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.