock biomass of the eastern Baltic cod stock in the mid 1980s. The eastern Baltic cod is genetically highly differentiated from the adjacent western Baltic cod and locally adapted to the brackish environmental conditions in the deeper Eastern basins of the Baltic Sea unsuitable for its marine counterparts. Our genotyping results show an increased proportion of eastern Baltic cod in western Baltic areas (Mecklenburg Bay and Arkona Basin)-indicative of a range expansion westwards-during the peak population abundance in the 1980s. Additionally, we detect high frequencies of potential hybrids (including F1, F2 and backcrosses), verified by whole genome sequencing data for a subset of individuals. Analysis of mitochondrial genomes further indicates directional gene flow from eastern Baltic cod males to western Baltic cod females. Our findings unravel that increased overlap in distribution can promote hybridization between highly divergent populations and that the hybrids can be viable and survive under specific and favourable environmental conditions. However, the observed hybridization had seemingly no long-lasting impact on the continuous separation and genetic differentiation between the unique Baltic cod stocks.

Factors influencing the downstream passage of European silver eels (Anguilla anguilla) through a tidal sluice.

Tidal sluices are a frequent element in the tidal regions of Europe's rivers and may hinder downstream migrating European eels Anguilla anguilla. Sea level rise will reduce the possibility for tidal sluices to freely discharge water, further compressing windows of opportunity for the passage of eels. Understanding how eels utilize the discharge events of tidal sluices and which conditions facilitate successful passage is pivotal for the design of effective fish migration measures. To investigate eel migration at a tidal sluice, acoustic receivers were placed at the tidal sluice Nieuwe Statenzijl and in its tributary of the Westerwoldse Aa, the Netherlands. Of the 30 tagged eels, 26 eels reached the tidal sluice and passage success was 100%. The mean migration speed of eels in the unobstructed part of the tributary was slow (0.14 m s-1 ). The eels were delayed in their migration by the sluice and delay was right-skewed distributed with most eels showing moderate delays (<2 days), while about 10% of the tagged individuals experienced extensive delays of more than 3 weeks. The number of missed sluicing events prior to successful passage was influenced by biological characteristics such as migration speed in the tributary, weight and condition. In addition, sluicing events with rapidly increasing and high maximum discharge levels increased the success rate of an individual eel to pass the sluice. Compromising sluicing duration in favour of higher and faster increasing discharge could facilitate eel migration at tidal barriers and contribute to the recovery of this endangered species.

Mare-MAGE curated reference database of fish mitochondrial genes.

Biodiversity assessment approaches based on molecular biology techniques such as metabarcoding, RAD-seq, or SnaPshot sequencing are increasingly applied in assessing marine and aquatic ecosystems. Here we present a new reference database for fish meta-barcoding based on mitochondrial genes. The Mare-MAGE database contains quality-checked sequences of the mitochondrial 12S ribosomal RNA and Cytochrome c Oxidase I gene. All sequences were obtained from the National Center for Biotechnology Information- GenBank (NBCI-GenBank), the European Nucleotide Archive (ENA), AquaGene Database and BOLD database, and have undergone intensive processing. They were checked for false annotations and non-target anomalies, according to the Integrated Taxonomic Information System (ITIS) and FishBase. The dataset is compiled in ARB-Home, FASTA and Qiime2 formats, and is publicly available from the Mare-MAGE database website ( http://mare-mage.weebly.com/ ). It includes altogether 231,333 COI and 12S rRNA gene sequences of fish, covering 19,506 species of 4,058 genera and 586 families.

The role of genomic signatures of directional selection and demographic history in the population structure of a marine teleost with high gene flow.

Recent studies have uncovered patterns of genomic divergence in marine teleosts where panmixia due to high gene flow has been the general paradigm. These signatures of divergent selection are often impacted by structural variants, acting as "supergenes" facilitating local adaptation. The highly dispersing European plaice (Pleuronectes platessa)-in which putative structural variants (i.e., inversions) have been identified-has successfully colonized the brackish water ecosystem of the Baltic Sea. Thus, the species represents an ideal opportunity to investigate how the interplay of gene flow, structural variants, natural selection, past demographic history, and gene flow impacts on population (sub)structuring in marine systems. Here, we report on the generation of an annotated draft plaice genome assembly in combination with population sequencing data-following the salinity gradient from the Baltic Sea into the North Sea together with samples from Icelandic waters-to illuminate genome-wide patterns of divergence. Neutral markers pointed at large-scale panmixia across the European continental shelf associated with high gene flow and a common postglacial colonization history of shelf populations. However, based on genome-wide outlier loci, we uncovered signatures of population substructuring among the European continental shelf populations, i.e., suggesting signs of ongoing selection. Genome-wide selection analyses (xp-EHH) and the identification of genes within genomic regions of recent selective sweeps-overlapping with the outlier loci-suggest that these represent the signs of divergent selection. Our findings provide support for genomic divergence driven by local adaptation in the face of high gene flow and elucidate the relative importance of demographic history versus adaptive divergence in shaping the contemporary population genetic structure of a marine teleost. The role of the putative inversion(s) in the substructuring-and potentially ongoing adaptation-was seemingly not substantial.

Less impact than suspected: Dietary exposure of three-spined sticklebacks to microplastic fibers does not affect their body condition and immune parameters.

Microplastic fibers are frequent anthropogenic contaminants in most aquatic environments and have consequently been detected in the digestive tract of many fish species. Upon ingestion, microplastic fibers pose risks of interference with nutrient uptake, impaired intestinal health, and as a consequence may alter growth performance and fitness. In addition, foreign particles such as fibers might cause tissue irritations and stress, and thus interfere with immune parameters. In nature, fish regularly encounter microplastic fibers as well as fiber debris from natural sources and materials. Thus, we wanted to test the potential impact of microplastic fibers on growth, organosomatic indices, and immune parameters of subadult fish and compare these to possible effects caused by natural fibers. We administered sticklebacks diets, which were supplemented with either polyester or cotton fibers (each at concentrations of 0.2 mg/g and 2 mg/g feed) or a control diet without fiber supplementation for nine weeks. Mortalities did not occur and sticklebacks grew equally well across treatments. Neither organosomatic indices nor immune parameters revealed significant differences between treatments. While natural differences between males and females were observed for some parameters, no treatment-related gender-specific effects were detected. Our results suggest that the dietary uptake of polyester fibers does not affect growth, body condition, gonad development, and immunity of sticklebacks - even at fiber concentrations higher than what can be encountered in the wild. Furthermore, virgin microplastic fibers do not seem to affect fish differently than fibers from natural origin. The present study implies that at least some species are resilient towards pollution with (virgin) microplastic fibers even at high concentrations.

A novel hyperbaric swimming respirometer allows the simulation of varying swimming depths in fish respirometry studies.

The understanding of swimming physiology and knowledge on the metabolic costs of swimming are important for assessing effects of environmental factors on migratory behavior. Swim tunnels are the most common experimental setups for measuring swimming performance and oxygen uptake rates in fishes; however, few can realistically simulate depth and the changes in hydrostatic pressure that many fishes experience, e.g. during diel vertical migrations. Here, we present a new hyperbaric swimming respirometer (HSR) that can simulate depths of up to 80 m. The system consists of three separate, identical swimming tunnels, each with a volume of 205 L, a control board and a storage tank with water treatment. The swimming chamber of each tunnel has a length of 1.40 m and a diameter of 20 cm. The HSR uses the principle of intermittent-flow respirometry and has here been tested with female European eels (Anguilla anguilla). Various pressure, temperature and flow velocity profiles can be programmed, and the effect on metabolic activity and oxygen consumption can be assessed. Thus, the HSR provides opportunities to study the physiology of fish during swimming in a simulated depth range that corresponds to many inland, coastal and shelf waters.

Visual Gene Expression Reveals a cone-to-rod Developmental Progression in Deep-Sea Fishes.

Vertebrates use cone cells in the retina for color vision and rod cells to see in dim light. Many deep-sea fishes have adapted to their environment to have only rod cells in the retina, while both rod and cone genes are still preserved in their genomes. As deep-sea fish larvae start their lives in the shallow, and only later submerge to the depth, they have to cope with diverse environmental conditions during ontogeny. Using a comparative transcriptomic approach in 20 deep-sea fish species from eight teleost orders, we report on a developmental cone-to-rod switch. While adults mostly rely on rod opsin (RH1) for vision in dim light, larvae almost exclusively express middle-wavelength-sensitive ("green") cone opsins (RH2) in their retinas. The phototransduction cascade genes follow a similar ontogenetic pattern of cone-followed by rod-specific gene expression in most species, except for the pearleye and sabretooth (Aulopiformes), in which the cone cascade remains dominant throughout development, casting doubts on the photoreceptor cell identity. By inspecting the whole genomes of five deep-sea species (four of them sequenced within this study: Idiacanthus fasciola, Chauliodus sloani; Stomiiformes; Coccorella atlantica, and Scopelarchus michaelsarsi; Aulopiformes), we found that they possess one or two copies of the rod RH1 opsin gene, and up to seven copies of the cone RH2 opsin genes in their genomes, while other cone opsin classes have been mostly lost. Our findings hence provide molecular evidence for a limited opsin gene repertoire in deep-sea fishes and a conserved vertebrate pattern whereby cone photoreceptors develop first and rod photoreceptors are added only at later developmental stages.

Positively buoyant but sinking: Polymer identification and composition of marine litter at the seafloor of the North Sea and Baltic Sea.

Different litter types accumulate in all marine environments. Plastics are of special interest because of their high abundance and possible threats to marine organisms. Polymer type is crucial for their distribution and fate in marine environments. Seafloor litter abundance and composition in the Baltic and North Sea were analysed based on three sampling campaigns according to the protocol of ICES International Bottom Trawl Survey. Polymers were identified via attenuated total reflection-Fourier transform infrared spectroscopy. General litter abundances differed significantly between the Baltic and North Sea with 9.6 items/km2 and 70.7 items/km2, respectively. Plastic built the dominating litter group in both seas (62.2% and 91.3%, respectively). Polymer identification revealed clear dominance of polyethylene, polypropylene and polyamide. Most polymers were positively buoyant in seawater (89.5%), thereby excluding polymer density as the main driver of vertical plastic litter transportation. Plastics at the seafloor basically reflected the entirety of polymers entering marine environments.

Dumped munitions: New insights into the metabolization of 2,4,6-trinitrotoluene in Baltic flatfish.

Livers from dab (Limanda limanda), plaice (Pleuronectes platessa) and flounder (Platichthys flesus) sampled from the Baltic Sea were used to determine the interaction of flatfish CYP1A enzymes with 2,4,6-trinitrotoluene (TNT) in vitro. Competitive inhibition of 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-deethylase (MROD) could be demonstrated for all three flatfish species. The highest inhibition of CYP1A activities was measured in liver samples of flounder resulting in a half maximal inhibitory concentration (IC50) of 28.1 µM TNT. Due to their lower inhibition (EROD IC50 65.2 µM TNT, MROD IC50 40.3 µM TNT), dab liver samples were used to conduct in vitro metabolization experiments with TNT. The metabolization of TNT in fish was investigated with post-mitochondrial fractions (PMF) of dab liver as a model system after adding different cofactors. Rapid and time-dependent enzymatic degradation of TNT was observed. The concentrations of 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene increased in the samples over time. Additionally, 2,2,6,6-tetranitro-4,4-azoxytoluene was detected in one sample. The results of this study indicate that in vitro experiments are useful to investigate the xenobiotic metabolism of fish under controlled conditions prior to field studies. The metabolites found can serve as target compounds for marine monitoring of TNT contamination in munition dumpsites.

Swimming under elevated hydrostatic pressure increases glycolytic activity in gas gland cells of the European eel.

In spite of many decades of research, the spawning migration of the European eel Anguilla anguilla from the European coast to the Sargasso Sea remains a mystery. In particular, the role of the swimbladder as a buoyancy regulating structure is not yet understood. In this study, we exercised silver eels in a swim tunnel under elevated hydrostatic pressure. The transcriptome of gas gland tissue of these exercised eels was then compared to the known transcriptome of not exercised (control) silver eel gas gland cells. Due to the high infection rate of the eel population with the swimbladder parasite Anguillicola crassus, the comparison also included an exercised group of silver eels with a heavily damaged swimbladder, and we compared the previously published transcriptome of not exercised silver eels with a highly damaged swimbladder with the exercised group of silver eels with a heavily damaged swimbladder. The comparisons of unexercised (control) silver eels with exercised silver eels with functional swimbladder (EF), as well as with exercised silver eels with damaged swimbladder (ED), both showed a significant elevation in transcripts related to glycolytic enzymes. This could also be observed within the comparison of unexercised silver eels with a highly infected swimbladder with exercised eels with a damaged swimbladder (DED). In contrast to EF, in ED a significant elevation in transcript numbers of mitochondrial NADH dehydrogenase was observed. While in EF the transcriptional changes suggested that acid production and secretion was enhanced, in ED these changes appeared to be related to thickened tissue and thus elevated diffusion distances. The remarkable number of differentially expressed transcripts coding for proteins connected to cAMP-dependent signaling pathways indicated that metabolic control in gas gland cells includes cAMP-dependent pathways. In contrast to ED, in EF significant transcriptional changes could be related to the reconstruction of the extracellular matrix, while in ED tissue repair and inflammation was more pronounced. Surprisingly, in exercised eels hypoxia inducible transcription factor expression was elevated. In EF, a large number of genes related to the circadian clock were transcriptionally modified, which may be connected to the circadian vertical migrations observed during the spawning migration.

Evolution of male pregnancy associated with remodeling of canonical vertebrate immunity in seahorses and pipefishes.

A fundamental problem for the evolution of pregnancy, the most specialized form of parental investment among vertebrates, is the rejection of the nonself-embryo. Mammals achieve immunological tolerance by down-regulating both major histocompatibility complex pathways (MHC I and II). Although pregnancy has evolved multiple times independently among vertebrates, knowledge of associated immune system adjustments is restricted to mammals. All of them (except monotremata) display full internal pregnancy, making evolutionary reconstructions within the class mammalia meaningless. Here, we study the seahorse and pipefish family (syngnathids) that have evolved male pregnancy across a gradient from external oviparity to internal gestation. We assess how immunological tolerance is achieved by reconstruction of the immune gene repertoire in a comprehensive sample of 12 seahorse and pipefish genomes along the "male pregnancy" gradient together with expression patterns of key immune and pregnancy genes in reproductive tissues. We found that the evolution of pregnancy coincided with a modification of the adaptive immune system. Divergent genomic rearrangements of the MHC II pathway among fully pregnant species were identified in both genera of the syngnathids: The pipefishes (Syngnathus) displayed loss of several genes of the MHC II pathway while seahorses (Hippocampus) featured a highly divergent invariant chain (CD74). Our findings suggest that a trade-off between immunological tolerance and embryo rejection accompanied the evolution of unique male pregnancy. That pipefishes survive in an ocean of microbes without one arm of the adaptive immune defense suggests a high degree of immunological flexibility among vertebrates, which may advance our understanding of immune-deficiency diseases.

First evidence of explosives and their degradation products in dab (Limanda limanda L.) from a munition dumpsite in the Baltic Sea.

Corrosion and disintegration of munition shells from the World Wars increase the risk that explosives are released into the marine environment, exposing a variety of organisms. Only few studies investigated contamination of fish with explosives in the field under environmental conditions. Here we present a comprehensive study on the contamination status of dab (Limanda limanda) from a munition dumpsite and from reference sites in the Baltic Sea. Bile of 236 dab from four different study sites, including a dumpsite for conventional munitions, was investigated and explosive compounds were detected by high performance liquid chromatography-mass spectrometry. Five explosive compounds were identified, including 2,4,6-trinitrotoluene, 4-amino-2,6-dinitrolouene, and hexahydro-1,3,5-trinitro-1,3,5-triazine. 48% of the samples from the dumpsite contained at least one explosive compound. The results prove that toxic explosive compounds from a dumpsite in the Baltic Sea are accumulated by flatfish and may therefore pose a risk to fish health and human food safety.

Assessing SNP-markers to study population mixing and ecological adaptation in Baltic cod.

Atlantic cod (Gadus morhua) is a species of great ecological and economical importance in the Baltic Sea. Here, two genetically differentiated stocks, the western and the eastern Baltic cod, display substantial mechanical mixing, hampering our understanding of cod ecology and impeding stock assessments and management. Based on whole-genome re-sequencing data from reference samples obtained from the study area, we designed two different panels of Single Nucleotide Polymorphisms markers (SNPs), which take into account the exceptional genome architecture of cod. A minimum panel of 20 diagnostic SNPs and an extended panel (20 diagnostic and 18 biologically informative SNPs, 38 in total) were developed and validated to distinguish unambiguously between the western and the eastern Baltic cod stocks and to enable studies of local adaptation to the specific environment in the Baltic Sea, respectively. We tested both panels on cod sampled from the southern Baltic Sea (n = 603) caught in 2015 and 2016. Genotyping results showed that catches from the mixing zone in the Arkona Sea, were composed of similar proportions of individuals of the western and the eastern stock. Catches from adjacent areas to the east, the Bornholm Basin and Gdansk Deep, were exclusively composed of eastern Baltic cod, whereas catches from adjacent western areas (Belt Sea and Öresund) were composed of western Baltic cod. Interestingly, the two Baltic cod stocks showed strong genetic differences at loci associated with life-history trait candidate genes, highlighting the species' potential for ecological adaptation even at small geographical scales. The minimum and the extended panel of SNP markers presented in this study provide powerful tools for future applications in research and fisheries management to further illuminate the mixing dynamics of cod in the Baltic Sea and to better understand Baltic cod ecology.

Bone resorption and body reorganization during maturation induce maternal transfer of toxic metals in anguillid eels.

During their once-in-a-lifetime transoceanic spawning migration, anguillid eels do not feed, instead rely on energy stores to fuel the demands of locomotion and reproduction while they reorganize their bodies by depleting body reserves and building up gonadal tissue. Here we show how the European eel (Anguilla anguilla) breaks down its skeleton to redistribute phosphorus and calcium from hard to soft tissues during its sexual development. Using multiple analytical and imaging techniques, we characterize the spatial and temporal degradation of the skeletal framework from initial to final gonadal maturation and use elemental mass ratios in bone, muscle, liver, and gonadal tissue to determine the fluxes and fates of selected minerals and metals in the eels' bodies. We find that bone loss is more pronounced in females than in males and eventually may reach a point at which the mechanical stability of the skeleton is challenged. P and Ca are released and translocated from skeletal tissues to muscle and gonads, leaving both elements in constant proportion in remaining bone structures. The depletion of internal stores from hard and soft tissues during maturation-induced body reorganization is accompanied by the recirculation, translocation, and maternal transfer of potentially toxic metals from bone and muscle to the ovaries in gravid females, which may have direct deleterious effects on health and hinder the reproductive success of individuals of this critically endangered species.

Vision using multiple distinct rod opsins in deep-sea fishes.

Vertebrate vision is accomplished through light-sensitive photopigments consisting of an opsin protein bound to a chromophore. In dim light, vertebrates generally rely on a single rod opsin [rhodopsin 1 (RH1)] for obtaining visual information. By inspecting 101 fish genomes, we found that three deep-sea teleost lineages have independently expanded their RH1 gene repertoires. Among these, the silver spinyfin (Diretmus argenteus) stands out as having the highest number of visual opsins in vertebrates (two cone opsins and 38 rod opsins). Spinyfins express up to 14 RH1s (including the most blueshifted rod photopigments known), which cover the range of the residual daylight as well as the bioluminescence spectrum present in the deep sea. Our findings present molecular and functional evidence for the recurrent evolution of multiple rod opsin-based vision in vertebrates.

Spawning by the European eel across 2000 km of the Sargasso Sea.

It has been known for about a century that European eels have a unique life history that includes offshore spawning in the Sargasso Sea about 5000-7000 km away from their juvenile and adult habitats in Europe and northern Africa. Recently hatched eel larvae were historically collected during Danish, German and American surveys in specific areas in the southern Sargasso Sea. During a 31 day period of March and April 2014, Danish and German research ships sampled for European eel larvae along 15 alternating transects of stations across the Sargasso Sea. The collection of recently hatched eel larvae (≤12 mm) from 70° W and eastward to 50° W showed that the European eel had been spawning across a 2000 km wide region of the North Atlantic Ocean. Historical collections made from 1921 to 2007 showed that small larvae had also previously been collected in this wide longitudinal zone, showing that the spatial extent of spawning has not diminished in recent decades, irrespective of the dramatic decline in recruitment. The use of such a wide spawning area may be related to variations in the onset of the silver eel spawning migration, individual differences in their long-term swimming ability, or aspects of larval drift.