Abstract
Disseminated neoplasia (DN) is one of the most challenging and
unrecognised diseases occurring in aquatic fauna. It has been diagnosed
in four bivalve species from the Gulf of Gdańsk (Southern Baltic Sea)
with the highest frequency in Limecola balthica (formerlyMacoma balthica) , reaching up to 94% in some populations. The
aetiology of DN in the Baltic Sea has not yet been identified, with
earlier studies trying to link its occurrence with environmental
pollution. Taking into account recent research providing evidence that
DN is horizontally transmitted as clonal cells between individuals in
some bivalve species, we aimed to test whether DN is a transmissible
cancer in the population of L. balthica from the Gulf of Gdańsk
highly affected with cancer. We examined mitochondrial cytochrome c
oxidase I (mtCOI ) and elongation factor 1α (EF1α )
sequences of genomes obtained from haemolymph and tissues of neoplastic
and healthy individuals. Sequence analysis resulted in detection of an
independent transmissible cancer lineage occurring in 4 neoplastic clams
that is not present in healthy animals. This paper describes the first
case of transmissible DN in the clam L. balthica providing
further insights for studies on this disease.
Introduction
Disseminated neoplasia (DN), also known as clam leukaemia, disseminated
sarcoma, and haemic/systematic/haematopoietic neoplasia, is a cancerous
disease observed in over 30 species of bivalves worldwide (Wołowicz et
al. 2005, Carballal et al. 2015). The disease can act as a dangerous
epizootic event leading to mass mortality in some populations (Farley et
al. 1991, Muttray et al. 2012, Benabdelmouna & Ledu 2016) affecting
commercially used species and leading to ecosystem unbalance (Barber et
al. 2004). DN is a proliferative disorder of circulating cells
predominantly found in the haemolymph. Neoplastic cells (NCs) are
morphologically and physiologically distinct from normal haemolymph
cells, which are gradually replaced with NCs during cancer progression.
NCs, most likely of haemocyte origin, are characterized by high
aneuploidy level, higher nucleus-cytoplasm volume ratio and increased
proliferative activity resulting in nuclear and cellular hypertrophy
(Thriot-Quiévreux & Wołowicz 1996, Smolarz et al. 2005ac, Smolarz et
al. 2006a, Carella et al. 2013, Carella et al. 2017). Yet, alteration
level in specific physiological and biochemical parameters of NCs differ
between species (Barber et al. 2004, Le Grand et al. 2014, Carballal et
al. 2015, Odintsova 2020). During cancer progression NCs are able to
disseminate into tissues of an organism, leading to histological
modifications, tissue/organ malfunction and eventually to death of an
animal (Barber 2004, Carella et al. 2015, Carballal et al. 2015).
DN has been diagnosed in four bivalve species in the Gulf of Gdańsk
(Southern Baltic Sea): Limecola (Macoma ) balthica ,Mya arenaria , Mytilus trossulus and Cerastoderma
glaucum (Thiriot-Quiévreux & Wołowicz 1996, Wołowicz et al. 2005,
Smolarz et al. 2005bd, Smolarz et al. 2006b, Ogrodowczyk 2017). The
frequency of cancer differs between species, population of a species,
sampling sites, and sampling year, with the highest prevalence inL. balthica (the Baltic clam) reaching as high as 94% in
populations inhabiting deeper (30-85 m) parts of the gulf
(Thiriot-Quiévreux & Wołowicz 1996, Thiriot-Quiévreux & Wołowicz
2001). L. balthica is one the most widespread bivalve species
found in the Baltic Sea area, but it is also commonly found in the
temperate and arctic coastal waters of the North Atlantic and North
Pacific (Budd & Rayment 2001). The Baltic clam plays a key role in
various ecosystems, but due to the species’ adaptive potential it is of
particular importance in ecosystems characterized by changeable and
unfavourable conditions such as low salinity, contamination and/or
temporal/seasonal oxygen depletion, all found in the Baltic Sea
(Segerstråle 1957, Janas et al. 2004, Janas et al. 2007). Earlier
studies linked DN incidence with adverse environmental conditions and/or
anthropogenic pollution as contaminants such as heavy metals, polycyclic
hydrocarbons (WWA) and persistent organic pollutants (POPs) were
previously proposed as ultimate carcinogenic factors in DN induction
(reviewed in: Barber 2004, Carballal et al. 2015). However, those
studies do not indicate any clear correlation between any specific
environmental factor and cancer occurrence (Smolarz et al. 2005,
Wołowicz et al. 2005, Smolarz et al. 2006) suggesting that DN in these
clams may have a different cause.
In recent years, it was discovered that some types of cancer in animals
can be explained by non-viral infectious aetiology, with cancerous cells
being transmitted between individuals as an allograph that further leads
to cancer development. Transmissible cancer based on clonal malignant
cell transmission is generally considered to be a rare phenomenon, until
now, observed only in exceptional and distinctive cases. To date there
are three recognized types of cancer with transmissible aetiology:
canine transmissible venereal tumour (CTVT) (Murgia et al. 2006, Rebbeck
et al. 2009), devil facial tumour disease (DFTD) (Pearse et al. 2006),
and bivalve transmissible neoplasia (BTN). BTN is a transmissible form
of DN, and BTN lineages have been found in seven bivalve species:
soft-shell clam M. arenaria from Atlantic coast of North America
(Metzger et al. 2015); Cerastoderma edule and Polititapes
aureus from Galician coast of Spain (Metzger et al. 2016); and four
species of Mytilus mussels from around the world, M.
trossulus , M. edulis , M. chilensis and M.
galloprovincialis (Yonemitsu et al. 2019, Skazina et al. 2021, Hammel
et al. 2021). DN transmission in the above-mentioned bivalve species was
confirmed using polymorphic microsatellite alleles of nuclear DNA and
sequence analysis of mitochondrial and nuclear genes. These analyses
found that the genomes of NCs were different from the genomes of the
hosts, yet nearly identical to each other, indicating infectious nature
of the disease.
The Gulf of Gdańsk, located in the southern part of the Baltic Sea, with
its specific geomorphological structure, low salinity and low diversity
constitutes an excellent biological model to study horizontal transfer
of cancer cells. One of the four bivalve species occurring here,L. balthica , due to its widespread occurrence and high cancer
prevalence, can be considered as a valuable model organism in DN and BTN
research. Taking into account the unsolved basis of this commonly
diagnosed cancer in L. balthica and the findings that DN in many
species have been found transmissible (Metzger & Goff 2016), in this
study we aim to use genomic analysis to test whether DN in L.
balthica inhabiting the southern Baltic Sea (Gulf of Gdańsk) is a BTN.
This will increase our knowledge of transmissible cancer and the ways in
which cancer can affect animals in the environment.