Centelleghe, C. | bioRxiv Science

bioRxiv2026

Isolation and preliminary characterization of extracellular vesicles from bottlenose dolphin (Tursiops truncatus) and long-finned pilot whale (Globicephala melas) blow

Cetaceans are key sentinel species for environmental health monitoring. Although sampling from free-ranging animals is challenging, the analysis of cetacean blow offers a minimally invasive approach to assess their health status. Extracellular vesicles (EVs) are cell-derived nanostructures present in biological fluids and widely studied as disease biomarkers in humans. Despite the potential for similar uses, EVs have not been studied in cetacean blow to date . This proof-of-concept study aims to assess the feasibility of the isolation and characterization of EVs from blow samples collected from five bottlenose dolphins (Tursiops truncatus) kept under human care and from a free ranging specimen of long-finned pilot whale (Globicephala melas). EVs were purified from bottlenose dolphin blows by ultracentrifugation (UC) or size exclusion chromatography (SEC) and from the long-finned pilot whale by SEC. Particle concentration and size distribution were assessed by Nanoparticle Tracking Analysis (NTA), morphology by Air-atomic force microscopy (AFM) and protein expression by Western Blotting (WB). NTA revealed a higher mean particle concentration in bottlenose dolphin EVs isolated by UC compared to SEC, while EVs isolated from the long-finned pilot whale presented a lower particle concentration. AFM confirmed the presence of EV-like particles within the typical EV size range in bottlenose dolphins EVs obtained both by SEC or UC. All EV samples were positive for CD9 and integrin-{beta} and negative to Calnexin. SEC was more sensitive to detect OmpA, a membrane protein of Gram-negative bacteria, in EVs from both species. Our pilot study demonstrates that EVs are present in cetacean blow and can be isolated and characterized. Future investigations focused on characterizing and quantifying a wider array of EV associated molecules may further the application of blow EV analysis for cetacean health assessments.

zoology↗

bioRxiv2021

SARS-CoV-2, a threat to marine mammals? A study from Italian seawaters

Zoonotically transmitted coronaviruses were responsible for three disease outbreaks since 2002, with the "Severe Acute Respiratory Syndrome Coronavirus-2" (SARS-CoV-2) causing the dramatic "Coronavirus Disease-2019" (CoViD-19) pandemic, which affected public health, economy, and society on a global scale. The impacts of the SARS-CoV-2 pandemic permeate into our environment and wildlife as well; in particular, concern has been raised about the viral occurrence and persistence in aquatic and marine ecosystems. The discharge of untreated wastewaters carrying infectious SARS-CoV-2 into natural water systems that are home of sea mammals may have dramatic consequences on vulnerable species. The efficient transmission of coronaviruses raise questions regarding the contributions of virus-receptors interactions. The main receptor of SARS-CoV-2 is Angiotensin Converting Enzyme-2 (ACE-2), serving as a functional receptor for the viral spike (S) protein. This study was aimed, through the comparative analysis of the ACE-2 receptor with the human one, at assessing the susceptibility to SARS-CoV-2 of the different species of marine mammals living in Italian waters. We also determined, by means of immunohistochemistry, ACE-2 receptor localization in the lung tissue from different cetacean species, in order to provide a preliminary characterization of ACE-.2 expression in the marine mammals respiratory tract. Furthermore, in order to evaluate if and how wastewater management in Italy may lead to susceptible marine mammal populations being exposed to the virus, geo-mapping data of wastewater plants, associated to the identification of specific stretches of coast more exposed to extreme weather events, overlapped to marine mammal population data, were carried out. Results showed the SARS-CoV-2 exposure for marine mammals inhabiting Italian coastal waters. Thus, we highlight the potential hazard of reverse zoonotic transmission of SARS-CoV-2 infection, along with its impact on marine mammals regularly inhabiting the Mediterranean Sea, whilst also stressing the need of appropriate action to prevent further damage to specific vulnerable populations. Significance StatementGrowing concern exists that SARS-CoV-2, as already ascertained for its SARS-CoV and MERS-CoV "predecessors", originated from an animal "reservoir", performing thereafter its spillover into mankind, that was possibly anticipated by viral "passage" into a secondary animal host. Within the dramatic SARS-CoV-2 pandemic context, hitherto characterized by over 110 million cases and almost 2,500,000 deaths on a global scale, several domestic and wild animal species have been reported as susceptible to natural and/or experimental SARS-CoV-2 infection. In this respect, while some marine mammal species are deemed as potentially susceptible to SARS-CoV-2 infection on the basis of the sequence homology of their ACE-2 viral receptor with the human one, this study addresses such a critical issue also in stranded sea mammal specimens.

pathology↗