In general, the conservation status of the Mediterranean cetaceans is under pressure from anthropogenic disturbance and the populations of the species highlighted in the study are greatly impacted by several sources of human activities. Incidental mortality in fishing operations, collisions with ship, ingestion of debris, seismic surveys, naval exercises, chemical pollution, and viral infections are just some of the threats faced by the cetaceans inhabiting the Mediterranean Sea Basin (Fossi et al., 2005; Cozar et al., 2015; Panigada et al., 2006 Di Guardo & Mazzariol, 2013). Most of the aforementioned factors can have a direct effect on the individuals, while indirect effects on the medium long-term period are represented by a general habitat degradation.
Marine mammals may be exposed to environmental stressors such as chemical pollutants, harmful algal biotoxins and emerging or resurging pathogens; this phenomenon may be related to complex factors such as climate change, toxins, and immunosuppression, with coastal marine mammals particularly at risk since many inhabit an environment more affected by human activity (Bossart, 2006). Besides threats of anthropic origin menacing their conservation, infectious diseases represent a global issues, in particular morbilliviruses (Van Bressem et al., 2014; Duignan et al. 2014). These RNA viruses, currently endemic in the Mediterranean, showed in the recent years an apparently increased tendency to cross species infection causing epidemic events in different species through spill-over events. Phylogenetic analyses support the idea of a common ancestor of morbillivirus affecting aquatic animals, namely cetacean morbillivirus (CeMV) with those reported in terrestrial ones and recent reports of this virus in more terrestrial animals (i.e. otters and seals) suggest a travel back to land of this virus (Di Guardo and Mazzariol 2019)
Impact of human on wildlife during a global pandemic may include the potential transmission of novel virus to susceptible animals (Mathavarajah et al. 2020). In our study we identified species of marine mammals living along italian coastline and we evaluete the conservation of the SARS-CoV-2 viral ACE-2 receptor across species.
It has been shown how ACE-2 variability explains why certain species are susceptible to SARS-CoV-2, while others are not (Mathavarajah and Dellaire, 2020); ACE-2, an extracellular peptidase originally characterized as SARS-CoV receptor, has been subsequently identified as the main SARS-CoV-2 receptor. The S protein binding region is located in the ACE-2 catalytic site (Li et al, 2005), with some amino acid residues at a particular position in the human ACE-2 sequence playing a crucial role in virus-host cell interaction (Li et al, 2020, Wan et al, 2020); these binding residues determine the degree of susceptibility, thus likely representing the main drivers for cross-species transmission (Wan et al, 2020).
Our analyses revealed that a group of closely related cetaceans (O orca, G melas, T truncatus and S coeruleoalba) is predicted to be highly susceptible to the virus, hence at potential risk of acquiring SARS-CoV-2 infection whenever exposed to the viral pathogen. One of the main aim of this study was to evaluate ACE-2 abundance and distribution, by means of IHC, in cetacean lungs, thereby supporting our parallel and comparative investigations on ACE-2 amino acid sequences, aimed at assessing the susceptibility of marine mammals to SARS-CoV-2 infection; in this respect, IHC analyses could identify a number of potential routes of infection for SARS-CoV-2, along with the viral spread and replication sites throughout the body. Given the IR patterns found, the expression of ACE-2 in macrophages and their role in antiviral defense mechanisms (as in the case of SARS-CoV-2 infection) should be emphasized; Abassi et al. have hypothesized that, while lung macrophages play an important role in antiviral defense mechanisms, they could also serve as a “Trojan horse” for SARS-CoV-2, thus enabling viral anchoring within the pulmonary parenchyma. A variable expression of ACE-2 on macrophages among individuals might also govern the severity of SARS-CoV-2 infection (Abassi et al, 2020), although additional studies are required. Our findings suggest that ACE-2 expression can vary between different lung regions and between individuals (in particular, if they belong to different species).
Reports of pathogens of terrestrial origins in marine mammals has already been observed, as the case of Toxoplasma gondii, Salmonella typhimurium, Listeria monocytogenes and often an exposure to untreated wastewater was deemed to be the possible source (Grattarola et al, 2019, Grattarola et al, 2016).
As other fecal pathogens, SARS-CoV-2 is through the sewage system, thereby gaining access into wastewater treatment plants, whenever existing, and/or, more in general, the aquatic environment.
In this respect, Italy is characterized by heterogeneous geographic systems, with sea surface waters surrounding the Italian peninsula. Spaces and distances granted to the hydrographic network by the mountains and by the sea are mostly very modest, making the territory particularly exposed and vulnerable to alluvial events, known as sudden floods or flash floods, often triggered by short and intense weather phenomena (https://www.isprambiente.gov.it/files2018/pubblicazioni/rapporti/rapporto-dissesto idrogeologico /Rapporto_Dissesto_Idrogeologico_ISPRA_287_2018_Web.pdf.). Compared to the unpredictability of flood events, there is still a sort of repetition in the occurrence of the events themselves, and some portions of our National territory, due to the morphological characteristics and use of soil, are configured as hydrological hazard-prone areas, including coastal areas.
The present study identified some high-risk areas where insufficient wastewater treatment may occur in the vicinity of marine mammals, putting them at risk for infection by a fecally transmitted zoonotic pathogen like SARS-CoV-2 when they swim and feed. It is clear that several data and information are still missing as the survival time into the marine environment and the effects of marine currents and dilution factors which can lower the possibility of infection. Furthermore it should be noted that SARS-CoV-2 has been found in wastewater, there is no data on its actual possibility of survival and dispersion in seawater.
Analyzing wastewater management practices in Italy, we identified that some wastewater treatment plants in the vicinity of marine mammals utilizing tertiary treatment, which rules out the possibility of virus exposure in these areas. However there were locations (the coastline of the Ligurian Sea and of the Central and South-East Thyrrenian Sea) that bordered marine mammals population at risk for occasionally over-flooding of sewage treatment plants and release of wastewater not fully treated. The number of areas identified at risk could be underestimated, as we considered areas characterized by inadequate treatments, while in some cases other coexisting conditions, like problematic sewage overflow or pipe exfiltration, represent a risk. Overall, considering all these factors we identified some high -risk areas for a potential virus spillover (Fig. 3), which correspond to North Adriatic Sea, Ligurian Sea, Central Adriatic Sea, North and South Ionian Sea, Central Tyrrhenian Sea, South-East Thyrrenian Sea, Sicilian Channel.
The impact of a possible viral spillover could have on coastal marine mammal communities remains to be determined. Our study, in fact, confirm a high susceptibility of sea mammals to SARS-CoV-2 infection based on the receptor homology, as reported in previous studies (Mathavarajah et al.,2020). The effects of SARS-CoV-2 on marine mammals are currently unknown, although coronavirus infections have been reported in marine mammals prior to the CoViD-19 pandemic (De Caro et al., 2020,). Infections with other coronaviruses are indeed recognized as a cause of liver and lung disease (Mihindukulasuriya et al., 2008), while gammacoronaviruses were retrieved from fecal samples of three Indo-Pacific bottlenose dolphins (Tursiops aduncus) (Woo et al., 2014) (Zappulli et al., 2020).
Since many cetacean species are social, such as Tursiops truncatus (bottlenose dolphin) and Stenella coeruleoalba (striped dolphin), their high susceptibility to SARS-CoV-2 suggests that their populations could be especially vulnerable to intra-species transmission of this novel coronavirus. Among the species with a high susceptibility to SARS-CoV-2 infection, the common bottlenose dolphin could result the most impacted cetacean, given its distribution along the continental shelf and along the entire Italian coastline. Moreover, the behaviour and the size of the pods of the species could play an important role in the spread of the virus among the specimens in the pod; both striped and common bottlenose dolphins are gregarious specie and this ecological feature may facilitate the SARS-CoV-2 spread among the animals through their close interactions. Along all the Italian coasts, the most common species is T. truncatus (Reeves & Notarbartolo, 2006); the species is the only regular for the Northern Adriatic Sea (Bearzi et al., 2004) and then distributed in the Ligurian Sea, the whole Tyrrhenian Sea (Lauriano et al., 2015; Gnone et al., 2011), the Sicily Strait, and the Ionian Sea. Since T. truncatus is an “inshore” species, the risk of acquiring SARS-CoV-2 infection, which like many others is characterized by a “land-to-sea” transmission eco-epidemiological pathway, appears to be greater than for “offshore” species. This would candidate bottlenose dolphins among the most reliable “sentinels” for an “early” detection of SARS-CoV-2 infection in marine mammals.
S. coeruleoalba, the most abundant species in the Mediterranean Sea (Aguilar, 2000), displays an offshore distribution (Notarbartolo di Sciara, 2016) and is then regular in the Southern Adriatic Sea, Ionian Sea, including the Sicily Strait, all the Tyrrhenian Sea, and the Ligurian Sea along with B. physalus (Panigada et al., 2017). The fin whale distribution ranges from north to south feeding grounds in summer and winter, respectively, as it has been described by satellite telemetry (Panigada et al., 2017) and boat-based observations (Canese et al., 2006).
Even if a decline in the population of D. delphis, has been reported for the Mediterranean Sea (Bearzi et al., 2003), the species is still regularly observed in the Tyrrhenian Sea.
Infection susceptibility can hinder the conservation status of the species; indeed, the fin whale is listed as Vulnerable in the IUCN (Mediterranean and Italian range), since a population abundance decline has been inferred in the last years. The decline is a major concern for the Mediterranean sub population (Panigada et al., 2017); hence epidemic disease, as already documented for morbilliviral infection (Mazzariol et al., 2016), would represent a plague. A worst situation would be expected for the Critically Endangered killer whale, given the extremely low number of individuals left in the subpopulation confined in the Gibraltar Strait (Esteban and Foote, 2019). Even if the analysis for the sperm whale revealed a medium susceptibility, the low number of individuals within the population and the Endangered IUCN Mediterranean and Italian status, foresee a high-risk degree for such species.
By highlighting the vulnerability of marine mammals to SARS-CoV-2 infection, the scientific community hopes to shape policy decisions regarding wastewater management around the world, in order to help protect at-risk marine mammal species that may be exposed to this coronavirus.
Regarding Italian situation, additional measures of treating the wastewater in those areas where they are insufficient or inadequate (Ligurian Sea, Central Adriatic Sea, South-Eastern Tyrrhenian Sea, Northern and Southern Ionian Sea and Sicilian Channel) would help protect the species and reduce the probability of them being exposed to SARS-CoV-2.
In summary, there is an urgent need to increase SARS-CoV-2 infection’s surveillance in free-ranging cetaceans and also to screen stranded specimens for such infection, particularly in cases of mass stranding or unusual behaviour.
Further studies are needed in cetaceans to provide a more in-depth insight into SARS-CoV-2 susceptibility, also in relation to the escalating levels of anthropogenic stressors to which they are exposed.
Considering the economic costs of the present pandemic, greater efforts to improve wastewater treatment, specifically with the removal or inactivation of viral contaminants on a global scale, should be regarded as a priority.
This is made even more urgent by the new SARS-CoV-2 variants which have emerged in the United Kingdom as well as in Brazil and South Africa, with the S protein’s mutations carried by them leading to greater viral transmission/transmissibility rates and, possibly, also to increased pathogenicity.
These variants, which are rapidly spreading around the world, have also been isolated and sequenced on the Italian territory; in this respect, viral genome sequencing from wastewater can detect new SARS-CoV-2 variants before they are detected in infected patients. Consequently, analyzing sewage and run-off waters can help detect and track the spread of new SARS-CoV-2 variants posing a risk to human health and, potentially, also to the health and conservation status of wild animal species and populations, including sea mammals.