Molecular and pathophysiological responses in non-healthy and healthy harbour porpoises (Phocoena phocoena)

Abstract

Harbour porpoises (Phocoena phocoena) in the North and Baltic Seas are constantly exposed to cumulative environmental and anthropogenic stressors that compromise their health. In particular, pathological lesions in the lungs that are caused or exacerbated by accumulating nematode infestations are a serious health issue. Such damages may not only impair respiratory function, but may also reduce oxygen delivery to end-organs, such as the skeletal muscles. Ultimately, this may negatively affect diving ability, resilience and overall fitness with potential population-level consequences. The aim of this dissertation was to investigate whether harbour porpoises with severe lung nematode infestations and associated secondary diseases possess molecular mechanisms to tolerate these pathological and parasitic insults to the respiratory system and whether the pathophysiological effects extend systemically to the skeletal muscles. To achieve this, lung and muscle tissue samples of harbour porpoises that died from stranding or as by-catch were used, and analysed by a combined omics approach, including transcriptomics, proteomics, metabolomics and lipidomics. Transcriptomic profiling and quantitative real time-PCR (qRT-PCR) were used to assess potential molecular changes in the muscles that may be indicative of reduced oxygen supply as a consequence of the pathological lung (Chapter I). The muscles showed signs of increased antioxidant activity, indicating elevated oxidative stress, but not a typical hypoxia response. Elevated transcripts involved in lipid metabolism and glycolysis pointed to energetic imbalance. A tightly regulated dynamic between tissue repair and turnover may be indicated by enhanced expression of transcripts related to regeneration and selective protein degradation, thus may support muscle maintenance despite ongoing stress exposure. These findings provided initial molecular insights that declining lung health may result in downstream effects in harbour porpoises. In Chapter II, the pathophysiological response in the lungs of non-healthy harbour porpoises was examined by using comparative transcriptomics, qRT-PCR, and de novo transcriptome assembly. The gene expression in the lungs of non-healthy individuals indicated a proinflammatory immune response, pointing to a persistent or exacerbating, and potentially compromising immune activation, underscoring findings of published studies. The similar regulation compared to humans suffering from lung diseases suggested that pulmonary immune responses may be conserved among mammals despite different life histories and habitats. Shared dysregulated expression patterns between the lungs and muscles indicated potential whole-organism consequences. The identified, significantly dysregulated non-coding RNAs via the de novo assembly may further regulate adaptive immunity, warranting future investigation to determine their function. Chapter III presents the integration of multiple omics datasets to obtain comprehensive insights into molecular processes across lungs and muscles in non-healthy harbour porpoises. Lungs of non-healthy porpoises displayed elevated antioxidant and anti-inflammatory features, including plasmalogen lipids, pointing to increased oxidative damage. Furthermore, dysregulation of immune responses underscored the results in Chapter II and indicated a chronic, systemic inflammation. Reduced collagen and increased cardiolipin levels in the lungs of non-healthy porpoises hinted at progressive tissue damage and impaired tissue function. In the skeletal muscles, reduced lipid metabolism and disrupted tissue maintenance suggested exceeding energy demand that may accelerate muscle catabolism and atrophy. Molecular alterations shared by both tissues supported a potential systemic impairment originating from the respiratory system. The integration of the omics data enabled the identification of potential biomarker panels, including proteins, metabolites, and lipids, which may support health monitoring of free-ranging harbour porpoises after validation in functional studies with larger sample sizes. Collectively, this dissertation reveals that respiratory disease and accumulating parasite infestations in free-ranging harbour porpoises extend beyond localised pulmonary damage to a systemic condition that affects immunity, energetic balance, and tissue maintenance. By linking declining pulmonary health with systemic stress, it highlights functional consequences and energetic trade-offs that may become critical for harbour porpoises under intensifying anthropogenic pressures and shifting ocean conditions. The identified molecular biomarker panels may support future non-invasive health monitoring and emphasise the growing potential of emerging omics technologies for cetacean health research. Together, these findings advance the understanding of how disease and anthropogenic pressures may shape the health, resilience and survival of harbour porpoise populations in the North and Baltic Seas.