BackgroundThe harbour porpoise is exposed to increasing pressure caused by anthropogenic activities in its marine environment. Numerous offshore wind farms are planned or under construction in the North and Baltic Seas, which will increase underwater noise during both construction and operation. A better understanding of how anthropogenic impacts affect the behaviour, health, endocrinology, immunology and physiology of the animals is thus needed. The present study compares levels of stress hormones and mRNA expression of cytokines and acute-phase proteins in blood samples of harbour porpoises exposed to different levels of stress during handling, in rehabilitation or permanent human care.Free-ranging harbour porpoises, incidentally caught in pound nets in Denmark, were compared to harbour porpoises in rehabilitation at SOS Dolfijn in Harderwijk, the Netherlands, and individuals permanently kept in human care in the Dolfinarium Harderwijk and Fjord & Belt Kerteminde, Denmark. Blood samples were investigated for catecholamines, adrenaline, noradrenaline and dopamine, as well as for adrenocorticotropic hormone (ACTH), cortisol, metanephrine and normetanephrine. mRNA expression levels of relevant cell mediators (cytokines IL-10 and TNFα, acute-phase proteins haptoglobin and C-reactive protein and the heat shock protein HSP70) were measured using real-time PCR.ResultsBiomarker expression levels varied between free-ranging animals and porpoises in human care. Hormone and cytokine ranges showed correlations to each other and to the health status of investigated harbour porpoises. Hormone concentrations were higher in free-ranging harbour porpoises than in animals in human care. Adrenaline can be used as a parameter for the initial reaction to acute stress situations; noradrenaline, dopamine, ACTH and cortisol are more likely indicators for the following minutes of acute stress. There is evidence for different correlations between production of normetanephrine, metanephrine, cortisol and the expression of IL-10, HSP70 and haptoglobin.ConclusionsThe expression patterns of the selected molecular biomarkers of the immune system are promising to reflect the health and immune status of the harbour porpoise under different levels of stress.

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Studies on the occurrence of fungal communities in the marine environment are still scarce, but mycotic infections in cetaceans are increasingly reported. Fungal disease following infection with Aspergillus species is most frequently reported, with the respiratory tract commonly affected in cetaceans and other taxa, like humans and birds. Infection with Aspergillus spp. is dependent on the characteristics of the fungus as well as the hosts immune status, with dissemination into other organs being relatively common. Along the southern North Sea, harbor porpoise (Phocoena phocoena) strandings increased significantly since 2005 and necropsies to determine causes of death have been conducted since 2008. Here we describe the post-mortem findings in stranded, free-ranging harbor porpoises on the Dutch coast which were diagnosed with fungal disease, to determine the prevalence of mycotic infections, and to compare them to those described in other species. A total of 18/754 (2.4%) harbor porpoises showed lesions compatible with localized or disseminated fungal disease as confirmed by histological examination. The respiratory tract was most commonly affected (67%), followed by the central nervous system (CNS, 33%), and auditory system (AS, 17%). Aspergillosis was confirmed for 11/18, by fungal growth (as A. fumigatus species complex, n = 9) and PCR (as Aspergillus spp., n = 1, and as A. fumigatus sensu strictu by sequence analysis, n = 1). One live stranded and euthanized animal presented partial hemiplegia of the blowhole and therefore an MRI was conducted, which resulted in a unique image of the extensive, fungus-induced lesion in the left cerebellar hemisphere, deforming and displacing the brainstem, and additionally affected the AS. The gross-and histologic lesions in the 18 porpoises diagnosed with fungal disease were similar to changes described in other mammalian species. The prevalence of fungal disease in free-living harbor porpoises is lower than seen in captive and rehabilitated animals, suggesting that captivity increases the

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Testing the hearing abilities of marine mammals under water is a challenging task. Sample sizes are usually low, thus limiting the ability to generalize findings of susceptibility towards noise influences. A method to measure harbor porpoise hearing thresholds in situ in outdoor conditions using auditory steady state responses of the brainstem was developed and tested. The method was used on 15 live-stranded animals from the North Sea during rehabilitation, shortly before release into the wild, and on 12 wild animals incidentally caught in pound nets in Denmark (inner Danish waters). Results indicated that although the variability between individuals is wide, the shape of the hearing curve is generally similar to previously published results from behavioral trials. Using 10-kHz frequency intervals between 10 and 160 kHz, best hearing was found between 120 and 130 kHz. Additional testing using one-third octave frequency intervals (from 16 to 160 kHz) allowed for a much faster hearing assessment, but eliminated the fine scale threshold characteristics. For further investigations, the method will be used to better understand the factors influencing sensitivity differences across individuals and to establish population-level parameters describing hearing abilities of harbor porpoises.