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A changing marine environment with emerging natural and anthropogenic stressors challenges the marine mammal immune system. The skin and adnexa form a first protective barrier in the immune response, although this is still relatively understudied in cetaceans. The cellular and tissue morphology of the nodular and diffuse lymphoid tissue are not fully charted and the physiological responses are not yet completely understood. The odontocete’s external ear canal has a complex relationship with the external environment, with an artificial lumen rendering the inside of the canal a relatively secluded environment. In this work, we studied the odontocete ear canal-associated lymphoid tissue (ECALT) by histo- and immunohistochemistry (HC, IHC) with anti-CD3, anti-CD20, anti-Iba-1, anti-HLA-DR, and anti-vimentin antibodies. The ECALT cellular composition consists mainly of B-lymphocytes with the occasional presence of T-lymphocytes and the dispersed distribution of the macrophages. In cases of activation, the cellular reaction showed a similar pattern with the occasional presence of T-cells, plasma cells, and neutrophils. Nodular lymphoid tissue was generally in line with the description in other odontocetes, although with abundant erythrocytes throughout the entire organ. This study contributes to the understanding of the cellular composition of diffuse and nodular lymphoid tissue in several species of odontocetes, and in association with inflammation of the external ear canal.

Long-finned pilot whale (Globicephala melas) mitochondrial (mtDNA) genetic diversity is considered low, especially in the North Atlantic, where only seven haplotypes have been recorded in previous studies using a 345 bp control region fragment. Such studies have not included samples from Ireland or the Netherlands. In this study we analyzed a longer sequence of the mtDNA control region (631 bp) from individuals stranded around Ireland, Scotland, and the Netherlands between 1995 and 2019 (n = 180). Nine haplotypes were identified, of which five were newly described (haplotype diversity h = 0.511). Pairwise tests revealed significant differentiation between the Irish and Scottish samples. Potential confounding factors are discussed but given that failure to recognize population structure may compromise conservation efforts, the findings show the need for further investigation using nuclear markers. Six mass stranding events were included, of which one event reported two haplotypes among individuals confirming a mixing of matrilineal groups. Although the permanence of this combination cannot be determined, this is the first record of such an occurrence within the North Atlantic. This study shows that stranding sample databases are a useful resource for genetic studies and provides new insights into genetic diversity of long-finned pilot whales in the eastern North Atlantic and adjacent waters.

The application of whole-body post-mortem computed tomography (PMCT) in veterinary and wildlife post-mortem research programs is advancing. A high incidence of pulmonary pathology is reported in the harbor porpoise (Phocoena phocoena). In this study, the value of PMCT focused on pulmonary assessment is evaluated. The objectives of this study were to describe pulmonary changes as well as autolytic features detected by PMCT examination and to compare those findings with conventional necropsy. Retrospective evaluation of whole-body PMCT images of 46 relatively fresh harbor porpoises and corresponding conventional necropsy reports was carried out, with a special focus on the respiratory tract. Common pulmonary PMCT findings included: moderate (24/46) to severe (19/46) increased pulmonary soft tissue attenuation, severe parasite burden (17/46), bronchial wall thickening (30/46), and mild autolysis (26/46). Compared to conventional necropsy, PMCT more frequently identified pneumothorax (5/46 vs. none), tracheal content (26/46 vs. 7/46), and macroscopic pulmonary mineralization (23/46 vs. 11/46), and provided more information of the distribution of pulmonary changes. These results indicate that PMCT adds information on pulmonary assessment and is a promising complementary technique for necropsy, despite the frequent presence of mild autolytic features.

Humans impact natural systems at an unprecedented rate. The North Sea is one of the regions in the world with the highest levels of anthropogenic activity. Here, the harbor porpoise (Phocoena phocoena) is an abundant species and is often regarded as an ecosystem sentinel. A post-mortem surveillance program was established in the Netherlands aimed at increasing knowledge of the effects of human activities on harbor porpoises. In this study, we describe the pathological findings related to anthropogenic and natural causes of death categories in 612 harbor porpoises that stranded between 2008 and 2019, and assess their relations to age, sex, season, and location. The largest anthropogenic category was bycatch (17%), with mainly juveniles affected and peak periods in March and September–October. Other, infrequently diagnosed anthropogenic causes of death were trauma (4%), largely most likely due to ship collisions, and marine debris ingestion and entanglement (0.3%). The risk of dying from anthropogenic causes was highest for juveniles. Lesions compatible with noise-induced hearing loss were investigated in carcasses which were fresh enough to do so (n = 50), with lesions apparent in two porpoises. Non-direct human-induced threats included infectious diseases, which were by far the largest cause of death category (32%), and affected mainly adults. Also, gray seal (Halichoerus grypus) attacks were a frequently assigned cause of death category (24%). There were more acute predation cases in the earlier study years, while porpoises with lesions that suggested escape from gray seal attacks were diagnosed more recently, which could suggest that porpoises adapted to this threat. Our study contributes to understanding porpoise health in response to persisting, new, emerging, and cumulative threats. Building up such knowledge is crucial for conservation management of this protected species.

Baleen from mysticete whales is a well-preserved proteinaceous material that can be used to identify migrations and feeding habits for species whose migration pathways are unknown. Analysis of δ 13 C and δ 15 N values from bulk baleen have been used to infer migration patterns for individuals. However, this approach has fallen short of identifying migrations between regions as it is difficult to determine variations in isotopic shifts without temporal sampling of prey items. Here, we apply analysis of δ 15 N values of amino acids to five baleen plates belonging to three species, revealing novel insights on trophic position, metabolic state and migration between regions. Humpback and minke whales had higher reconstructed trophic levels than fin whales (3.7–3.8 versus 3–3.2, respectively) as expected due to different feeding specialization. Isotopic niche areas between baleen minima and maxima were well separated, indicating regional resource use for individuals during migration that aligned with isotopic gradients in Atlantic Ocean particulate organic matter. Phenylanine δ 15 N values confirmed regional separation between the niche areas for two fin whales as migrations occurred and elevated glycine and threonine δ 15 N values suggested physiological changes due to fasting. Simultaneous resolution of trophic level and physiological changes allow for identification of regional migrations in mysticetes.

Evidence of hearing impairment was identified in a harbour porpoise (Phocoena phocoena) on the basis of scanning electron microscopy. In addition, based on histopathology and immuno-histochemistry, there were signs of unrelated cerebral toxoplasmosis. The six‐year old individual live stranded on the Dutch coast at Domburg in 2016 and died a few hours later. The most significant gross lesion was multifocal necrosis and haemorrhage of the cerebrum. Histopathology of the brain revealed extensive necrosis and haemorrhage in the cerebrum with multifocal accumulations of de-generated neutrophils, lymphocytes and macrophages, and perivascular lymphocytic cuffing. The diagnosis of cerebral toxoplasmosis was confirmed by positive staining of protozoa with anti‐Toxo-plasma gondii antibodies. Tachyzoites were not observed histologically in any of the examined tis-sues. Ultrastructural evaluation of the inner ear revealed evidence of scattered loss of outer hair cells in a 290 μm long segment of the apical turn of the cochlea, and in a focal region of ~ 1.5 mm from the apex of the cochlea, which was compatible with noise‐induced hearing loss. This is the first case of concurrent presumptive noise‐induced hearing loss and toxoplasmosis in a free‐ranging harbour porpoise from the North Sea.

This report presents a validated list of stranded cetaceans in the Netherlands, as well as some cetaceans accidentally brought in on ship bulbs, between 2015-2019. During this period 2701 cetaceans representing eleven species were reported. The list also includes a few bones and skulls, among others of killer whale (Orcinus orca) and bottlenose dolphin (Tursiops truncatus), species not reported otherwise during this period. The most common species was harbour porpoise (Phocoena phocoena), with 2651 individuals. The average number of porpoises per year was 530, lower than the preceding seven-year average, but numbers fluctuated largely between years and there has been no particular trend after the strong increase in the early 2000s. The monthly pattern of strandings as recorded during the previous periods, with peaks in March and July-September, remained the same. The number of stranded porpoise is equally spread along the entire coastline. The density, expressed as the number of stranded porpoise per kilometre per year, is 0.6 for the entire coastline, or 1.2 if the extensive and less well surveyed areas of Western Scheldt, Eastern Scheldt and Wadden Sea proper are omitted. Sex ratio remained stable over the years and is in line with results from before 2015, with a preponderance of males. On the basis of length, over half were immature and 8.7% neonate. Among neonates and immatures there was a preponderance of males as well, but not in adults. In the Wadden Sea area more neonates and adults were found than in the other two subareas. The major cause of death was infectious disease, followed by predation by grey seal (Halichoerus grypus). Accidental by-catch was identified as the cause of death of 11% of the stranded animals. Of particular interest was the stranding of six sperm whales (Physeter macrocephalus) in January 2016, the largest stranding event of this species in the Netherlands. It was part of a stranding event spread out over the Central and southern North Sea, involving thirty individuals. During 2015-2019 four dead fin whales (Balaenoptera physalus) and eight minke whales (B. acutorostrata) were reported, several of which were hit by ships. There seems to be a slight increase in the strandings of fin whale and Sowerby’s beaked whale (Mesoplodon bidens) since 2000, while white-beaked dolphin (Lagenorhynchus albirostris) shows a decrease. There is no temporal trend for any of the other species since 2000.

Recent population growth of the harbour porpoise ( Phocoena phocoena ), grey seal ( Halichoerus grypus ) and common seal ( Phoca vitulina ) in the North Sea has increased potential interaction between these species. Grey seals are known to attack harbour porpoises. Some harbour porpoises survive initially, but succumb eventually, often showing severely infected skin lesions. Bacteria transferred from the grey seal oral cavity may be involved in these infections and eventual death of the animal. In humans, seal bites are known to cause severe infections. In this study, a 16S rRNA-based microbiome sequencing approach is used to identify the oral bacterial diversity in harbour porpoises, grey seals and common seals; detect the potential transfer of bacteria from grey seals to harbour porpoises by biting and provide insights in the bacteria with zoonotic potential present in the seal oral cavity. β-diversity analysis showed that 12.9% (4/31) of the harbour porpoise skin lesion microbiomes resembled seal oral microbiomes, while most of the other skin lesion microbiomes also showed seal-associated bacterial species, including potential pathogens. In conclusion, this study shows that bacterial transmission from grey seals to harbour porpoises by biting is highly likely and that seal oral cavities harbour many bacterial pathogens with zoonotic potential.

33 pages.-- 9th Meeting of the Parties, online 7-11 September 2020, The ASCOBANS area hosts a high diversity of small cetaceans (36 species – Evans, 2020). This includes a large number of delphinids, the most common of which arecommon dolphin (Delphinus delphis), striped dolphin (Stenella coeruleoalba), bottlenose dolphin (Tursiops truncatus),white-beaked dolphin (Lagenorhynchus albirostris),Atlantic white-sided dolphin (Lagenorhynchus acutus), Risso’s dolphin (Grampus griseus), long-finned pilot whale (Globicephala melas), and killer whale (Orcinus orca). Other small odontocetes present include several beaked-whales: northern bottlenose whale (Hyperoodon ampullatus); Sowerby’s beaked whale (Mesoplodon bidens); and Cuvier’s beaked whale (Ziphius cavirostris). Within the family Phocoenidae, only one member is present: the harbour porpoise (Phocoena phocoena)

The 2019 iteration of the ASCOBANS and ACCOBAMS joint best practice for cetacean stranding investigations and tissue sampling .\ud Monitoring dead stranded cetaceans offers an often unique opportunity to gain insights into the health of, and threats and stressors affecting, marine ecosystems. Information derived from the systematic examination of stranded carcases can provide insights into the at-sea population not easily acquired through other means, indeed strandings data is the major source of information available for some species. Detailed investigation of carcases can assist in the determination of causes of death, offer surveillance on the incidence of trauma and disease and provide tissues and data for subsequent analysis into a range of biological and ecological parameters. The collection of data and samples for ancillary investigations over a range of disciplines can provide information on the general population ecology, helping countries evaluate and mitigate possible threats affecting species conservation and the marine ecosystem.\ud Many EU countries operate cetacean strandings investigation networks as part of their obligations to international agreements. These include the Agreement on the Conservation of Small Cetaceans of the Baltic, North East Atlantic, Irish and North Seas (ASCOBANS), the Agreement for the Conservation of Cetaceans in the Black Sea, Mediterranean Sea and contiguous Atlantic Area (ACCOBAMS), the EU Habitats Directive (NATURA2000), the OSPAR Commission and the Baltic Marine Environment Protection Commission (HELCOM). The Marine Strategy Framework Directive (MSFD) obliges the member states to develop indicators and descriptors for the surveillance of Good Environmental Status of cetaceans.\ud Many of these strandings networks are well established and have long-term datasets comprising extensive biological and pathological information from the systematic collation and investigation of mortalities. In addition, many European strandings networks curate uniquely important tissue and pathogen archives. Most networks follow the original protocol by Kuiken and Hartmann, published in 1993. In the subsequent decades, stranding networks throughout Europe have developed, and new analytical techniques (e.g. assessment of the effects of barotrauma) or health impacts e.g. grey seal predation, have been incorporated into operational methods.\ud The aim of this document is to update the protocol with the currently available techniques and methodologies agreed between all member countries of ACCOBAMS and ASCOBANS. It is hoped that this updated protocol can serve three overall aims:\ud 1. To provide a reference document for veterinarians and biologists currently engaged in cetacean post-mortem investigation, summarising a recognised approach to stranding investigation across European networks;\ud 2. To highlight areas where harmonisation of data from existing networks could allow for analysis and inference to be made between networks, of particular relevance for the transboundary, mobile species;\ud 3. Provide a start-up guide for researchers attempting to instigate new stranding monitoring programmes, particularly in regions of the world with limited resources for extensive, top-down surveillance programmes.\ud It should be emphasised that this document is not designed to replace existing protocols, particularly those of longstanding and well-established laboratories and stranding networks, but offers a post mortem framework aiming for consistency across Europe when conducting examinations on dead cetaceans.

Vertebral series in the harbor porpoise (Phocoena phocoena) include cervical, thoracic, lumbar, and caudal. In contrast to studying skeletons from museums, in which small bones can be missed, evaluation of full body computed tomography (CT) scans provides an overview of the vertebral column, while maintaining interrelationship of all structures. The aim of this study was to document variations in vertebral patterning of the harbor porpoise via evaluation of CT images of intact stranded harbor porpoises. The harbor porpoises were divided into age classes, based on developmental stage of reproductive organs on postmortem examination and closure of proximal humeral physis on CT. Numbers of vertebrae per series, fusion state of the syncervical, type of first hemal arch, number of double articulating ribs, and floating ribs were recorded based on CT images. Included in the study were 48 harbor porpoises (27 males and 21 females), which were divided in two age classes (27 immatures and 21 adults). Total vertebral count varied from 63 to 68 with vertebral formula range C7T12‐14L12‐16Cd29‐33. Twenty‐five different vertebral formulas were found, of which C7T13L14Ca30 was the most common (n = 8, 17%). Thoracic vertebrae with six, seven, or eight double articulating ribs and zero, one, or two vertebrae with floating ribs were seen. Four different fusion states of the syncervical and four types of hemal arches were recognized. This study showed a great variation in vertebral patterning in the harbor porpoise, with homeotic and meristic variation in the thoracic, lumbar, and caudal vertebral series.

Harbour porpoises are often found to be infected by endoparasites in several organs including the lungs and stomach as well as the heart, liver and ears. Nevertheless there is still little knowledge about the impact, ecology, transmission, and virulence of these parasitic infections. Here, we profile the presence of parasites in 4 frequently infected organs (lungs, stomach, liver and ears) in relation to biological parameters of harbour porpoises stranded along the Dutch coastline between December 2008 and December 2013. We found that parasites were common, with prevalence of 68% in lungs, 74.4% in ears, 26% in stomach and 23.5% in liver. We used generalised linear models to further quantify parasite presence in relation to biological data gathered during necropsy (sex, body length and nutritive condition). Body length (used as a proxy for age) was significant in explaining parasite presence for all organs with increasing probability of having the parasite with increasing body length. For the parasitic infections in the ears and stomach the nutritive condition was an additional significant factor, with a higher probability of parasite presence in porpoises in a poorer nutritive condition. The results of this study can be used as a baseline for assessing parasite presence in harbour porpoises and are a first step towards linking parasite infections to basic biological data gathered during necropsy.

Harbour porpoises (Phocoena phocoena), the smallest of cetaceans, need to consume quantities of prey that amount to ca. 10% of their own body mass per day. They mostly feed on small fish, with the main prey species differing geographically. The δ¹³C muscle signature of harbour porpoises sampled in the Eastern Scheldt, SW Netherlands, has indicated that animals tend to stay here for some time after they entered this semi-enclosed basin, and that they thus must feed on local prey. A relatively low primary production and low local fish biomassraises the question what there is for harbour porpoises to feed on in the Eastern Scheldt. This study reveals that there are no big differences between biological or stranding parameters of harbour porpoises found dead in the Eastern Scheldt compared with the adjacent North Sea (the “Voordelta”), but some differences in diet were found. Still, despite the low fish biomass in the Eastern Scheldt, no evidence of excessive harbour porpoise starvation was found. The main prey species for juvenile porpoises, both in the North Sea and in the Eastern Scheldt, were gobies. Gadoids were important prey for adults in both regions. Gadoid prey was supplemented by gobies and sandeels in the North Sea, and by squid and estuarine roundfish in the Eastern Scheldt. Our results demonstrate that harbor porpoises that stay in the Eastern Scheldt for a longer period of time may develop specialised feeding skills, to cope with the relatively poor prey base. Juveniles on the other hand, must settle for small and lean prey (gobies and smallsepiolids) and may face competition from adults.

Cetaceans are well known definitive hosts of parasitic nematodes of the genus Anisakis (Nematoda: Anisakidae). Anisakid nematodes are also a health hazard for humans, potentially causing gastrointestinal infections or allergic reactions following the consumption of infected fish. In marine mammals, the nematodes develop from third-stage larvae to adults in the stomachs. In the first (or fore-) stomach, these parasites are typically associated with mucosal ulceration; parasites have not been identified in other organs. Two small cetaceans, a bottlenose dolphin Tursiops truncatus and a harbour porpoise Phocoena phocoena, presented marked gastric A. simplex infection, as well as chronic granulomatous and ulcerative dermatitis with intralesional nematodes, bordered by epithelial hyperplasia. Nematodes in the skin of the bottlenose dolphin were morphologically similar to Anisakis spp. Morphology of the parasitic remnants in the skin lesion of the harbour porpoise was indistinct, but molecular identification confirmed the presence of A. simplex. This is the first report of Anisakis spp. infection in the skin of marine mammals.

Long-finned pilot whales (Globicephala melas) are rare visitors to the southern North Sea, but recently two individual strandings occurred on the Dutch coast. Both animals shared the same, unusual cause of death: asphyxiation from a common sole (Solea solea) stuck in their nasal cavity. This is a rare cause of death in cetaceans. Whilst asphyxiation has been reported in smaller odontocetes, there are no recent records of this occurring in Globicephala species. Here we report the stranding, necropsy and diet study results as well as discuss the unusual nature of this phenomenon. Flatfish are not a primary prey species for pilot whales and are rarely eaten by other cetaceans, such as harbour porpoises (Phocoena phocoena), in which there are several reports of asphyxiation due to airway obstruction by soles. This risk may be due to the fish’s flexible bodies which can enter small cavities either actively in an attempt to escape or passively due to the whale ‘coughing’ or ‘sneezing’ to rid itself of the blockage of the trachea. It is also possible that the fish enter the airways whilst the whale is re-articulating the larynx after trying to ingest large, oddly shaped prey. It is unlikely that the soles entered the airways after the death of the whales and we believe therefore that they are responsible for the death of these animals.

Peribullar sinuses of harbour porpoises Phocoena phocoena are parasitized with high prevalence by the nematode Stenurus minor. The effect of S. minor on the hearing ability of this species is still undetermined. Here, we review the occurrence of S. minor in the inner ear of harbour porpoises recovered from strandings in the North and Baltic Seas. In particular, we present the results from ears collected in German and Danish waters from 2002 to 2016 and from Dutch waters from 2010 to 2016. While the prevalence of S. minor in pterygoid and peribullar sinuses and tympanic cavity was high in harbour porpoises (66.67% in our cases), its prevalence in the cochlea was rare. Only 1 case out of 129 analysed by either histology, electron microscopy or immunofluorescence showed the presence of a nematode parasite morphologically consistent with S. minor at the most basal portion of the right cochlea. This individual also had severe haemorrhage along the right cochlear spiral, which was likely caused by ectopic S. minor migration. Although this animal might have had impaired hearing in the right ear, it was otherwise in good body condition with evidence of recent feeding. These findings highlight the need to study the effect of parasites on hearing, and other pathological changes that might impair appropriate processing of acoustic information.