Evolutionary history and seascape genomics of Harbour porpoises (Phocoena phocoena) across environmental gradients in the North Atlantic and adjacent waters.

The Harbour porpoise (Phocoena phocoena) is a highly mobile cetacean species primarily occurring in coastal and shelf waters across the Northern hemisphere. It inhabits heterogeneous seascapes broadly varying in salinity and temperature. Here, we produced 74 whole genomes at intermediate coverage to study Harbour porpoise's evolutionary history and investigate the role of local adaptation in the diversification into subspecies and populations. We identified ~6 million high quality SNPs sampled at eight localities across the North Atlantic and adjacent waters, which we used for population structure, demographic and genotype-environment association analyses. Our results suggest a genetic differentiation between three subspecies (P.p. relicta, P.p. phocoena and P.p. meridionalis), and three distinct populations within P.p. phocoena: Atlantic, Belt Sea and Proper Baltic Sea. Effective population size and Tajima's D suggest population contraction in Black Sea and Iberian porpoises, but expansion in the P.p. phocoena populations. Phylogenetic trees indicate post-glacial colonization from a southern refugium. Genotype-environment association analysis identified salinity as major driver in genomic variation and we identified candidate genes putatively underlying adaptation to different salinity. Our study highlights the value of whole genome resequencing to unravel subtle population structure in highly mobile species, shows how strong environmental gradients and local adaptation may lead to population differentiation, and how neutral and adaptive markers can give different perspectives on population subdivision. The results have great conservation implications as we found inbreeding and low genetic diversity in the endangered Black Sea subspecies and identified the critically endangered Proper Baltic Sea porpoises as a separate population.

Variation of blubber thickness for three marine mammal species in the southern Baltic Sea.

Evaluating populational trends of health condition has become an important topic for marine mammal populations under the Marine Strategy Framework Directive (MSFD). In the Baltic Sea, under the recommendation of Helsinki Commission (HELCOM), efforts have been undertaken to use blubber thickness as an indicator of energy reserves in marine mammals. Current values lack geographical representation from the entire Baltic Sea area and a large dataset is only available for grey seals (Halichoerus grypus) from Sweden and Finland. Knowledge on variation of blubber thickness related to geography throughout the Baltic Sea is important for its usage as an indicator. Such evaluation can provide important information about the energy reserves, and hence, food availability. It is expected that methodological standardization under HELCOM should include relevant datasets with good geographical coverage that can also account for natural variability in the resident marine mammal populations. In this study, seasonal and temporal trends of blubber thickness were evaluated for three marine mammal species-harbor seal (Phoca vitulina), grey seal (Halichoerus grypus) and harbor porpoise (Phocoena phocoena)-resident in the southern Baltic Sea collected and investigated under stranding networks. Additionally, the effects of age, season and sex were analyzed. Seasonal variation of blubber thickness was evident for all species, with harbor seals presenting more pronounced effects in adults and grey seals and harbor porpoises presenting more pronounced effects in juveniles. For harbor seals and porpoises, fluctuations were present over the years included in the analysis. In the seal species, blubber thickness values were generally higher in males. In harbor seals and porpoises, blubber thickness values differed between the age classes: while adult harbor seals displayed thicker blubber layers than juveniles, the opposite was observed for harbor porpoises. Furthermore, while an important initial screening tool, blubber thickness assessment cannot be considered a valid methodology for overall health assessment in marine mammals and should be complemented with data on specific health parameters developed for each species.

Estimating the abundance of the critically endangered Baltic Proper harbour porpoise (Phocoena phocoena) population using passive acoustic monitoring.

Knowing the abundance of a population is a crucial component to assess its conservation status and develop effective conservation plans. For most cetaceans, abundance estimation is difficult given their cryptic and mobile nature, especially when the population is small and has a transnational distribution. In the Baltic Sea, the number of harbour porpoises (Phocoena phocoena) has collapsed since the mid-20th century and the Baltic Proper harbour porpoise is listed as Critically Endangered by the IUCN and HELCOM; however, its abundance remains unknown. Here, one of the largest ever passive acoustic monitoring studies was carried out by eight Baltic Sea nations to estimate the abundance of the Baltic Proper harbour porpoise for the first time. By logging porpoise echolocation signals at 298 stations during May 2011-April 2013, calibrating the loggers' spatial detection performance at sea, and measuring the click rate of tagged individuals, we estimated an abundance of 71-1105 individuals (95% CI, point estimate 491) during May-October within the population's proposed management border. The small abundance estimate strongly supports that the Baltic Proper harbour porpoise is facing an extremely high risk of extinction, and highlights the need for immediate and efficient conservation actions through international cooperation. It also provides a starting point in monitoring the trend of the population abundance to evaluate the effectiveness of management measures and determine its interactions with the larger neighboring Belt Sea population. Further, we offer evidence that design-based passive acoustic monitoring can generate reliable estimates of the abundance of rare and cryptic animal populations across large spatial scales.

Anisakid nematodes in dolphins (Cetacea: Delphinidae) from the Baltic Sea area.

Dolphins are rarely observed in the Baltic Sea, making only sporadic appearances as a result of their migrations. The study included six specimens: four white-beaked dolphins Lagenorhynchus albirostris and two striped dolphins Stenella coeruleoalba. Their gastrointestinal tracts were found to contain the nematodes Anisakis simplex (in both species) and Contracaecum sp. (only in the white-beaked dolphins). This is the first record of Anisakidae nematodes in dolphins found in the Baltic Sea.

A global checklist of the parasites of the harbor porpoise Phocoena phocoena, a critically-endangered species, including new findings from the Baltic Sea.

The common harbor porpoise is a widely-distributed marine mammal with three known subspecies, including P. phocoena phocoena, with a clearly distinct and critically endangered (CR) subpopulation from the Baltic Sea (Baltic Proper). As part of an assessment of the condition and health threats of these mammals, it is important to conduct parasitological monitoring. The aim of the study was therefore to compare the data on porpoise parasitofauna from this subpopulation with those on porpoises from other areas. The study included 37 individuals from 1995 to 2019; eight species of parasites were found (prevalence 83.8%, mean intensity 724.2, range 2-3940), with a predominance of lung nematodes - Stenurus minor (94.7%), Torynurus convolutus (69.4%), Pseudalius inflexus (63.8%), Halocercus invaginatus (22.2%); the highest intensity was recorded for S. minor (989, 53-2928). Two species of Anisakidae (Anisakis simplex - 33.3%, Contracaecum sp. - 20.0%) were found in the digestive tracts, which were a new record for this population. The fluke Campula oblonga was found in the livers of 31.3% of porpoises. The tapeworm Diphylobothrium stemmacephalum was also recorded in the intestine of one individual; this is typical for these hosts, but previously undetected in the Baltic subpopulation. Parasites coexisted in numerous hosts, constituting a heavy burden for them. The obtained data were compared with those from the P. phocoena parasitofauna from other regions, based on a compiled checklist (1809-2021) including all species of porpoise parasites (55 taxa). Compared to the worldwide porpoise parasitofauna checklist, the number of parasites found in the nominative subspecies (Baltic Proper subpopulation) is small: including only 10 taxa (eight in the current study). These species are typical of porpoises and usually the most common; however, the level of infection of Baltic porpoises (intensity and total parasite load) is very high, which can undoubtedly have a negative impact on their condition and overall health.

Fur and faeces - Routes of mercury elimination in the Baltic grey seal (Halichoerus grypus grypus).

This study focused on evaluating the elimination of Hg by Baltic grey seals (Halichoerus grypus grypus) via faeces and pelage. In addition, we investigated the potential for ecosystem contamination via these routes. Faeces and fur were collected in 2014-2017 from captive adult grey seals and their pups. The concentrations of total mercury (THg) and methylmercury (MeHg) were measured in the samples. The amount of mercury eliminated in a labile form (the sum of the bioavailable Hg(II) and methylmercury) was also determined. An adult seal removed about 46% of mercury supplied with food via the faeces, of which only 17% was MeHg. Considering that mercury is mainly supplied to the body as MeHg, it can be assumed that mercury excreted along with faeces has undergone transformation inside the animal body. Despite the much higher THg and MeHg concentrations measured in fur, the incorporation of mercury into newly formed fur is a less effective method of Hg elimination removing just 4% of mercury entering the body via the alimentary route. The presence of mercury in lanugo is evidence of maternal transfer. First droppings of the pups were characterised by the highest content of MeHg and a low THg concentration. Then, despite the limited supply of mercury with food, and the rapid growth of the pup, the concentration of THg increased, suggesting that mercury started to be transformed into less toxic forms. It was estimated that faeces and fur expelled by seals could deliver about 800 g of mercury to the Baltic Sea. For both faeces and fur, most of the mercury (>95% for excrements and >85% for fur) was expelled in a labile form that can be quickly recycled.

Pathological findings in North Sea and Baltic grey seal and harbour seal intestines associated with acanthocephalan infections.

Grey seals Halichoerus grypus and harbour seals Phoca vitulina are common seal species in the North and Baltic seas and final hosts of Corynosoma acanthocephalans. C. strumosum and C. magdaleni infect the small intestines of both seal species. In contrast to harbour seals, Baltic grey seals in the past have regularly displayed severe C. semerme infections in the caecum and colon, with associated tunica muscularis hypertrophy, inflammation and ulcerations as part of the Baltic seal disease complex (BSDC). Pathogenesis and correlation of acanthocephalan infections with these lesions are still unknown. This study describes the intestinal pathology and parasitic distribution in each seal species. Grey seal (n = 83) and harbour seal (n = 1156) intestines of all age groups and sexes, collected in Poland, Germany and Estonia from 1998 to 2017, were investigated. Most harbour seals came from the North Sea, whereas grey seals were predominantly derived from the Baltic Sea. Both species featured mild to moderate small intestinal infections. Grey seals showed colonic infections not found in harbour seals and featured a chronic erosive to ulcerative, eosinophilic or lympho-plasmacytic colitis with tunica muscularis hypertrophy, indicating still prevailing clinical signs of the BSDC. Harbour seals displayed granulomatous, eosinophilic, lympho-plasmacytic or catarrhal enteritis. The prevalence of acanthocephalan infections in harbour seals increased from 2012 onwards. Furthermore, significant associations between acanthocephalan infection and the presence of intestinal inflammation were found for both seal species. This study suggests that the level of acanthocephalan infection and associated lesions are suitable seal population health indicators, with the colon being a specific target organ for Baltic grey seal health monitoring.

Highly Pathogenic Avian Influenza A(H5N8) Virus in Gray Seals, Baltic Sea.

We detected a highly pathogenic avian influenza A(H5N8) virus in lung samples of 2 gray seals (Halichoerus grypus) stranded on the Baltic coast of Poland in 2016 and 2017. This virus, clade 2.3.4.4 B, was closely related to avian H5N8 viruses circulating in Europe at the time.

Changes in total mercury, methylmercury, and selenium blood levels during different life history stages of the Baltic grey seal (Halichoerus grypus grypus).

Using the blood of grey seal pups, the blood and milk of female grey seals inhabiting the Hel Marine Station of Gdansk University's Institute of Oceanography (HMS), we monitored the transfer of total mercury (THg), methylmercury (MeHg), and selenium (Se) with blood during foetal life and nursing. Changes in the concentration of mercury and selenium were characterised in the pups' blood during their first three months of life when they transition from suckling, to a post-weaning fast, to eating fish. In the blood of pregnant females, there was a significant decrease in THg and MeHg concentrations throughout the gestation, indicating the transfer of these toxins through the placenta into the foetus. At no other stage of the pup's development was there such a high level of THg and MeHg as on the day of birth, despite the incorporation of mercury into the lanugo during foetal growth. This suggests that the maternal transfer of mercury during gestation may be the time of greatest mercury exposure for a young seal pup. The consumption of milk caused a rapid increase in weight and a lowering of the mercury level in the blood in the subsequent days of the pups' life. The postweaning fast was the period of the lowest mercury concentration. The switch to a diet consisting of fish caused a systematic increase in the concentration of mercury in the blood of the pups. Milk was the significant source of selenium for pups and the selenium concentration in females' blood was reduced during lactation. The nursing period seemed to have the greatest impact on the mercury and selenium blood levels in examined seals. Natural development of the grey seal pup created an opportunity to decrease the levels of toxic substances obtained through the maternal transfer during foetal growth.

137Cs and 40K in gray seals Halichoerus grypus in the southern Baltic Sea.

This study presents levels of 137Cs and 40K concentrations in the placentas of seals gathered in the period 2007-2015. The mean activity of 137Cs and 40K was 5.49 Bq kg-1w.w. and 136.6 Bq kg-1 ww respectively. Statistically significant correlation was observed between the 137Cs activities in placenta and in herring-the staple food for seals. The concentrations of 137Cs and 40K were also determined in other tissues (muscle, liver, lung, and brain) of wild seals. The concentrations of 137Cs were from 2.59 Bq-1 ww (lungs) to 24.3 Bq kg-1 ww (muscles). The transfer factor values for 137Cs (seal tissue/fish) ranged from 0.89 to 2.42 in the case of the placentas and from 1.35 to 8.17 in the case of the muscle. For adults seal, the effective dose from 137Cs was 2.98 nGy h-1. The mean external radiation dose to pup was 0.77 nGy h-1 from 137Cs and 6.69 nGy h-1 from 40K.

Digenetic trematode Ogmogaster antarcticus (Notocotylidae) in a fin whale Balaenoptera physalus (Balaenopteridae) stranded in the Baltic Sea.

On 28 August 2015, a dead fin whale Balaenoptera physalus (Linnaeus, 1758) was collected from the waters of the Gulf of Gdan΄%%KERN_ERR%%sk (Baltic Sea). The individual was a male in a poor nutritional condition with an empty stomach. Over 34000 Ogmogaster antarcticus flukes were found in the colon and rectum. This is the first observation of O. antarcticus in the Baltic Sea.

Maternal transfer of phenol derivatives in the Baltic grey seal Halichoerus grypus grypus.

Studies of circulating levels in difference sex and age classes, and maternal transfer of bisphenol A, 4-tert-octylphenol and 4- nonylphenol in the Baltic grey seal were performed from 2014-2017. Blood was collected from long-term captive adult males, pregnant females and pups. Milk was collected from nursing females. The aim of this study was not only to determine the concentrations of phenol derivatives, i.e. bisphenol A (BPA), 4-tert-octylphenol (OP) and 4-nonylphenol (NP), but also to try to evaluate the transfer of these compounds to the next generation in the final stage of foetal life and in the first few weeks of life in juvenile marine mammals. The measurements were carried out using high performance liquid chromatography. The obtained data show that all phenol derivatives are present in the blood of males, females and pups (range <0.07-101 ng·cm-3) and in female milk (range <0.1-406.3 ng·cm-3). The main source of phenol derivatives in organisms is food exposure. Gender, age, or number of births were not observed to have a significant effect on changes in phenol derivative levels in seal blood within the breeding group. In the prenatal stage of life, a small amount of BPA and alkylphenols was passed on to the offspring through the placenta. In the blood of the offspring the concentration of these compounds exceeded the concentration in the mother's blood 1.5-fold. During nursing, females detoxified their systems. Level of phenol derivatives in the pups blood increased linearly with its increasing concentrations in the mother's milk. On the other hand, the seafood diet which started after the physiological fasting stage of the pup, stabilised the levels of phenol derivatives below 10 ng ∙ cm-3.

Prevalence and molecular characterisation of Acanthocephala in pinnipedia of the North and Baltic Seas.

Harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) are final hosts of acanthocephalans in the German North and Baltic Seas. Parasitic infections in seals can cause pathological changes, which may result in deteriorated health of the host. Common gastrointestinal parasites of harbour and grey seals are acanthocephalans and a number of 275 of 2460 (11.2%) investigated seals from 1996 to 2013 were infected with Corynosoma spp. (Acanthocephala, Polymorphidae). The prevalence showed a wave-like pattern: it increased from 1.2% and 0.4% in 1996 and 1997, respectively, to 23.9% during the second phocine distemper epizootic in 2002 and decreased to 6.2% in 2004. In 2005, prevalence peaked again with 25.0% followed by a decrease to 9.3% in 2009 and an increase to 38.5% in 2012. Statistical analysis revealed that harbour seals originating from the North Sea showed a higher prevalence than grey seals, whereas no significant difference between grey and harbour seals from the Baltic Sea was observed. Furthermore, juvenile pinnipedia from the North Sea were significantly less infected with Corynosoma spp. than seals older than seven month. Molecular species identification as well as phylogenetic relationship analysis among the detected Corynosoma species were achieved by sequencing and comparisons of the ribosomal ITS1-5.8S-ITS2-complex and cytochrome-c-oxidase I gene. Molecular analysis resulted in a newly arranged distribution of Acanthocephala in the North Sea as in contrast to previous studies, C. strumosum could not be confirmed as predominant species. Instead, C. magdaleni and a C. magdaleni isolate (isolate Pv1NS) with an atypical number of longitudinal rows of hooks at the proboscis were detected. Furthermore, morphological and molecular analyses indicate the possible finding of a cryptic species (Candidatus Corynosoma nortmeri sp. nov.).

Transfer of mercury and phenol derivatives across the placenta of Baltic grey seals (Halichoerus grypus grypus).

The placenta is an intermediary organ between the female and the developing foetus. Some chemical substances, including the most harmful ones, exhibit the ability to accumulate in or penetrate through the placenta. The aim of the study was to determine the role of the placenta of the Baltic grey seal (Halichoerus grypus grypus) in the transfer of endocrine disrupting compounds (EDCs) - (bisphenol A, 4-tert- octylphenol, 4- nonylphenol), as well as total and organic mercury. 30 placentas were collected from grey seals pupping under human care at the Hel Marine Station in the years 2007-2016. The assays were conducted using the technique of high-preformance liquid chromatography (phenol derivatives) and atomic absorption spectrometry (mercury and selenium). A measurable level of EDCs was indicated in the placentas of grey seals. It was established that the inorganic Hg form was accumulated in the placenta, and that its concentrations were an order of magnitude higher than the concentrations of the organic form, which penetrated to the foetus. Similar observations were made for phenol derivatives - bisphenol A, 4-tert- octylphenol and 4-nonylphenol. For this compound group the placenta was a barrier, but the properties of phenol derivatives suggest the possibility of their penetration through this organ.

Spatially Explicit Analysis of Genome-Wide SNPs Detects Subtle Population Structure in a Mobile Marine Mammal, the Harbor Porpoise.

The population structure of the highly mobile marine mammal, the harbor porpoise (Phocoena phocoena), in the Atlantic shelf waters follows a pattern of significant isolation-by-distance. The population structure of harbor porpoises from the Baltic Sea, which is connected with the North Sea through a series of basins separated by shallow underwater ridges, however, is more complex. Here, we investigated the population differentiation of harbor porpoises in European Seas with a special focus on the Baltic Sea and adjacent waters, using a population genomics approach. We used 2872 single nucleotide polymorphisms (SNPs), derived from double digest restriction-site associated DNA sequencing (ddRAD-seq), as well as 13 microsatellite loci and mitochondrial haplotypes for the same set of individuals. Spatial principal components analysis (sPCA), and Bayesian clustering on a subset of SNPs suggest three main groupings at the level of all studied regions: the Black Sea, the North Atlantic, and the Baltic Sea. Furthermore, we observed a distinct separation of the North Sea harbor porpoises from the Baltic Sea populations, and identified splits between porpoise populations within the Baltic Sea. We observed a notable distinction between the Belt Sea and the Inner Baltic Sea sub-regions. Improved delineation of harbor porpoise population assignments for the Baltic based on genomic evidence is important for conservation management of this endangered cetacean in threatened habitats, particularly in the Baltic Sea proper. In addition, we show that SNPs outperform microsatellite markers and demonstrate the utility of RAD-tags from a relatively small, opportunistically sampled cetacean sample set for population diversity and divergence analysis.

Impacts of Underwater Noise on Marine Vertebrates: Project Introduction and First Results.

The project conducts application-oriented research on impacts of underwater noise on marine vertebrates in the North and Baltic Seas. In distinct subprojects, the hearing sensitivity of harbor porpoises and gray seals as well as the acoustic tolerance limit of harbor porpoises to impulsive noise from pile driving and stress reactions caused by anthropogenic noise is investigated. Animals are equipped with DTAGs capable of recording the actual surrounding noise field of free-swimming harbor porpoises and seals. Acoustic noise mapping including porpoise detectors in the Natura 2000 sites of the North and Baltic Seas will help to fully understand current noise impacts.

In-air evoked potential audiometry of grey seals (Halichoerus grypus) from the North and Baltic Seas.

In-air anthropogenic sound has the potential to affect grey seal (Halichoerus grypus) behaviour and interfere with acoustic communication. In this study, a new method was used to deliver acoustic signals to grey seals as part of an in-air hearing assessment. Using in-ear headphones with adapted ear inserts allowed for the measurement of auditory brainstem responses (ABR) on sedated grey seals exposed to 5-cycle (2-1-2) tone pips. Thresholds were measured at 10 frequencies between 1-20 kHz. Measurements were made using subcutaneous electrodes on wild seals from the Baltic and North Seas. Thresholds were determined by both visual and statistical approaches (single point F-test) and good agreement was obtained between the results using both methods. The mean auditory thresholds were ≤40 dB re 20 µPa peak equivalent sound pressure level (peSPL) between 4-20 kHz and showed similar patterns to in-air behavioural hearing tests of other phocid seals between 3 and 20 kHz. Below 3 kHz, a steep reduction in hearing sensitivity was observed, which differed from the rate of decline in sensitivity obtained in behavioural studies on other phocids. Differences in the rate of decline may reflect influence of the ear inserts on the ability to reliably transmit lower frequencies or interference from the structure of the distal end of the ear canal.

Anisakids of seals found on the southern coast of Baltic Sea.

In the present study 5 grey seals (Halichoerus grypus), 3 common seals (Phoca vitulina) and 1 ringed seal (Pusa hispida) bycaught or stranded on the Polish Baltic Sea coast in years 2000-2006 were investigated for the infestation of parasitic anisakid nematodes. 749 of anisakids were found. The most common were: Contracaecum osculatum (59.3%) and Pseudoterranova decipiens (31.0%). There were also small numbers of Anisakis simplex (0.8%). After performing RFLP three sibling species were found. C. osculatum was identified as C. osculatum C, P decipiens was identified as P. decipiens sensu stricto and A. simplex - A. simplex sensu stricto. Nematodes found in seals were mostly in L4 and adult life stage - both of them were equal with some minor variations among the specimens. Sex ratio was also equal, but there was slight excess of males in some cases. There was a minority of L3 larvae belonging to A. simplex species (0.8%).