4. Discussion
Results of phylogenetic analysis of both mitochondrial (mtCOI ) and nuclear (EF1α ) genes show that genomes of haemolymph and host tissue of neoplastic clams are different. Moreover, mtCOI andEF1α alleles found in haemolymph DNA of individuals diagnosed with DN are common between all of them, suggesting the existence of an independent cell line shared between these cancerous clams. Such results indicate the existence of at least one horizontally transmitted cancer lineage in the L. balthica population from the Baltic Sea, in accordance with original studies documenting first evidence of BTN in bivalves (Metzger et al. 2015, Metzger et al. 2016) that also formulated the basis of molecular identification of BTN. All tested neoplastic clams were discovered to share a common allele in both loci investigated, but one of the individuals was also identified with a distinct EF1α allele in the haemolymph genotype that was not present in host genome. This most likely indicates the presence of somatic mutation in the NCs and the loss of the second allele. Yet, at this stage the possible occurrence of another cancer lineage in the tested population cannot be excluded either. Genome variation between different types of tissue within the same organism can occur, especially in tissues characterized by elevated mitotic activity, such as hepatopancreas or gills, due to a potential accumulation of mutations resulting in somatic mosaicism (O’Huallachain 2012), but the chance of different individuals developing the same consistent alleles within multiple conserved genes is very low. Variations in coding sequences can be observed in cases of cancer development in higher organisms (O’Huallachain 2012, GTEx Consortium 2017). Genomic rearrangement within single individuals has not been described yet in invertebrates, although some genetic variability is reasonably expected in proliferative disorders such as DN, due to poly- and aneuploidy of cancerous cells and their high mutative potential (Diaz et al. 2010, Ruiz et al. 2013). However, taking into the account the nearly perfect similarity of alleles occurring in haemolymph of neoplastic L. balthicaindividuals we conclude that the recognized lineage originated from a single organism and are now transmitted within the gulf population of this species.
Phylogenetics also revealed natural polymorphism occurring within the population, as alleles found in both mtCOI and EF1α loci of healthy clams differ between individuals. L. balthica is generally characterized as a species of high genetic variability among all of its populations worldwide, mostly associated with its local adaptations for environmental conditions (Yurchenko et al. 2018). Such variability is also observed in populations inhabiting the Gulf of Gdańsk, where distinct genetic structures are observed between shallow and deep sites (Becquet et al. 2013, Lasota et al. 2018) as populations inhabiting deeper areas are partially isolated by straitened water mixing, sea currents, and seasonally by thermocline and halocline (Kowalewski 1997, Kruk-Dowgiałło & Szaniawska 2008). A possible bottleneck effect that some Baltic L. balthica populations may have undergone (Belov 2011) can affect their susceptibility for evolving transmissible cancer lineages. These results suggest that BTN affecting the Baltic clam may also be present in other clam populations on a worldwide scale due to an extensive geographical range of the species and its evolutionary history (Väinölä 2003, Pante et al. 2012). DN has been already diagnosed in various L. balthica populations, e.g. from Finnish coast of Baltic Sea (Pekkarinen 1993), Wadden Sea (Dairain et al. 2020), and Chesapeake Bay (Christensen et al. 1974), although this paper is the first confirming clonal aetiology of this disease.
The L. balthica population chosen for our study comes from a site (H45) that is considered a relatively deep (45 m) sampling area in the gulf and is characterized by historically highest DN frequency in this species, ranging from 25 to 94%, depending on sampling year (Thriot-Quiévreux & Wołowicz 2001, Smolarz et al. 2005bd, Ogrodowczyk 2017). This population has been under investigation for many years, not only in terms of DN occurrence, but also because of adverse environmental conditions that occur in the site area, such as oxygen depletion and/or presence of toxic hydrogen sulphide, either temporal, seasonal or constant in some years, as well as anthropogenic pollution consisting of, among others, heavy metals, aromatic polycyclic hydrocarbons (WWAs), or polychlorinated biphenyls (PCBs), staying at a relevant level in water and/or sediments throughout the years (Renner et al. 1998; Pazdro et al. 2004, Kot-Wasik et al. 2004, Zaborska et al. 2019). Such challenging environmental characteristics, along with ecological, individual and genetic variations (e.g. trophic position, sex, fitness, genetic structure) were previously proposed to be carcinogenic factors in DN induction in the Baltic Sea (Wołowicz et al. 2005). Although our study confirms the transmissible character of DN inL. balthica , the role of potential pollution and other environmental factors on cancer development and susceptibility should not be overlooked. Chronic and acute exposure to various pollutants, temporal anoxic conditions, immunosuppression and co-occurring oxidative stress may potentially increase vulnerability of bivalves to contagious cancer cells (Metzger & Goff 2016) and/or induce the expression of genome-integrated retrovirus elements, as some studies also suggest the role of transmissible retroelements in BTN induction (Arriagada et al. 2014).
NCs isolated from L. balthica are described as highly aneuploid with high disseminating potential, enormous and pleomorphic nuclei, and low amount of cytoplasm (Thriot-Quiévreux & Wołowicz 1996, Thriot-Quiévreux & Wołowicz 2001, Smolarz et al. 2005ac; Smolarz et al. 2006a). These features of NCs are similar in all bivalve species (Barber et al. 2004, Carballal et al. 2015) suggesting some universal characteristics of DN within Bivalvia. Most probably, the cellular mechanism by which NCs are able to be transplanted between individuals evading immunological signalling is also common between taxa. Due to the absence of adaptive immunity in bivalves, biochemically changed NCs do not provoke effective pathogen-directed defence systems in these animals and those cells are able to clone themselves and disseminate into the tissues of other hosts (Metzger & Goff 2016, Ujvari et al. 2016). The mechanism of NCs transmission is not fully described yet, but it is believed that single clonal cancerous cells that originate in one neoplastic individual are expelled from its body in, either by direct release of DNA from heavily neoplastic animals (Giersch et al. 2021) or possibly through spawning or death events, and are then transmitted to other individual(s) via seawater uptake. This hypothesis is supported by studies showing successful inoculation of NCs and/or haemolymph from neoplastic to healthy bivalve through injection which resulted in further DN development and also transmission of NCs through cohabitation in different bivalve species (thoroughly discussed in Carballal et al. 2015 and Metzger et al. 2015). The studies of viability of NCs fromM. arenaria shows that these cells are able to survive in the water column for several hours (Sunila & Farley 1989) or even up to 8 weeks in lower temperatures (Giersch et al. 2021). The success of implantation to another organism is determined by the water circulation and density of animals (Elston et al. 1990) and most probably with the filtrating potential of species. It was also reported that NCs from theMytilus BTN cell line (MtrBTN2) can survive outside of host’s organism up to 6 days (Burioli et al. 2021) giving plenty of time for potential transmission via water filtration. To date, there is no published paper considering L. balthica NCs viability, but in our routine lab work we observe that those cells can survive at least two hours when kept in an isotonic solution (Czajkowska 2021). Ecological consequences of DN/BTN can be extremely severe as it was documented that some populations affected with this cancer experience increased mortality, even mass mortality in some cases (Farley et al. 1986, Farley et al. 1991, Muttray et al. 2012, Benabdelmouna & Ledu 2016) with critical impact on surrounding ecosystems. Such events are also observed in L. balthica from Gulf of Gdańsk, especially from the H45 population that is investigated here, which is affected by mass mortality occurring in bi- or triennial periods lead by an increase in DN prevalence (Sokołowski et al. 2004, Wołowicz et al. 2005).
As stated previously, disseminated neoplasia has also been diagnosed in three other bivalve species from the Gulf of Gdańsk, Poland, namelyM. arenaria , C. glaucum , and M. trossulus , although at a much lower prevalence (Smolarz et al. 2005d; Smolarz et al. 2006b; Ogrodowczyk 2017). Further studies are needed to determine if DN in these species is also a BTN and if cancer contagiousness is related to intra- or interspecies transmission.
Our study presents the first evidence of transmissible aetiology of this cancer in L. balthica , and provides a base for further investigation of the severity of this transmissible cancer in otherL. balthica populations as well as in other Baltic species affected with DN. Adding L. balthica to the list of BTN-affected species resulting in overall eight BTN-affected species and nine cancer lineages, making the transmissible cancer even more common in the biological world than it was thought earlier. The widespread occurrence of BTN in multiple genera is an interesting phenomenon in cancer biology, and this phenomenon has potential to be a model disease (Aguilera 2017, Fernández Robledo et al. 2019) for in-depth understanding of leukemic diseases in other organisms, including humans.