ArticlePDF Available

The role of invasive tunicates as reservoirs of molluscan pathogens


Abstract and Figures

Ascidian tunicates frequently display rapid expansion when introduced beyond their native range and are considered successful invaders. This invasive potential may be exacerbated by a warming climate, allowing for the occupation of environmental niches previously held by native species. Research into tunicate invasion ecology is prevalent, but less is known about their role in pathogen maintenance. This study investigated the impact of invasive tunicates on the maintenance of pathogens that affect commercial bivalves, including the cultured species Ostrea edulis (European flat oyster) and Crassostrea gigas (Pacific oyster), and the fished species Cerastoderma edule (Common cockle). Focal pathogens included ostreid herpesvirus OsHV-1 μVar, Vibrio aestuarianus, Bonamia ostreae and Minchinia spp. The range of pathogens in their molluscan hosts was determined and the tunicates Botrylloides violaceus, Didemnum vexillum and Styela clava were then screened for these same pathogens, using both field samples from oyster culture sites and marinas and a series of laboratory cohabitation trials. Sample sites reflected areas close to and further away from known pathogen sources. PCR, Sanger sequencing and histology confirmed the presence of B. ostreae and Minchinia mercenariae-like in S. clava, and V. aestuarianus was confirmed by qPCR in B. violaceus and D. vexillum. Furthermore, histology confirmed Minchinia mercenariae-like sporonts in S. clava suggesting that the tunicate can facilitate replication of this species. S. clava also maintained B. ostreae in tanks with no oysters present. The results indicate that tunicates can act as reservoirs of infection in areas where disease occurs and potentially transport diseases to uninfected sites.
Content may be subject to copyright.
The role of invasive tunicates as reservoirs of molluscan
Katie E. Costello .Sharon A. Lynch .Rob McAllen .Ruth M. O’Riordan .
Sarah C. Culloty
Received: 4 March 2020 / Accepted: 15 October 2020
ÓSpringer Nature Switzerland AG 2020, corrected publication 2020
Abstract Ascidian tunicates frequently display
rapid expansion when introduced beyond their native
range and are considered successful invaders. This
invasive potential may be exacerbated by a warming
climate, allowing for the occupation of environmental
niches previously held by native species. Research
into tunicate invasion ecology is prevalent, but less is
known about their role in pathogen maintenance. This
study investigated the impact of invasive tunicates on
the maintenance of pathogens that affect commercial
bivalves, including the cultured species Ostrea edulis
(European flat oyster) and Crassostrea gigas (Pacific
oyster), and the fished species Cerastoderma edule
(Common cockle). Focal pathogens included ostreid
herpesvirus OsHV-1 lVar, Vibrio aestuarianus,Bon-
amia ostreae and Minchinia spp. The range of
pathogens in their molluscan hosts was determined
and the tunicates Botrylloides violaceus,Didemnum
vexillum and Styela clava were then screened for these
same pathogens, using both field samples from oyster
culture sites and marinas and a series of laboratory
cohabitation trials. Sample sites reflected areas close
to and further away from known pathogen sources.
PCR, Sanger sequencing and histology confirmed the
presence of B. ostreae and Minchinia mercenariae-
like in S. clava, and V. aestuarianus was confirmed by
qPCR in B. violaceus and D. vexillum. Furthermore,
histology confirmed Minchinia mercenariae-like
sporonts in S. clava suggesting that the tunicate can
facilitate replication of this species. S. clava also
maintained B. ostreae in tanks with no oysters present.
The results indicate that tunicates can act as reservoirs
of infection in areas where disease occurs and
potentially transport diseases to uninfected sites.
Keywords Invasive Tunicates Oysters
Aquaculture Pathogens
The Subphylum Tunicata is a diverse group of
invertebrates belonging to the Phylum Chordata, and
members of this group are globally recognised as
significant contributors to biofouling communities,
particularly species within the Class Ascidiacea; the
sea squirts (Rosa et al. 2013; Comeau et al. 2015; Zhan
K. E. Costello S. A. Lynch R. McAllen
R. M. O’Riordan S. C. Culloty
School of Biological, Earth and Environmental Sciences,
University College Cork, Cork, Ireland
K. E. Costello (&)S. A. Lynch
R. M. O’Riordan S. C. Culloty
Aquaculture and Fisheries Development Centre,
Environmental Research Institute, University College
Cork, Cork, Ireland
S. C. Culloty
MaREI Centre, Environmental Research Institute,
University College Cork, Cork, Ireland
Biol Invasions,-volV)(0123456789().,-volV)
et al. 2015). A number of tunicate species, both
colonial and solitary, have become high profile
invasive species worldwide due to their significant
impacts on the aquaculture sector, including costs
associated with their control and removal when
fouling occurs, and their ability to compete with
commercial shellfish for food resources (Hillock and
Costello 2013; Casso et al. 2018).
Botrylloides violaceus (orange sheath tunicate) is a
colonial tunicate native to the northwest Pacific but
now established in temperate regions around the globe
(Simkanin et al. 2013) and known for its invasive
tendencies. This species can become competitively
dominant over commercial species, thus impeding
settlement (Cordell et al. 2012) and fouling cultured
stock (Paetzold et al. 2012). Didemnum vexillum
(carpet sea squirt) is another high-profile colonial
tunicate that has steadily increased its global range
from Japan (thought to be its native range) to New
Zealand, North America and Europe. Similar to B.
violaceus,D. vexillum can impact commercial aqua-
culture practices, for example heavy growth results in
shellfish valve openings or siphons becoming
obstructed (Ferguson et al. 2017).
Styela clava (leathery sea squirt/club tunicate) is a
solitary ascidian native to the Northwest Pacific and its
high tolerance of fluctuating environmental conditions
has seen it become a successful invader, expanding its
range from the Pacific coasts of Asia and Russia to
extend throughout the northern and southern hemi-
spheres (Goldstien et al. 2010). This tunicate attaches
to hard substrates such as rocks and bivalve shells, and
when natural substrates are unavailable it acts as a
biofouling organism on artificial structures and aqua-
culture gear. Mature adults are hermaphroditic and
oviparous, and densities can reach 500–1000 individ-
(Wong et al. 2011), as such the cleaning of
culture gear has been known to make shellfish
production more labour intensive (Bourque et al.
Interactions between invasive species, pathogens
and native communities are complex, as invaders that
have not lost their associated parasites along the
invasion pathway may transmit novel parasites to
native hosts (Dunn and Hatcher 2015) or become
infected by native pathogens in the invaded range.
Furthermore, invasive species may also inhibit or
promote the transmission of pathogens by native
species (Goedknegt et al. 2016).
While many studies have focused on tunicates as
model organisms for understanding invasion success,
less is known about their role in the maintenance of
parasites and pathogens and their subsequent interac-
tions with commercial bivalves. Franchi and Ballarin
(2017) noted that tunicate physiology results in natural
defences against parasites and pathogens, as the tunic
acts as a physical barrier and the vascularised oral
siphon and pharynx have circulating haemocytes that
trigger immune responses that can lead to inflamma-
tion and phagocytosis of foreign material. Despite
these defences, tunicates have been shown to be
susceptible to specific pathogens, for example Styela
clava has been established as a potential carrier of the
flagellated protozoan Azumiobodo hoyamushi—the
causative agent of Soft Tunic Syndrome (Kumagai
et al. 2014). This disease widely affects cultured
tunicates such as the edible ascidian Halocynthia
roretzi and has caused economic losses in the aqua-
culture sector in Korea and Japan.
Rosa et al. (2013) tested the ability of biofouling
ascidians including Styela clava and Botrylloides
violaceus to distribute potentially harmful algal cells
to new geographic ranges by exposing them to
cultured strains of algae capable of forming harmful
algal blooms. Algal cells were found to pass through
the ascidian digestive system and remain capable of
re-establishing bloom populations. Rueckert et al.
(2015) also detected a number of apicomplexan
parasites within the genus Lankesteria infecting
Pacific ascidians and Lynch et al. (2016) found four
parasite groups including ciliates and trematodes in
the European sea squirt Ascidiella aspersa.
Determining the ability of bacteria, protists and
viruses to maintain themselves in the environment can
inform how they contribute to disease cycling, partic-
ularly in commercially important bivalves. However,
this can prove difficult due to free-living life stages,
the potential ability to persist short-term outside a host
and the use of alternative hosts. Haplosporidia
belonging to the genus Minchinia have traditionally
been detected in a range of bivalves including clams,
cockles and mussels (Ramilo et al. 2018). Other
haplosporidian species, including Bonamia ostreae
and Haplosporidium nelsoni, are well studied within
their hosts but knowledge of their life-history, diver-
sity and mechanisms of transmission is limited (Ward
et al. 2019). B. ostreae is an intra-haemocytic parasite
of the European flat oyster Ostrea edulis that induces
K. E. Costello et al.
physiological disorder and mortality. All oyster life-
stages are susceptible, although individuals over two
years are more adversely affected, and the pathogen is
present throughout the year but infections appear to
increase from Autumn and peak in late winter/early
Spring. Laboratory experiments suggest that an inter-
mediate host is not required, and the pathogen is
capable of surviving in seawater for up to one week
(OIE 2019). It is also known that B. ostreae is capable
of utilising alternative hosts, for example in a labora-
tory setting the brittle star Ophiothrix fragilis caused
transmission of B. ostreae to naive O. edulis (Lynch
et al. 2007).
The bacterium Vibrio aestuarianus is associated
with mortality events in populations of the Pacific
cupped oyster Crassostrea gigas in Europe, with adult
oysters of marketable size primarily affected and
mortalities mainly occurring in Summer (Lupo et al.
2019). The ostreid herpesvirus OsHV-1 lVar is
another pathogen associated with C. gigas mortality
events, with larval, spat and juvenile life-stages the
most susceptible. Transmission is primarily direct
although the virus is also hypothesised to travel via
vector particles in the water column. Mortalities occur
more frequently in summer water temperatures
although temperature effects differ between geo-
graphic locations, for example Europe and Australia
(OIE 2019). The virus has also been detected in other
phyla, for example the shore crab Carcinus maenas
can transmit OsHV-1 lVar from crabs exposed to the
virus in the wild to naive crabs in a laboratory setting
(Bookelaar et al. 2018).
Comprehensive information on the entire host
range for pathogens remains difficult to achieve, and
this can have implications for monitoring pro-
grammes, as these tend to focus on known susceptible
hosts and may therefore miss other potential hosts.
Accordingly, applied research of potential alternative
host species could provide more robust conclusions
about the host range of pathogens and also potential
carriers, reservoirs and vectors (Carnegie et al. 2016).
Climate change is predicted to increase introduc-
tions of problematic species, and affect the range,
abundance and impacts of invasive species (Beaury
et al. 2019). This study selected tunicates as a model
taxon that is heavily represented by species with
invasive tendencies, which may expand their range
under shifting climate conditions. Three species of
tunicate that are considered invasive outside of their
native range, Botrylloides violaceus,Didemnum vex-
illum and Styela clava, were screened for parasites and
pathogens that are known to affect commercial bivalve
species. These pathogens included the herpesvirus
OsHV-1 lVar, a virus that can cause mass mortalities
in C. gigas (Prado-Alvarez et al. 2016), and Vibrio
aestuarianus, a bacterium also linked to C. gigas
mortality events (McCleary and Henshilwood 2015).
Screening was also conducted for the Phylum
Haplosporidia, in particular Bonamia ostreae, the
intra-haemocytic parasite that affects O. edulis and is
the causative agent of bonamiosis (Lynch et al. 2010,
Prado-Alvarez et al. 2015), and Minchinia spp. as the
haplosporidia Minchinia tapetis and Minchinia merce-
nariae-like have also been detected in common
cockles (Cerastoderma edule) in Cork Harbour and
are known to cause losses for the cockle sector
´et al. 2020). Screening was also
conducted for a broader range of haplosporidian
species, as previous research conducted in similar
sample sites to this study has indicated the presence of
Haplosporidium nelsoni in C. gigas and O. edulis, and
a further Haplosporidium sp., most likely
Haplosporidium armoricanum,inO. edulis (Lynch
et al. 2013a).
The aim of this study was to focus on the potential
impact of invasive tunicates on the maintenance of
pathogens known to be problematic within the aqua-
culture and shellfishery sectors. This was investigated
by determining the current status of pathogens known
to cause significant mortalities in Ostrea edulis and
Crassostrea gigas sampled from aquaculture sites in
Ireland, and also by reviewing the pathogens currently
affecting the fished species Cerastoderma edule; then
establishing if these same pathogens are present in
invasive tunicates cohabiting with the oysters at the
aquaculture sites. The study also aimed to determine
whether pathogens can be detected in invasive tuni-
cates sampled from an area with no aquaculture but
potentially other sources of introduction such as heavy
recreational shipping traffic. The final aim was to
establish the potential role of tunicates as reservoirs of
infection using laboratory cohabitation trials.
The role of invasive tunicates as reservoirs of molluscan pathogens
Materials and methods
Study sites
Sampling for tunicates and oysters was conducted
under licence from Ireland’s National Parks and
Wildlife Service and the Marine Institute. Sampling
in 2018 was conducted in Summer (July) and late
Autumn/Winter (October-November) to ensure that
pathogen screening encompassed both cold winter and
elevated summer water temperatures. Sampling was
conducted in two locations within Cork Harbour and
sea temperatures were sourced from Copernicus
(Fig. 1and Table 1). The first sample site was an
oyster culture site in Rossmore, Co. Cork where both
the oysters Ostrea edulis (n = 60) and Crassostrea
gigas (n = 60) and tunicate Styela clava (n = 90) were
Sampling of the colonial tunicate Botrylloides
violaceus in 2018 was conducted in the second
location in Co. Cork, a marina in Crosshaven, to
ascertain whether growth in an area with heavy
recreational shipping would result in the presence of
pathogens due to incidental transport.
Summary of pathogen screening
The oysters, as natural hosts of the pathogens of
interest, were screened to determine presence/absence
of the pathogens for which they are the primary host
and prevalence if present, before screening the tuni-
cates to determine if proximity to the oysters resulted
in a positive detection. In cases where the number of
tissue samples is higher than the number of individuals
(Table 1) it is because multiple tissue types were
collected per individual (see Sample Processing—
2018 and 2019 Samples for a detailed description).
Ostrea edulis was screened using specific primers
for Bonamia ostreae and Haplosporidium nelsoni
(MSX). General haplosporidian primers were then
also used to screen O. edulis and ensure that any
further haplosporidian species (e.g. Haplosporidium
spp., Minchinia spp.) were detected if present. Cras-
sostrea gigas was screened for Vibrio aestuarianus
and OsHV-1 lVar, as these are both pathogenic to C.
gigas but not O. edulis.C. gigas were also screened
with general primers for haplosporidian spp., to ensure
that any pathogens within this phylum were detected.
Styela clava was then screened for B. ostreae, OsHV-1
lVar, general haplosporidian spp. and V. aestuari-
anus.Botrylloides violaceus was screened for V.
aestuarianus, OsHV-1 lVar and haplosporidian spp.
using general primers (Table 2) (see Molecular
Screening for a detailed description).
Laboratory trials: 2019
Samples for the subsequent laboratory cohabitation
trials in 2019 were obtained from both Co. Cork
(Rossmore & Rostellan), Dungarvan, Co. Waterford
and Carlingford, Co. Louth (Fig. 1and Table 3). All
sampling locations were oyster aquaculture sites and
tunicate species were collected from the trestles.
Trial 1: Cohabitation trial between Bonamia
ostreae infected Ostrea edulis and Styela clava
to determine if S. clava is a reservoir or carrier
of infection
150 Ostrea edulis were randomly sampled from
Rossmore, Cork Harbour and of these an initial
sample (n = 30) was screened to assess the prevalence
of Bonamia ostreae. The remaining oysters were
divided into three experimental 50 L tanks, each
Fig. 1 Map of sample sites for 2018 and 2019 samples
K. E. Costello et al.
Table 1 Summary of oyster and tunicate samples collected from Cork Harbour in 2018
Species Site Date collected Avg. monthly sea temperature (°C) Individuals n tissue samples
Cork Harbour
Crassostrea gigas Rossmore 11-07-2018 16.47 30 30
Crassostrea gigas Rossmore 2-11-2018 11.93 30 30
Ostrea edulis Rossmore 11-07-2018 16.47 30 30
Ostrea edulis Rossmore 23-11-2018 11.93 30 60
Styela clava (intertidal) Rossmore 4-07-2018 16.47 30 76
Styela clava (subtidal) Rossmore 11-07-2018 16.47 30 144
Styela clava (subtidal) Rossmore 2-11-2018 11.93 30 162
Crosshaven Marina
Botrylloides violaceus Crosshaven 16-07-2018 16.57 30
Botrylloides violaceus Crosshaven 25-10-2018 14.38 10
See Sample Processing—2018 and 2019 Samples for detailed explanation of column ‘n tissue samples’
Botrylloides violaceus is a colonial organism, therefore ‘individuals’ refers to the different lobes of three large colonies that
extended along the underside of marina pontoons
Table 2 Summary of the pathogens screened in each oyster and tunicate species in 2018
Species Crassostrea gigas
(n = 60)
Ostrea edulis
(n = 60)
Botrylloides violaceus
(n = 40
Styela clava
(n = 90)
Bonamia ostreae X4X4
Haplosporidian spp. 444 4
OsHV-1 lVar 4X44
Vibrio aestuarianus 4X44
Botrylloides violaceus is a colonial organism, therefore ‘individuals’ refers to the different lobes of three large colonies that extended
along the underside of marina pontoons
Table 3 Summary of samples for laboratory trials 2019
Species Site Date collected Avg. monthly sea temperature (°C) n individuals n tissue samples
Ostrea edulis Rossmore 5-04-2019 10.08 150 300
Styela clava (intertidal) Rostellan 18-06-2019 12.07 32 248
Crassostrea gigas Dungarvan 4-07-2019 15.37 150 150
Didemnum vexillum Carlingford 6-08-2019 14.72 28
See Sample Processing—2018 and 2019 Samples for detailed explanation of column ‘n tissue samples’
Didemnum vexillum is a colonial organism with an amorphous shape, therefore ‘individuals’ refers to different (and often
heterogeneous) colonies that extended along the underside of oyster trestles
The role of invasive tunicates as reservoirs of molluscan pathogens
containing 40 oysters, and held in a constant temper-
ature room at 14 °C, a salinity of 33–35 and a 12:12 h
light/dark regime.
Ostrea edulis were held for 75 days to allow the
intensity and prevalence of infection with Bonamia
ostreae to increase. After 75 days, 32 Styela clava of
all sizes were randomly sampled from Rostellan, Cork
Harbour. Tunicates growing on algae rather than
directly on the oyster trestles were collected, and a
piece of the algal substrate was taken too, as this
ensured the tunicates were not damaged in the
collection process and could survive the transfer to
the tanks. An initial sample of the tunicates (n = 8)
was screened for B. ostreae and the remaining 24
placed in three 50 L tanks, with eight tunicates per
tank; this resulted in one control tank with eight S.
clava only, and two experimental tanks each contain-
ing the B. ostreae-infected oysters and tunicates (8 S.
clava and 35 O. edulis 92 tanks). The final control
tank contained 35 O. edulis only. The trial ran for 60
As both Minchinia tapetis and Minchinia merce-
nariae-like have been recorded in Cerastoderma edule
from Cork Harbour (Albuixech-Martı
´et al. 2020), a
subsample of eight Styela clava was also screened for
both Minchinia tapetis and Minchinia mercenariae-
like at the end of the trial, using PCR with primers
specific to each species, histology as per Ford et al.
(2009) and Sanger sequencing. This subsample
encompassed two tunicates from the initial baseline
screening, three from the oyster cohabitation tanks and
three from the control tank with tunicates only.
Trial 2: Cohabitation trial between Vibrio
aestuarianus infected Crassostrea gigas
and Didemnum vexillum
150 Crassostrea gigas were randomly sampled from
Dungarvan, Waterford (selected because OsHV-1
lVar and Vibrio aestuarianus are both present at this
location (Bookelaar 2018)) and of these an initial
sample (n = 30) was screened to assess the prevalence
of V. aestuarianus. The remaining oysters were
divided into three experimental 50 L tanks, each
containing 40 oysters, and held in a constant temper-
ature room at 16 °C and a salinity of 33–35. After 34
days there were 42 oyster mortalities and a further 10
were removed and screened for V. aestuarianus. The
remaining oysters (n = 68) were subsequently divided
into two tanks, 34 per tank.
28 colonies of Didemnum vexillum/colonial spp.
were collected from Carlingford, Co. Louth. The
target species was D. vexillum as this is a prolific
global invader, and ID guides were used to identify
this species in the field, however given the morpho-
logical similarities between colonial tunicate species,
and the heterogeneous nature of colonies fouling the
oyster trestles, other species were also collected and
included in the trials. A tissue sample from all 28
colonies were sequenced at the end of the trial using
the primer pair F16–R497 (Price et al. 2005)to
confirm the community composition. An initial sam-
ple (n = 7 colonies) was removed to assess the
baseline prevalence of V. aestuarianus. The remaining
21 colonies were placed in three 50 L tanks with seven
colonies per tank; this resulted in one control tank with
seven D. vexillum/colonial tunicate spp. colonies only,
and two experimental tanks each containing the V.
aestuarianus-infected oysters and tunicates (7 D.
vexillum/colonial tunicate spp. colonies and 34 C.
gigas 92 tanks). The trial ran for 11 days.
In all laboratory experiments tanks were checked
twice daily and any moribund or dead oysters and
tunicates removed and processed. Tanks were con-
stantly aerated using airlines with airstones and
oysters and tunicates were fed standard marine
invertebrate food 3–4 times per week.
Sample processing—2018 and 2019 samples
Oyster species were processed by carefully opening
the shell without disrupting the tissue within and
draining the excess cavity fluid and seawater onto
clean tissue. In Ostrea edulis, heart and gill smears
were prepared as per Bonamia ostreae screening
(Flannery et al. 2014). Smears were screened for B.
ostreae using a Leica DM500 microscope at the 409
lens. Each slide was observed for five minutes and the
results categorised into class 0: no cells visible after
five minutes, class 1: 1–10 cells, class 2: 11–100 cells
and class 3: heavy infection throughout the slide. Gill
smears were used as a second reference on occasions
where cells were difficult to distinguish within the
heart smears.
The Ostrea edulis heart and piece of gill tissue were
then used for DNA extraction (after smearing) using
10% Chelex Ò100 resin and subsequent PCR
K. E. Costello et al.
analysis. In Crassostrea gigas a piece of gill tissue
only was removed for DNA extraction. Oyster sam-
pling in 2018 consisted of 60 O. edulis individuals
with 90 tissue samples screened (gill only in July 2018,
heart and gill for all future screening), and 60 C. gigas
individuals with 60 tissue samples screened (Table 1).
Oyster sampling in 2019 trials consisted of 150 O.
edulis individuals with 300 tissue samples screened,
and 150 C. gigas individuals with 150 tissue samples
screened (Table 3). In both oyster species a transverse
section consisting of gill, gonad, digestive tract and
mantle was then preserved in Davidson’s fixative as
per the OIE (2019) and transferred to 70% ethanol for
long-term storage.
Solitary tunicate samples (Styela clava) were
prepared for molecular work by measuring the length,
wet weight and drained weight then removing the
tunic and dissecting the soft tissue. Tunicates ranged
from 2.7 cm to 11.8 cm in length, therefore the
number of tissue samples was scaled to the size of
the individual (minimum 2 tissue samples and max-
imum 12) and taken from base to oral siphon to ensure
all tissue types were preserved. DNA extraction was
performed in the same manner as the oyster tissue. S.
clava sampling in 2018 consisted of 90 individuals
with 382 tissue samples screened (Table 1). S. clava
sampling in 2019 trials consisted of 32 individuals
with 248 tissue samples screened (Table 3). Samples
also had a histological section consisting of one piece
from base to oral siphon fixed in Davidson’s solution
and prepared as per Flannery et al. (2014), and samples
from 2019 also had a sample (again base to oral
siphon) prepared for in situ hybridisation (ISH) as per
Lynch et al. (2008). Images were captured on a Leica
DM500 microscope using LAS 4.12.0 software.
Colonial samples were also preserved for molecular
work and long-term storage for histology. In the case
of Botrylloides violaceus each small lobe had 3 tissue
sections removed for DNA extraction (120 tissue
samples in total) and 1 section per lobe fixed in
Davidson’s solution (40 in total). In the case of
Didemnum vexillum each colony had 5 tissue sections
removed for DNA extraction (140 tissue samples in
total) and 5 sections fixed in Davidson’s solution (140
in total).
Molecular screening
Prior to molecular screening the quality and concen-
tration of DNA was tested using a NanoDrop
Spectrophotometer. Conventional PCR assays were
initially conducted on oysters, for pathogens of which
they are the natural hosts (see Table 2). If positive
results were obtained from the oysters, the tunicates
were then screened for those pathogens. Screening for
Bonamia ostreae consisted of the BO-BOAS primer
pair (Cochennec et al. 2000) and amplifications were
carried out in a total volume of 50 ll, including 1 ll
DNA, 25.75 ll ddH
0, 10 ll59Green GoTaqÒ
Reaction Buffer (Promega), 10 ll dNTPs (1.25 mM
stock solution), 2 ll MgCl
(25 mM), 0.25 ll
GoTaqÒDNA Polymerase (Promega) and 0.5 ll each
of forward and reverse primers. Reactions were
carried out on a thermocycler at 94 °C for five minutes
(one cycle), then one minute each at 94 °C, 55 °C and
72 °C for 35 cycles and finally 72 °C for ten minutes.
Haplosporidian primer pairs consisted of MSXA-
MSXB for Haplosporidium nelsoni (causative agent
of multinucleate sphere X disease (MSX)), and the
HapF1-HapR3 pair for generic haplosporidian species.
Haplosporidian amplifications were carried out in a
total volume of 20 ll, including 1 ll DNA, 10 ll
0, 4 ll59Green GoTaqÒReaction Buffer
(Promega), 4 ll dNTPs (1.25 mM stock solution),
0.2 ll GoTaqÒDNA Polymerase (Promega) and
0.4 ll of forward and reverse primers. Reactions were
carried out on a thermocycler at 95 °C for three
minutes, then 95 °C (30 s), 59 °C (MSXA-MSXB)/
48 °C (HapF1-HapR3) (30 s) and 72 °C (30 s HapF1-
HapR3/one minute MSXA-MSXB) for 35 cycles and
finally 72 °C for five minutes.
The primer pair OHVA-OHVB was used to detect
the presence of OsHV-1 lVar as per Lynch et al.
(2013b), with the addition of 1.5 ll dMSO to the mix.
The primer pair DnaJf420-DnaJr456 along with the
probe DnaJp441 were used to detect Vibrio aestuar-
ianus using qPCR with a total volume of 25 ll,
including 5 ll DNA, 5.62 ll ddH
0, 12.5 ll Taq-
ManÒ, 0.38 ll probe and 0.75 ll each of forward and
reverse primers. The qPCR thermal profile was as per
McCleary and Henshilwood (2015).
The primer pairs TAP-For/Rev and MER-For/Rev
were used for Minchinia tapetis and Minchinia
mercenariae-like respectively (on a subset of Styela
clava from 2019) as per Albuixech-Martı
´et al. (2020).
The role of invasive tunicates as reservoirs of molluscan pathogens
PCR negative controls consisted of double distilled
O (ddH
O) and two positive controls for each
screen. Positive controls for V. aestuarianus were
obtained from the Marine Institute in the form of a
stock solution and dilutions had Ct (cycle threshold)
values ranging from 27 to 34, a mean Ct below 37 was
considered positive. In the case of B. ostreae, positive
controls were not used in the initial PCR on O. edulis
samples but DNA that was isolated in those samples
was used as a positive control in all subsequent
screenings. Results were visualised using gel elec-
trophoresis on a 2% agarose gel. All primer pairs are
listed in Table 4and on occasions where Sanger
sequencing was utilised, samples were sent to Source
BioSciences, Co. Waterford.
2018 field samples (Cork Harbour)
Table 5outlines the screening for pathogens in the two
oyster species over the summer and winter samples. In
Cork Harbour Crassostrea gigas samples were nega-
tive for OsHV-1 lVar in both July 2018 and Novem-
ber 2018, but positive for Vibrio aestuarianus, with a
higher prevalence in July (10%) than November
(3.3%). Haplosporidian spp. were detected in C. gigas
samples using general haplosporidian primers, with
3.3% and 10% prevalence of infection for July and
November respectively. Subsequently, one sample
from July and three samples from November were
sequenced but were unsuccessful in identifying the
haplosporidian species present in C. gigas. Pacific
oysters were not screened for Bonamia ostreae as they
are not the primary host for the pathogen.
26.6% of Ostrea edulis from the November samples
were positive for haplosporidian spp. using general
primers (HapF1-HapR3), and Sanger sequencing of
one sample (after purification using a Qiagen QIA-
quickÒGel Extraction Kit) identified it as Bonamia
ostreae (100% BLASTn). Bonamia ostreae-specific
primers then detected this pathogen in O. edulis
samples from both months, with a higher prevalence in
November (46%) compared to July (3.3%). O. edulis
samples were all negative for Haplosporidium nelsoni
(MSX). Flat oysters were not screened for OsHV-1
lVar or Vibrio aestuarianus.
Microscopic examination of O. edulis heart smears
determined that 56.6% of oysters had no visible
Bonamia ostreae cells, with 70% infections class 0 in
July and 43.3% class 0 in November. For oysters with
a visible infection, class 1 infections were the most
prevalent, again in both months. A single class 3
infection was detected in November (Fig. 2).
Styela clava were screened from Cork Harbour and
were negative for both haplosporidian spp. and OsHV-
1lVar (Table 6). Samples were not screened for
Haplosporidium nelsoni (MSX) as it had not been
detected in the oyster samples. Vibrio aestuarianus
Table 4 List of forward
and reverse primers used for
PCR/qPCR in this study
Primer Sequence 50-30Amplicon
(Cochennec et al. 2000)
300 bp
(Renault et al. 2000)
190 bp
(Lynch et al. 2013b)
385 bp
(Renault et al. 2000)
573 bp
´et al. 2020)
165 bp
´et al. 2020)
170 bp
DnaJp441 (probe) FAM-AGG GCA CGT CGG C-MGB N/A
(McCleary and Henshilwood 2015)
K. E. Costello et al.
was detected in S. clava in the July samples (6.6%,
lowest Ct 35.5). Bonamia ostreae was also detected in
S. clava in July (5%) and November (10%), and four
samples were sequenced with one confirmed as B.
ostreae (90.6% match, BLASTn). This study also
calculated the wet weight and drained weight of S.
clava to ascertain the potential for tunicates to
transport water containing B. ostreae cells, with 27%
of tunicate biomass from 62 samples (from field trials
and subsequent laboratory cohabitation trials) com-
prised of water contained within the tunic.
Botrylloides violaceus sampled from the marina in
Crosshaven were negative for haplosporidian spp. and
OsHV-1 lVar. However, Vibrio aestuarianus was
detected in B. violaceus samples from July (33.3%,
lowest Ct 35.5). B. violaceus samples were not
screened for H. nelsoni (MSX).
2019 laboratory cohabitation trials
Trial 1: Cohabitation trial between Bonamia ostreae
infected Ostrea edulis and Styela clava
Of the 150 Ostrea edulis collected on 5th April 2019,
30 were instantly removed and screened for Bonamia
ostreae using PCR, with the prevalence of infection
recorded at 13.3%. Oysters were then maintained in
the laboratory for 75 days to allow a higher prevalence
and intensity of infection to develop before cohabita-
tion trials began. 15 live and moribund animals were
screened before the addition of tunicates. B. ostreae
prevalence was 100%, and 46.6% of these were
animals with class 3 infections as evidenced with the
heart smears. This 100% infection was considered the
baseline oyster infection when adding S. clava. The
prevalence of B. ostreae in O. edulis decreased from
the start to the end of the trial, from 100% to 84.3%,
and the prevalence of infection in the control O. edulis
tank (oysters only) was 60% (Fig. 3).
Styela clava screened at the start of the study
showed a 25% prevalence of Bonamia ostreae and this
was considered the baseline tunicate infection. At the
end of the trial, after cohabiting with infected oysters
for two months, this prevalence was 18.75%. The
prevalence of infection in the S. clava in the control
tank, i.e. tunicates only, was 37.5%.
Table 5 PCR (Haplosporidian spp., Haplosporidium nelsoni,
Bonamia ostreae and OsHV-1 lVar), qPCR (Vibrio aestuar-
ianus) and B. ostreae/Ostrea edulis heart smear results for the
detection of pathogens in Crassostrea gigas and O. edulis (NS
= Not Screened, NA = Not Applicable)
Species Date Generic Haplosporidian
spp. PCR (%)
B. ostreae
PCR (%)
smears (%)
H. nelsoni (MSX)
PCR (%)
OsHV-1 lVar
PCR (%)
V. aestuarianus
qPCR (%)
3.3 NS NA NS 0 10.0
10.0 NS NA NS 0 3.3
0 3.3 30.0 0 NS NS
26.6 46.6 56.6 0 NS NS
Fig. 2 Different classes of Bonamia ostreae infection in Ostrea
edulis heart smears with a sample size of 30 in each month (see
text for each class category)
The role of invasive tunicates as reservoirs of molluscan pathogens
All S. clava screened for Minchinia spp. (samples
that were positive for Bonamia ostreae, n = 8) had
100% prevalence of infection of M. mercenariae-like
using PCR, with six samples sequenced for confirma-
tion (100% BLASTn: Minchinia sp. ex Cerastoderma
edule clone 3 small subunit ribosomal RNA gene,
partial sequence; 96.34% BLASTn: Minchinia merce-
nariae small subunit ribosomal RNA gene, partial
sequence). These positive samples encompassed indi-
vidual tunicates from the trial baseline sample,
experimental tanks and control tank, and these same
samples were all negative for M. tapetis. Histology
also revealed sporonts and spore-like structures
belonging to Minchinia mercenariae-like (Fig. 4).
Haplosporidian cells were visualised in a subsam-
ple of Styela clava (n = 5) from the trials using in situ
hybridisation and histology. Bonamia ostreae-specific
in situ hybridisation revealed cells that fit the profile of
B. ostreae, but there was also the potential for them to
be considered aspecific ‘loose’ content due to deteri-
oration of the tunicate tissue during the hybridisation
process. However histological examination of the
coinfected tunicate samples revealed uninucleate
haplosporidian cells consistent with either B. ostreae
or Minchinia mercenariae-like in addition to the
sporonts and spore-like structures belonging to the M.
mercenariae-like species (Fig. 4).
Trial 2: Cohabitation trial between Vibrio
aestuarianus infected Crassostrea gigas
and Didemnum vexillum
Of the 150 Crassostrea gigas collected on 4th July
2019, 30 were instantly removed and screened for
Vibrio aestuarianus using qPCR, with the prevalence
of infection recorded at 93.3%. In the 32 days prior to
the addition of the tunicates there were 42 oyster
mortalities, all of which were screened and had 100%
V. aestuarianus infection. Tunicates were added to
tanks on 6th August and prior to addition of tunicates a
further ten oysters were screened, with 50% oysters
infected with V. aestuarianus. This 50% was consid-
ered the baseline infection of oysters at the start of the
cohabitation trial. Due to the limited number of oysters
as a result of the early high mortality rate there was no
Table 6 Molecular results for the detection of pathogens in Botrylloides violaceus and Styela clava (NS = Not Screened)
Species Date Haplosporidian spp.
PCR (%)
Bonamia ostreae
PCR (%)
H. nelsoni (MSX)
PCR (%)
OsHV-1 lVar
PCR (%)
Vibrio aestuarianus
qPCR (%)
0 NS NS 0 33.3
S. clava 4-07-
0 6.6 NS 0 0
S. clava 11-07-
0 3.3 NS 0 6.6
S. clava 2-11-
Fig. 3 % prevalence of Bonamia ostreae in Ostrea edulis and
Styela clava using PCR (control screened at end of cohabitation
K. E. Costello et al.
tank of oysters only as a control. There were no further
oyster mortalities while the cohabitation trial was
running and at the end of the trial all remaining oysters
were screened (n = 68), with prevalence of intensity
just 0.03%.
The total prevalence of infection for the baseline
tunicate sample (7 heterogeneous colonies) was 71.4%
and the total prevalence of infection in the experi-
mental sample (14 heterogeneous colonies) was 50%.
No Vibrio aestuarianus was detected in the control
tunicates (7 heterogeneous colonies) that were held
alone in a tank without any oysters being present.
Given the heterogeneous nature of the tunicate
colonies in Carlingford, and the morphological sim-
ilarities between species, a sample from each of the 28
colonies was sequenced at the end of the trial to
determine the full species composition of the 28
colonies. Sanger sequencing confirmed 11 Didemnum
vexillum (invasive) colonies and 9 Aplidium glabrum
(native) colonies. There was also one sample each of
the tunicate genera Botryllus/Botrylloides sp., Styela
sp. and Ascidiella sp. The remaining five sequences
failed but were visually identified (by comparing
morphology to other sequenced individuals) as D.
vexillum (94) and A. glabrum (91). Infection was
higher in D. vexillum than A. glabrum/other at both the
baseline sample (100% in D. vexillum, 33.3% in A.
glabrum/other species) and at the end of the cohab-
itation trial with mortalities included (55.5% in D.
vexillum, 40% in A. glabrum/other). In the control tank
with no oysters present there was no infection detected
in any tunicate species either from mortalities or at
completion of the trial (Fig. 5).
Fig. 4 (a) and (b) uninucleate haplosporidian ‘fried egg’ cells
consistent with Bonamia ostreae or Minchinia mercenariae-like
in the connective tissue of Styela clava (c) vacuoles in Styela
clava epithelial tissue arising from Minchinia mercenariae-like
spore-like stage with polar nuclei visible (d)Minchinia merce-
nariae-like sporonts in Styela clava connective tissue
The role of invasive tunicates as reservoirs of molluscan pathogens
This study used OIE-recommended diagnostic tech-
niques and current literature to demonstrate that of a
number of molluscan pathogens recorded in Cork
Harbour, Bonamia ostreae,Minchinia mercenariae-
like and Vibrio aestuarianus are currently present in
commercial bivalves. Furthermore, field surveys and
laboratory trials indicated that these same pathogens
are present in invasive tunicates cohabiting with
oysters at the aquaculture sites. Field surveys also
demonstrated that V. aestuarianus can occur in
invasive tunicates present in marinas removed from
aquaculture sites, raising the possibility that transport
of pathogens may occur from aquaculture sites and
remain in reservoirs outside the zone of infection, for
example via currents or recreational shipping. Addi-
tionally, the haplosporidian M. mercenariae-like was
capable of replicating in Styela clava, suggesting that
this tunicate is not only a carrier of the pathogen but
potentially a viable host. S. clava were also capable of
maintaining a second haplosporidian pathogen, B.
ostreae, without the presence of the primary host
Ostrea edulis. The presence of both B. ostreae and M.
mercenariae-like in S. clava individuals also demon-
strates that the tunicates are susceptible to coinfection.
For both Styela clava/Bonamia ostreae and Didem-
num vexillum/Vibrio aestuarianus the prevalence of
intensity of the respective pathogens was higher at the
start of the trial than at the end of the trial. The filter
feeding mechanism of the tunicates may explain how
they take in the disease and it is possible that in a larger
aquaculture area the tunicates are filtering more so that
although the geographic spread is significantly larger
than in an enclosed tank the increased filtering could
result in a greater intake of pathogenic cells and a
higher infection.
In the case of Vibrio aestuarianus it is possible that
Didemnum vexillum and other colonial spp. may need
to be in contact with oysters to maintain the disease, as
both the baseline sample and the experimental treat-
ments proved positive despite a low prevalence of V.
aestuarianus in the cohabitating Crassostrea gigas at
the end of the trial which suggested that the remaining
oysters were ‘survivors’ that had not suffered Vibrio-
induced mortality. Conversely the control treatment of
tunicates only was negative for V. aestuarianus.
In the case of Bonamia ostreae,Styela clava in
control tanks maintained the disease for two months
without the presence of oysters. Furthermore, the
percentage prevalence of infection was higher in the
control than the baseline and experimental samples,
which indicates that replication in the system may be
possible and the pathogen may have been transmitting.
This would again suggest that S. clava is potentially a
host, not just a carrier or reservoir. The fact that B.
ostreae prevalence was higher in control tunicates
than tunicates cohabiting with oysters may also be
because the oysters were filtering more quickly and
therefore picking up B. ostreae cells so the infection
was reduced in tunicates.
Another key point is that the percentage prevalence
of B. ostreae was higher in O. edulis cohabiting with S.
clava, rather than in the O. edulis control tank,
meaning a cumulative effect of S. clava and O. edulis
on B. ostreae percentage prevalence cannot be ruled
out. O. edulis in this study came from a site where a
selective breeding programme commenced in 1988 for
over 30 years (Lynch et al. 2014) and the slight
reduction in B. ostreae in the oysters from the baseline
to the end of the trial may support that they have been
bred to be resistant to B. ostreae and can maintain it at
a sub-lethal level.
This study focused on Didemnum vexillum as the
target species for laboratory trials, however this
species was growing in heterogeneous colonies and
it is therefore possible that the interwoven nature of
colonial tunicates means they may circle pathogens
Fig. 5 % prevalence of Vibrio aestuarianus in Crassostrea
gigas,Didemnum vexillum and Aplidium glabrum/other species
K. E. Costello et al.
both from zooid to zooid within the colony, but also
from one species to another thus heightening the
potential for disease transmission. Furthermore, given
the detection of Vibrio aestuarianus it is highly likely
that other Vibrio spp. that are potentially pathogenic to
commercial bivalves could also be carried by
Although the impact of these pathogens on tunicate
health was not investigated in this study, the combined
results across the solitary and colonial forms indicate
that invasive tunicates can potentially act as reservoirs
of pathogens. Other studies have indicated that in
many cases such reservoirs can transmit the infection
back to susceptible bivalves, for example when
Crassostrea gigas infected with Bonamia ostreae
were held with naive Ostrea edulis, the O. edulis
cavity fluid then tested positive for the pathogen
(Lynch et al. 2010). B. ostreae has also been detected
in a number of benthic macroinvertebrates, including
the native European sea squirt Ascidiella aspersa and
other phyla including phylum Crustacea (Carcinus
maenas) and phylum Cnidaria (Actinia equina)
(Lynch et al. 2007). This circling of the disease
through diverse invertebrate groups could suggest that
there are multiple avenues for reservoirs to come in
contact with the disease.
Small and Pagenkopp (2011), noted that the
environment itself can act as a reservoir and this is
demonstrated by Bonamia ostreae, as the OIE states
that it can live for up to one week in the water column.
This also means that Styela clava could potentially act
as a mechanical vector and carry water with the protist
present to different sites if transferred on improperly
cleaned aquaculture gear or non-biosecure stock
Tunicates were used as a model taxon in this study,
however it is important to note that invasive species
across different phyla may be also able to move
significant pathogens. Davies et al. (2019) investigated
the globally invasive shore crab (Carcinus maenas)in
Wales and found it infected with Hematodinium sp.
and Hematodinium perezi. The disease was also
present in seawater eDNA samples, possibly due to
the release of infectious stage dinospores from mori-
bund individuals. Shore crabs in the study site were
cohabiting with the commercially valuable edible
brown crab, and this demonstrates that ability of
disease reservoirs to enable pathogens to persist in
habitats utilised by commercial shellfish.
The presence of disease in invasive tunicates is of
significant interest to the aquaculture sector, particu-
larly in the context of climate change, as species
potentially expand their invaded range (Hellmann
et al. 2008). When coupling the mechanical impacts of
the tunicates themselves, for example competition for
space, with their potential to interact with pathogens
affecting commercial bivalve species, they emerge as
a threat that warrants serious consideration and
enhanced biosecurity. The Food and Agriculture
Organization of the United Nations recognised the
risk of invasive species in the 2030 Agenda for
Sustainable Development, and noted the need to
introduce measures to eradicate or control such
species (FAO 2017). A number of aspects need to be
addressed to lessen invasive impacts; reproductive
strategies need to be identified, transport via shipping
or other pathways such as aquaculture needs to be
minimised and mechanisms identified to remove and
inactivate fouling organisms. Examples of these
mechanisms include controls such as those described
in Hillock and Costello (2013) with a study on
desiccation methods for Styela clava, or Turrell et al.
(2018) with a study of different bath treatments that
could be applied to commercial bivalve consignments
to induce full mortality in Didemnum vexillum.
It is necessary to develop a global approach to
ensure aquaculture transfer takes management of both
invasive species and disease into account, thereby
developing legislation and codes of practice. In 2016
shelled molluscs constituted 58.8% of the combined
production of marine and coastal aquaculture (FAO
2018) and growth projections for the future are
positive, meaning that molluscan aquaculture will
play an essential role in global food resources
(Rodgers et al. 2015; Arzul et al. 2017). However,
this sector is intrinsically linked to the movement of
invasive species and disease cycling is a further
constraint on growth. Guidelines and legislation are
only as good as the knowledge that informs them, and
it is not feasible to protect against species on which
there is little knowledge about health impacts or
potential roles in disease cycling. Accordingly, it is
necessary to broaden the understanding of the how
pathogens utilise invasive species, and the subsequent
impact on the commercial sector.
This study raises questions relating to the viability
of tunicates as sources of infection and suggests that
the taxon should be taken into account in risk
The role of invasive tunicates as reservoirs of molluscan pathogens
assessments and disease management, particularly as
pathogens can endure in species that are not their true
host (Lynch et al. 2010). Screening of the tunicates
focused specifically on pathogens that had previously
been detected in the natural hosts, however further
work could expand on the diversity and seasonality of
pathogen species, as if tunicates are reservoirs then a
range of pathogens might be present in them at times
of the year not found in oysters. It also demonstrates
that it is important to have good monitoring networks
and collaborative efforts so that information on
invasive species with potential impacts is widely
shared and available within the aquaculture sector
(Brenner et al. 2014).
Acknowledgements This work was supported by the Bluefish
Project (Grant Agreement No. 80991), part-funded by the
European Regional Development Fund (ERDF) through the
Ireland Wales Co-operation Programme. The authors would like
to thank Gavin Deane of the Royal Cork Yacht Club, the Hugh-
Jones of Atlantic Shellfish Ltd. and all at Carlingford Oyster
Company and Dungarvan Shellfish Ltd. for their assistance
during fieldwork. We would also like to thank Sara Albuixech-
´, Kate Mahony, Gary Kett, Ashley Bennison and Alex
McGreer for their support during the laboratory trials. Lastly, we
would like to thank Dr Eileen Dillane, Dr Elizabeth Cotter, Luke
Harman and Allen Whitaker for their technical assistance.
´S, Lynch SA, Culloty SC (2020) Biotic and
abiotic factors influencing haplosporidian species distri-
bution in the cockle Cerastoderma edule in Ireland. J In-
vertebr Pathol 174:1–12
Arzul I, Corbeil S, Morga B, Renault T (2017) Viruses infecting
marine molluscs. J Invertebr Pathol 147:118–135
Beaury EM, Fusco EJ, Jackson MR, Laginhas BB, Morelli TL,
Allen JM, Pasquarella VJ, Bradley BA (2019) Incorpo-
rating climate change into invasive species management:
insights from managers. Biol Invasions 22:233–252 https://
Bookelaar B (2018) Understanding and minimizing the impacts
of host-pathogen-environment interactions in the Pacific
oyster Crassostrea gigas. PhD Thesis, University College
Bookelaar B, O’Reilly AJ, Lynch SA, Culloty SC (2018) Role of
the intertidal predatory shore crab Carcinus maenas in
transmission dynamics of ostreid herpesvirus-1
microvariant. Dis Aquat Org 130:221–233
Bourque D, Davidson J, MacNair NG, Arsenault G, LeBlanc
AR, Landry T, Mirron G (2007) Reproduction and early
life history of an invasive ascidian Styela clava Herdman,
in Prince Edward Island, Canada. J Exp Mar Biol Ecol
Brenner M, Fraser D, Van Nieuwenhove K, O’Brien F, Buck
BH, Mazurie
´J, Thorarinsdottir G, Dolmer P, Sanchez-
Mata A, Strand O, Flimlin G, Miossec L, Kamermans P
(2014) Bivalve aquaculture transfers in Atlantic Europe.
Part B: environmental impacts of transfer activities. Ocean
Coast Manag 89:139–146
Carnegie RB, Arzul I, Bushek D (2016) Managing marine
mollusc diseases in the context of regional and interna-
tional commerce: policy issues and emerging concerns.
Philos Trans R Soc B 371:20150215
Casso M, Navarro M, Ordo
˜ez V, Ferna
´ndez-Tejedor M, Pas-
cual M, Turon X (2018) Seasonal patterns of settlement and
growth of introduced and native ascidians in bivalve cul-
tures in the Ebro Delta (NE Iberian Peninsula). Reg Stud
Mar Sci 23:12–22
Cochennec N, Le Roux F, Berthe F, Ge
´rard A (2000) Detection
of Bonamia ostreae based on small subunit ribosomal
probe. J Invertebr Pathol 76:26–32
Comeau LA, Filgueira R, Guyondet T, Sonier R (2015) The
impact of invasive tunicates on the demand for phyto-
plankton in longline mussel farms. Aquaculture
Cordell JR, Levy C, Toft JD (2012) Ecological implications of
invasive tunicates associated with artificial structures in
Puget Sound, Washington, USA. Biol Invasions
Davies CE, Batista FM, Malkin SH, Thomas JE, Bryan CC,
Crocombe P, Coates CJ, Rowley AF (2019) Spatial and
temporal disease dynamics of the parasite Hematodinium
sp. in shore crabs, Carcinus maenas. Parasit Vectors
Dunn AM, Hatcher MJ (2015) Parasites and biological inva-
sions: parallels, interactions, and control. Trends Parasitol
FAO (2017) The 2030 agenda and the sustainable development
goals: the challenge for aquaculture development and
management. FAO Fisheries and Aquaculture Circular No.
1141, Rome, Italy
FAO (2018) The state of world fisheries and aquaculture 2018.
Meeting the sustainable development goals. Rome,
Licence: CC BY-NC-SA 3.0 IGO
Ferguson LF, Davidson JDP, Landry T, Clements JC, Therriault
TW (2017) Didemnum vexillum: invasion potential via
harvesting and processing of the Pacific oyster (Cras-
sostrea gigas) in British Columbia, Canada. Manage Biol
Invasion 8(4):553–558
Flannery G, Lynch SA, Longshaw M, Stone D, Martin P,
Ramilo A, Villalba A, Culloty S (2014) Interlaboratory
variability in screening for Bonamia ostreae, a protistan
parasite of the European flat oyster Ostrea edulis. Dis
Aquat Org 110:93–99
Ford SE, Stokes NA, Burreson EM, Scarpa E, Carnegie RB,
Kraeuter JN, Bushek D (2009) Minchinia mercenariae n.
sp. (Haplosporidia) in the Hard Clam Mercenaria merce-
naria: implications of a rare parasite in a commercially
important host. J Eukaryot Microbiol 56(6):542–551
Franchi N, Ballarin L (2017) Immunity in protochordates: the
tunicate perspective. Front Immunol 8(674):1–16
Goedknegt MA, Feis ME, Wegner KM, Luttikhuizen PC,
Buschbaum C, Camphuysen K, van der Meer J, Thieltges
K. E. Costello et al.
DW (2016) Parasites and marine invasions: ecological and
evolutionary perspectives. J Sea Res 113:11–27
Goldstien SJ, Schiel DR, Gemmell NJ (2010) Regional con-
nectivity and coastal expansion: differentiating pre-border
and post-border vectors for the invasive tunicate Styela
clava. Mol Ecol 19:874–885
Hellmann JJ, Byers JE, Bierwagen BG, Dukes JS (2008) Five
potential consequences of climate change for invasive
species. Conserv Biol 22(3):534–543
Hillock KA, Costello MJ (2013) Tolerance of the invasive
tunicate Styela clava to air exposure. Biofouling
Kumagai A, Sakai K, Miwa S (2014) The sea squirt Styela clava
is a potential carrier of the kinetoplastid Azumiobodo
hoyamushi, the causative agent of soft tunicate syndrome in
the edible ascidian Halocynthia roretzi. Fish Pathol
Lupo C, Travers M-A, Tourbiez D, Barthe
´my CF, Beaune
Ezanno P (2019) Modeling the transmission of Vibrio
aestuarianus in pacific oysters using experimental infec-
tion data. Front Vet Sci 6(142):1–15
Lynch SA, Armitage DV, Coughlan J, Mulcahy MF, Culloty SC
(2007) Investigating the possible role of benthic macroin-
vertebrates and zooplankton in the life cycle of the hap-
losporidian Bonamia ostreae. Exp Parasitol 115:359–368
Lynch SA, Mulcahy MF, Culloty SC (2008) Efficiency of
diagnostic techniques for the parasite, Bonamia ostreae, in
the flat oyster, Ostrea edulis. Aquaculture 281:17–21
Lynch SA, Abollo E, Ramilo A, Cao A, Culloty SC, Villalba A
(2010) Observations raise the question if the Pacific oyster,
Crassostrea gigas, can act as either a carrier or a reservoir
for Bonamia ostreae or Bonamia exitiosa. Parasitology
Lynch SA, Villalba A, Abollo E, Engelsma M, Stokes NA,
Culloty SC (2013a) The occurrence of haplosporidian
parasites, Haplosporidium nelsoni and Haplosporidium
sp., in oysters in Ireland. J Invertebr Pathol 112:208–212
Lynch S, Dillane E, Carlsson J, Culloty SC (2013b) Develop-
ment and assessment of a sensitive and cost-effective
polymerase chain reaction to detect ostreid herpesvirus 1
and variants. J Shellfish Res 32(3):657–664
Lynch SA, Flannery G, Hugh-Jones T, Hugh-Jones D, Culloty
SC (2014) Thirty-year history of Irish (Rossmore) Ostrea
edulis selectively bred for disease resistance to Bonamia
ostreae. Dis Aquat Org 110:113–121
Lynch SA, Darmody G, O’Dwyer K, Gallagher MC, Nolan S,
McAllen R, Culloty SC (2016) Biology of the invasive
ascidian Ascidiella aspersa in its native habitat: Repro-
ductive patterns and parasite load. Estuarine Coastal Shelf
Sci 181:249–255
McCleary S, Henshilwood K (2015) Novel quantitative Taq-
ManÒMGB real-time PCR for sensitive detection of
Vibrio aestuarianus in Crassostrea gigas. Dis Aquat Org
OIE Manual of Diagnostic tests for Aquatic Animals (2019)
aquatic-manual/access-online. Accessed 26 May 2020
Paetzold SC, Hill J, Davidson J (2012) Efficacy of high-pressure
seawater spray against colonial tunicate fouling in mussel
aquaculture: inter-annual variation. Aquat Invasions
Prado-Alvarez M, Couraleau Y, Chollet B, Tourbiez D, Arzul I
(2015) Whole-genome amplification: a useful approach to
characterize new genes in unculturable protozoan parasites
such as Bonamia exitiosa. Parasitology 142:1523–1534
Prado-Alvarez M, Darmody G, Hutton S, O’Reilly A, Lynch
SA, Culloty SC (2016) Occurrence of OsHV1 in Cras-
sostrea gigas cultured in Ireland during an exceptionally
warm Summer. Front Physiol 7(492):1–14
Price A, Collie J, Smith D (2005) 18S ribosomal RNA and
cytochrome oxidase gene sequences of Didemnum sp., an
invasive colonial tunicate. SURFO Technical Reports, No.
Ramilo A, Abollo E, Villalba A, Carballal MJ (2018) A Min-
chinia mercenariae-like parasite infects cockles Cerasto-
derma edule in Galicia (NW Spain). J Fish Dis 41:41–48
Renault T, Stokes NA, Chollet B, Cochennec N, Berthe F,
´rard A, Burreson EM (2000) Haplosporidiosis in the
Pacific oyster Crassostrea gigas from the French Atlantic
coast. Dis Aquat Org 42:207–214
Rodgers CJ, Carnegie RB, Cha
´nchez MC, Martı
´nchez CC, Furones Nozal MD, Hine PM (2015) Leg-
islative and regulatory aspects of molluscan health man-
agement. J Invertebr Pathol 131:242–255
Rosa M, Holohan BA, Shumway SE, Bullard SG, Wikfors GH,
Morton S, Getchis T (2013) Biofouling ascidians on
aquaculture gear as potential vectors of harmful algal
introductions. Harmful Algae 23:1–7
Rueckert S, Wakeman KC, Jenke-Kodama H, Leander BS
(2015) Molecular systematics of marine gregarine api-
complexans from Pacific tunicates, with descriptions of
five novel species of Lankesteria. Int J Syst Evol Microbiol
Simkanin C, Dower JF, Filip N, Jamieson G, Therriault TW
(2013) Biotic resistance to the infiltration of natural benthic
habitats: Examining the role of predation in the distribution
of the invasive ascidian Botrylloides violaceus. J Exp Mar
Biol Ecol 439:76–83
Small HJ, Pagenkopp KM (2011) Reservoirs and alternate hosts
for pathogens of commercially important crustaceans: a
review. J Invertebr Pathol 106:153–164
Turrell WR, Brown L, Graham J, Gubbins MJ, Hermann G,
Matejusova I, Robinson C (2018) Selecting a bath treat-
ment for the marine carpet sea squirt Didemnum vexillum,
Kott 2002 in Scottish shellfish aquaculture. Scott Mar
Freshwater Sci Rep 9(12):1–94
Ward GM, Feist SW, Noguera P, Marcos-Lo
´pez M, Ross S,
Green M, Urrutia A, Bignell JP, Bass D (2019) Detection
and characterisation of haplosporidian parasites of the blue
mussel Mytilus edulis, including description of the novel
parasite Minchinia mytili n. sp. Dis Aquat Org 133:57–68
Wong NA, McClary D, Sewell MA (2011) The reproductive
ecology of the invasive ascidian, Styela clava, in Auckland
Harbour, New Zealand. Mar Biol 158:2775–2785
Zhan A, Briski E, Bock DG, Ghabooli S, MacIsaac HJ (2015)
Ascidians as models for studying invasion success. Mar
Biol 162:2449–2470
Publisher’s Note Springer Nature remains neutral with
regard to jurisdictional claims in published maps and
institutional affiliations.
The role of invasive tunicates as reservoirs of molluscan pathogens
... Introduced species that have not lost their associated pathogens or parasites can potentially transmit novel parasites to native hosts (Dunn andHatcher 2015, Costello et al. 2021). Further, introduced invasive species may promote transmission of pathogens by native species and act as reservoirs and facilitate replication in areas where disease occurs (Costello et al. 2021). Introduced ascidians have been shown to transmit viruses, bacteria and haplosporidian protists, such as Bonamia ostreae, Minchinia to oysters and cockles that in turn can cause mass mortalities of bivalves. ...
... Introduced ascidians have been shown to transmit viruses, bacteria and haplosporidian protists, such as Bonamia ostreae, Minchinia to oysters and cockles that in turn can cause mass mortalities of bivalves. Costello et al. (2021) showed from Ireland that D. vexillum was infected by the bacterium Vibrio aestuarianus in the vicinity of infected oysters at oyster aquaculture sites. It appeared that D. vexillum had to be in contact with oysters to maintain the disease, but it could function as a reservoir for reinfection or contribute to circulate Vibrio among other species, thus heightening disease transmission. ...
... It appeared that D. vexillum had to be in contact with oysters to maintain the disease, but it could function as a reservoir for reinfection or contribute to circulate Vibrio among other species, thus heightening disease transmission. It should also be considered that the combined effect of the physical impact from overgrowth and the maintenance of potentially lethal pathogens increases the threat that D. vexillum represents for susceptible species of bivalves (Costello et al. 2021). ...
Technical Report
Full-text available
This is an assessment of risk and risk-reducing measures related to the introduction and dispersal of the invasive alien carpet tunicate Didemnum vexillum in Norway.
... The branchial sac is also likely to be the entry point of potential pathogens and toxic algae inhabiting anthropogenic habitats where S. plicata thrives. 59,60 The gill microbiome can therefore be a crucial factor to aid the ascidian to cope with environmental conditions and to mediate immune responses, akin to the role assumed for the skin of other groups. 61,62 It has been shown that microbes can play a role in detoxifying potential toxic compounds in the diet of metazoans (''gut microbial facilitation hypothesis'' 63 ). ...
... In combination with opportunistic bacterial infections, OsHv-1 can cause mass mortalities (de Logeril et al. 2018), and the virus was also found in resident species in contact with introduced Pacific oysters (O'Reilly et al. 2018; Morga et al. 2021). Introduced ascidians can serve as a reservoir for such diseases (Costello et al. 2021). So far, introduced predators seem to have no striking impacts on the food web in the Wadden Sea. ...
Full-text available
For about a century, biodiversity in the tidal Wadden Sea (North Sea, European Atlantic) has increased by more than one hundred introduced species from overseas. Most originate from warmer waters and could facilitate the transformation of this coastal ecosystem to comply with climate warming. Some introduced species promote sediment stabilization and mud accretion. This could help tidal flats to keep up with sea level rise. Although some introduced species also entail negative effects, introductions have diversified lower food web levels, and may benefit foraging birds. So far, no resident populations have gone extinct because an introduced species had established. Rather than degrading the ecosystem, the establishment of introduced species seems to have raised the capacity to follow environmental change. We support increasing efforts against introductions to avoid risk. However, once species are integrated, the common condemnation attitude against “non-natives” or “aliens” ought to be reconsidered for tidal ecosystems of low biodiversity.
... Par exemple, Bonamia ostreae (Pichot, 1980), parasite de la famille des Haplosporiidae et responsable de mortalités massives chez les huitres, peut rester dans le milieu des années après avoir décimé une population et engendrer de nouvelles mortalités si les huîtres sont réintroduites(Lynch et al., 2007). Une multitude d'espèces peut servir de réservoir au parasite de façon à ce que, finalement, il ne disparaisse jamais du milieu(Costello et al., 2021). Le parasite H. pinnae a été décrit récemment et reste très mal connu. ...
Les systèmes marins côtiers sont généralement discontinus et constitués d’une mosaïque de paysages sous-marins différents, créant ainsi des distributions parfois très fragmentées chez les espèces qui les colonisent. Les espèces marines côtières sont donc structurées en réseaux de populations connectées entre elles via la dispersion larvaire. Comprendre le fonctionnement et la connectivité entre les populations d’une espèce est indispensable pour adapter les stratégies de conservation. La grande nacre, Pinna nobilis, est une espèce endémique de la mer Méditerranée qui fait aujourd’hui face à une crise majeure qui menace sa survie. Depuis Octobre 2016, des mortalités de masse sont signalées sur ses populations, à travers toutes la mer Méditerranée, causées par un protozoaire parasite, Haplosporidium pinnae. Il s’agit d’un évènement sans précédent, que ce soit par le taux de mortalité (près de 100 %) ou la vitesse de propagation, et qui pourrait conduire à l’extinction de l’espèce. En se focalisant sur le littoral Occitan, cette thèse apporte des connaissances sur la biologie et l’écologie de l’espèce mais aussi sur son fonctionnement et les processus qui permettent le maintien de ses populations afin de proposer des priorités de conservation. Ainsi, nous avons mis en évidence la diversité d’habitats colonisés par l’espèce ainsi que l’importance des lagunes car elles abritent près de 90 % des grandes nacres, sur le littoral Occitan, et semblent servir d’habitat refuge à l’espèce en limitant l’infestation par le parasite. A l’aide de marqueurs microsatellites nouvellement développés, nous avons montré une structure génétique très homogène sur toute la côte, ce qui implique un certain niveau de connectivité et laisse penser qu’une grande partie de la diversité génétique de l’espèce reste préservée dans les lagunes. En se focalisant sur la population de la baie de Peyrefite, dans la Réserve Naturelle Marine de Cerbère-Banyuls, et grâce à une analyse de parenté, nous avons apporté des connaissances sur la dynamique démographique et les processus de repeuplement de l’espèce. L’ensemble de cette thèse permet de définir des recommandations qui seront utiles à la mise en place de mesures de conservation adaptées, indispensables pour la survie de l’espèce.
... The life cycle of haplosporidan parasites is a complex issue. Binary fission is the only known proliferation process (red dotted frame in Figure 8) of some haplosporidan species infecting oysters, such as Bonamia ostreae and Bonamia exitiosa, in which uninucleate and binucleate cells can leave the host with potential to infect new hosts, thus making possible direct parasite transmission among individuals of the type host species (Sas et al., 2020); additionally other potential transmission vehicles or reservoirs have been identified, including oyster larvae (Arzul et al., 2011) and other invertebrate species (Lynch et al., 2007(Lynch et al., , 2010Costello et al., 2020). In other haplosporidan species, such as Haplosporidium nelsoni, the only known stages are plasmodia and those of sporogonia (area enclosed with blue dashed outline in Figure 8); for these haplosporidan species, their transmission way is unknown; direct transmission has never been experimentally confirmed and the need of a host has been suggested (Burreson and Ford, 2004;Arzul and Carnegie, 2015); the spores are likely enduring stages whose infective potential last longer than that of naked uninucleate and binucleate stages. ...
Full-text available
A mass mortality event (MME) affecting the fan mussel Pinna nobilis was first detected in Spain in autumn 2016 and spread north-and eastward through the Mediterranean Sea. Various pathogens have been blamed for contributing to the MME, with emphasis in Haplosporidium pinnae, Mycobacterium sp. and Vibrio spp. In this study, samples from 762 fan mussels (necropsies from 263 individuals, mantle biopsies from 499) of various health conditions, with wide geographic and age range, taken before and during the MME spread from various environments along Mediterranean Sea, were used to assess the role of pathogens in the MME. The number of samples processed by both histological and molecular methods was 83. The most important factor playing a main role on the onset of the mass mortality of P. nobilis throughout the Mediterranean Sea
... Bloecher and Floerl (2020) calculated direct biofouling management costs for a typical Norwegian salmon farm (eight production pens) to be US$420,000 to $493,600 per production cycle (excluding farm personnel costs), equating to 2.2% of production costs for individual sites. Biofouling species common to aquaculture structures can act as reservoirs and amplifiers of pathogens (Costello et al., 2021), which can impact stock or broader disease dynamics within a seascape. There is growing recognition of the role of aquaculture in spreading marine pests and pathogens (Sim-Smith et al., 2016), and industry codes of practice and government guidance documents have been developed to improve outcomes in New Zealand and elsewhere (Georgiades et al., 2016(Georgiades et al., , 2020aMinistry for Primary Industries and Aquaculture New Zealand, 2016). ...
Full-text available
The number, extent, diversity, and global reach of submerged static artificial structures (SSAS) in the marine environment is increasing. These structures are prone to the accumulation of biofouling that can result in unwanted impacts, both immediate and long-term. Therefore, management of biofouling on SSAS has a range of potential benefits that can improve structure functions, cost-efficiency, sustainability, productivity, and biosecurity. This review and synthesis collates the range of methods and tools that exist or are emerging for managing SSAS biofouling for a variety of sectors, highlighting key criteria and knowledge gaps that affect development, and uptake to improve operational and environmental outcomes. The most common methods to manage biofouling on SSAS are mechanical and are applied reactively to manage biofouling assemblages after they have developed to substantial levels. Effective application of reactive methods is logistically challenging, occurs after impacts have accumulated, can pose health and safety risks, and is costly at large scales. Emerging technologies aim to shift this paradigm to a more proactive and preventive management approach, but uncertainty remains regarding their long-term efficacy, feasibility, and environmental effects at operational scales. Key priorities to promote more widespread biofouling management of SSAS include rigorous and transparent independent testing of emerging treatment systems, with more holistic cost-benefit analyses where efficacy is demonstrated.
... Tunicates usually occur in relatively low abundance in coastal waters. However, some tunicates are reported as invasive species in some coastal waters [171] and are known to cause space competition [172], damage to aquaculture [173,174] by harboring pathogenic viruses and bacteria [175], and ecosystem alteration within the spread area [176]. Few non-invasive tunicate species of the coral reef environment have also been reported to overgrow on massive corals and caused minimal [112] or partial inhibition or delayed development of coral polyps [177]. ...
Full-text available
Marine tunicates are identified as a potential source of marine natural products (MNPs), demonstrating a wide range of biological properties, like antimicrobial and anticancer activities. The symbiotic relationship between tunicates and specific microbial groups has revealed the acquisition of microbial compounds by tunicates for defensive purpose. For instance, yellow pigmented compounds, “tambjamines”, produced by the tunicate, Sigillina signifera (Sluiter, 1909), primarily originated from their bacterial symbionts, which are involved in their chemical defense function, indicating the ecological role of symbiotic microbial association with tunicates. This review has garnered comprehensive literature on MNPs produced by tunicates and their symbiotic microbionts. Various sections covered in this review include tunicates’ ecological functions, biological activities, such as antimicrobial, antitumor, and anticancer activities, metabolic origins, utilization of invasive tunicates, and research gaps. Apart from the literature content, 20 different chemical databases were explored to identify tunicates-derived MNPs. In addition, the management and exploitation of tunicate resources in the global oceans are detailed for their ecological and biotechnological implications
Full-text available
Background Animals should be viewed as holobionts, complex entities composed of an animal host and their associated symbionts. This integrated perspective recognizes that the interaction between these two components is vital for the survival of the host. This synergy can be particularly relevant in the case of invasive species, as they constantly face habitat changes, and thus play a crucial role in their introduction and adaptation success. However, our understanding on the microbiome changes of invasive species from juveniles to adults associated with different tissues remains limited. Results We reveal that the introduced ascidian Styela plicata has a highly variable microbiome, which undergoes significant changes from juvenile to adult individuals and is highly specialised for each compartment analysed (tunic, gill, and gut) in different localities. This is the first time that a distinct gill microbiome is identified in an ascidian species. The variability observed is attributed to S. plicata’s ability to acquire its resident bacteria from the surrounding water, with a subsequent differential proliferation leading to the development of clearly differentiated microbiomes in each tissue. We also observe that the microbiome varies across harbours, suggesting adaptation to local environmental conditions. Furthermore, we find that each tissue’s microbiome is strongly correlated with environmental trace element concentrations, especially in adults, where trace element levels are higher. This fact suggests that the microbiome of S. plicata can play a role by either taking advantage or disposing of trace elements, many of which are toxic. Finally, we examine the metabolic pathways attributable to each microbiome, revealing that adult microbiomes have specific metabolic pathways that might enhance tissue functionality, conferring a high adaptive potential to S. plicata to withstand new conditions during the introduction process, turning this species into one of the most successful invasive holobionts worldwide. Conclusions Our findings emphasise the need for integrative approaches to investigate animal microbiomes, considering multiple variables to fully comprehend the relationship between host and microbiome, as well as their implications for host integrity. Thus, this work confirms the complex interplay between S. plicata and its microbiomes, and how it has significant implications for the invasive success of this species.
Bonamia exitiosa and B. ostreae are small (typically, 2–5 μm) single-celled parasites that infect and replicate within oyster hemocytes causing a disease called bonamiasis. Four Bonamia species have been identified, but only B. exitiosa and B. ostreae have caused significant and consequential marine disease epizootics of farmed and wild oysters throughout the world. Due to the severity of disease that these parasites can cause, B. exitiosa and B. ostreae are listed as notifiable pathogens to the World Organization for Animal Health. Bonamia exitiosa and B. ostreae are directly transmitted between oysters and can quickly spread through oyster populations. Their small size can make diagnosis challenging, especially by histology or cytology alone. Because of their importance to oyster health, more sensitive and species-specific molecular tests are now available. This chapter provides background of these enigmatic parasites and the tools available to detect them.
Full-text available
Abstract Background The parasitic dinoflagellates of the genus Hematodinium represent the causative agent of so-called bitter or pink crab disease in a broad range of shellfish taxa. Outbreaks of Hematodinium-associated disease can devastate local fishing and aquaculture efforts. The goal of our study was to examine the potential role of the common shore (green) crab Carcinus maenas as a reservoir for Hematodinium. Carcinus maenas is native to all shores of the UK and Ireland and the North East Atlantic but has been introduced to, and subsequently invaded waters of, the USA, South Africa and Australia. This species is notable for its capacity to harbour a range of micro- and macro-parasites, and therefore may act as a vector for disease transfer. Methods Over a 12-month period, we interrogated 1191 crabs across two distinct locations (intertidal pier, semi-closed dock) in Swansea Bay (Wales, UK) for the presence and severity of Hematodinium in the haemolymph, gills, hepatopancreas and surrounding waters (eDNA) using PCR-based methods, haemolymph preparations and histopathology. Results Overall, 13.6% were Hematodinium-positive via PCR and confirmed via tissue examination. Only a small difference was observed between locations with 14.4% and 12.8% infected crabs in the Dock and Pier, respectively. Binomial logistic regression models revealed seasonality (P
Full-text available
Invasive alien species are likely to interact with climate change, thus necessitating management that proactively addresses both global changes. However, invasive species managers’ concerns about the effects of climate change, the degree to which they incorporate climate change into their management, and what stops them from doing so remain unknown. Therefore, we surveyed natural resource managers addressing invasive species across the U.S. about their priorities, concerns, and management strategies in a changing climate. Of the 211 managers we surveyed, most were very concerned about the influence of climate change on invasive species management, but their organizations were significantly less so. Managers reported that lack of funding and personnel limited their ability to effectively manage invasive species, while lack of information limited their consideration of climate change in decision-making. Additionally, managers prioritized research that identifies range-shifting invasive species and native communities resilient to invasions and climate change. Managers also reported that this information would be most effectively communicated through conversations, research summaries, and meetings/symposia. Despite the need for more information, 65% of managers incorporate climate change into their invasive species management through strategic planning, preventative management, changing treatment and control, and increasing education and outreach. These results show the potential for incorporating climate change into management, but also highlight a clear and pressing need for more targeted research, accessible science communication, and two-way dialogue between researchers and managers focused on invasive species and climate change.
Full-text available
Vibrio aestuarianus is a bacterium related to mortality outbreaks in Pacific oysters, Crassostrea gigas, in France, Ireland, and Scotland since 2011. Knowledge about its transmission dynamics is still lacking, impairing guidance to prevent and control the related disease spread. Mathematical modeling is a relevant approach to better understand the determinants of a disease and predict its dynamics in imperfectly observed pathosystems. We developed here the first marine epidemiological model to estimate the key parameters of V. aestuarianus infection at a local scale in a small and closed oyster population under controlled laboratory conditions. Using a compartmental model accounting for free-living bacteria in seawater, we predicted the infection dynamics using dedicated and model-driven collected laboratory experimental transmission data. We estimated parameters and showed that waterborne transmission of V. aestuarianus is possible under experimental conditions, with a basic reproduction number R0 of 2.88 (95% CI: 1.86; 3.35), and a generation time of 5.5 days. Our results highlighted a bacterial dose–dependent transmission of vibriosis at local scale. Global sensitivity analyses indicated that the bacteria shedding rate, the concentration of bacteria in seawater that yields a 50% chance of catching the infection, and the initial bacterial exposure dose W0 were three critical parameters explaining most of the variation in the selected model outputs related to disease spread, i.e., R0, the maximum prevalence, oyster survival curve, and bacteria concentration in seawater. Prevention and control should target the exposure of oysters to bacterial concentration in seawater. This combined laboratory–modeling approach enabled us to maximize the use of information obtained through experiments. The identified key epidemiological parameters should be better refined by further dedicated laboratory experiments. These results revealed the importance of multidisciplinary approaches to gain consistent insights into the marine epidemiology of oyster diseases.
Full-text available
The edible mussel Mytilus edulis is a major aquaculture commodity in Europe, with 168000 t produced in 2015. A number of abundant, well characterised parasites of the species are known, though none are considered to cause significant mortality. Haplosporida (Rhizaria, Endomyxa) is an order of protistan parasites of aquatic invertebrates, the best studied of which are the oyster pathogens Haplosporidium nelsoni and Bonamia ostreae. While these species are well characterised within their hosts, the diversity, life-cycle and modes of transmission of haplosporidians are very poorly understood. Haplosporidian parasites have previously been reported from Mytilus spp., however the majority of these remain uncharacterised, and no molecular data exist for any species. In this study, we identified 2 novel haplosporidian parasites of M. edulis present in the UK. The first of these, observed by light microscopy and in situ hybridisation infecting the gills, mantle, gonadal tubules and digestive connective tissues of mussels in the Tamar estuary, England, we describe as Minchinia mytili on the basis of 18S sequence data. The second, observed infecting a single archive specimen collected in Loch Spelve, Mull, Scotland, infects the foot muscle, gills and connective tissue of the digestive gland. Sequence data places this parasite in an uncharacterised clade of sequences amplified from tropical bivalve guts and water samples, sister to H. nelsoni. Screening of water and sediment samples collected at the sample site in the Tamar estuary revealed the presence of both sequence types in the water column, suggesting host-free or planktonic life stages.
Technical Report
Full-text available
Following the identification of the invasive non-native species Didemnum vexillum, Kott 2002, on a Scottish shellfish aquaculture unit, a control method was needed to permit the legal movement of live Pacific oysters (Magallana gigas) off from the farm, and which would kill the pest species but result in acceptable mortality in the aquaculture species. Following a literature review, the most relevant control method in our case was determined to be a bath treatment. Twelve relevant published studies were found describing bath treatments for fouled shellfish, although some results were contradictory and their interpretation was complex. This report presents a review and analysis of the evidence needed to have a control method accepted for initial trial by the Scottish shellfish industry and by the relevant national regulatory authorities. The control method that will go forward for field trial is immersion in freshwater for a minimum of 24 hours.
Full-text available
Routine shellfish aquaculture practices in British Columbia (BC) result in cultured Pacific oysters (Crassostrea gigas (Thunberg, 1793)) being moved from tunicate infested culture areas to non-infested areas for processing, thereby posing a potential risk of spreading the colonial ascidian Didemnum vexillum Kott, 2002 and other nuisance species, to new areas. Three intervention points (IPs; i.e., stages of the processing regime) were identified in existing aquaculture practices where clusters of C. gigas received manipulation or stress that could alter the cover of epibionts, notably D. vexillum. These IPs were: IP1) harvesting, IP2) transportation (from harvesting areas to processing plants), and IP3) processing (shucking of the oysters). The percentage coverage of D. vexillum on oyster clusters was evaluated at each IP for products originating from two aquaculture sites in BC, Lemmens Inlet and Okeover Inlet. A total of 60 clusters were sampled from Lemmens Inlet and 46 from Okeover Inlet. Results demonstrated a significant loss (P < 0.05) of D. vexillum coverage on C. gigas clusters from IP1 to IP3 for both sites. Although variations existed between the sites, the mean percentage coverage decreased from 48% post-harvest to 30% post-transportation and 17% post-shucking. Since shucked oyster shells still have a substantial cover of D. vexillum on them and are disposed of in areas exposed to tidal waters of un-infested bays, the risk of secondary introduction related to shellfish aquaculture practices remains high. Based on these results, thresholds could be set to reduce the risk of introducing D. vexillum into new areas, and new research is recommended to determine the risk of dispersal of D. vexillum should it be inadvertently introduced via shellfish movements.
The Phylum Haplosporidia consists of four genera (Minchinia, Haplosporidium, Urosporidium and Bonamia) that are endoparasitic protists of a wide range of marine invertebrates including commercial bivalve species. Characterization of haplosporidian species remains a challenge due to their patchy spatial and temporal distributions, host-restricted occurrence, and poorly known life cycles. However, they are commonly associated with significant mortality events in bivalves. Due to the recent sporadic mortality events that have occurred in cockles in Europe, the objectives of this study were to determine the diversity, distribution and seasonality of haplosporidian species in Cerastoderma edule populations at several Irish sites. The role of abiotic (temperature, salinity and dissolved oxygen in water) and biotic (cockle size and age) factors as drivers or inhibitors of haplosporidian infection were also assessed. Cockles (n = 998) from the intertidal were sampled from April/July 2018 to April 2019 at three sites with no commercial fishing activity on the south coast (Celtic Sea) and one site on the northeast coast (Irish Sea) with an active commercial fishery. Screening of the cockles by molecular techniques (PCR, Sanger sequencing) and by histopathology was carried out. Two species were identified and confirmed in Irish C. edule for the first time, Minchinia mercenariae -like (14.8%) and Minchinia tapetis (29.6%). Similar to other haplosporidian parasites, the Minchinia spp. detected in our study were present year-round at all sites, except for M. tapetis in Youghal Bay (Celtic Sea). Coinfection of both Minchinia species was only observed in Cork Harbour (Celtic Sea) and Dundalk Bay (Irish Sea), where Minchinia spp. showed a higher presence compared to Youghal Bay and Dungarvan Harbour (Celtic Sea). Moreover, haplosporidians detected with generic primers, were present at all of the sample sites throughout the year but had a higher occurrence during the winter months and were positively correlated with dissolved oxygen. Likewise, smaller and older C.edule seemed to be more vulnerable to the haplosporidian infection. Furthermore, haplosporidian distribution displayed spatial variability between and within sample sites, with the highest presence being observed in cockles at one of the commercially fished Dundalk beds, while the lowest presence was observed in cockles at the second Dundalk bed that was more influenced by freshwater runoff when the tide was out. Findings from this study provide additional information on the distribution and seasonal presence of novel haplosporidian species and their potential abiotic and biotic drivers/inhibitors of infection.
Ostreid herpesvirus-1 microVar (OsHV-1 µVar) has been responsible for significant mortalities globally in the Pacific oyster Crassostrea gigas. While the impact of this virus on the Pacific oyster has been significant, this pathogen may have wider ecosystem consequences. It has not been definitively determined how the virus is sustaining itself in the marine environment and whether other species are susceptible. The shore crab Carcinus maenas is a mobile predator and scavenger of C. gigas, commonly found at Pacific oyster culture sites. The aim of this study was to investigate the role of the crab in viral maintenance and transmission to the Pacific oyster. A field trial took place over 1 summer at different shore heights at 2 Irish Pacific oyster culture sites that are endemic for OsHV-1 µVar. Infection of OsHV-1 µVar in tissues of C. maenas at both shore heights of both sites was detected by polymerase chain reaction (PCR), quantitative PCR (qPCR), in situ hybridization and direct Sanger sequencing. In addition, a laboratory trial demonstrated that transmission of the virus could occur to naïve C. gigas within 4 d, from C. maenas previously exposed to the virus in the wild. These findings provide some insight into the possibility that the virus can be transmitted through marine food webs. The results also suggest viral plasticity in the hosts required by the virus and potential impacts on a range of crustacean species with wider ecosystem impacts if transmission to other species occurs.
Ascidians are important both as invasive species and as a fouling group in artificial marine habitats, causing negative impacts in aquaculture settings and the surrounding environment. The Ebro Delta is one of the major centres of bivalve production in the Mediterranean and is affected by proliferation of ascidian species (mostly introduced forms). Knowledge of the patterns of settlement and growth of the fouling species is mandatory to attempt mitigation measures. Settlement PVC plates were deployed from May to September 2015 at different depths (0.2, 1 and 2 m) in the Ebro Delta oyster aquaculture facilities. The occurrences of all species and the area cover of a selected subset of 6 species were monitored on a monthly basis from June 2015 to December 2016. Fifteen species were found, of which 10 are introduced. There were some differences between the deployed plates and the oyster ropes in species abundance and composition, likely due to differences in substrate complexity. For instance, Didemnum vexillum and Clavelina oblonga occurred in few plates in contrast to their abundance on oysters. The most abundant species were Styela plicata and Clavelina lepadiformis, which together with Ecteinascidia turbinata showed a preference to grow on plates deployed in May and June. Most of the species grew more at 0.2 m depth than at deeper plates. Thus, to minimise fouling on bivalves, spat immersion during fall and below 1 m depth is recommended. The number of occurrences and cover of the species was found to be similarly informative; suggesting that a periodic monitoring of species occurrence on replicate plates is sufficient for detecting new introduced species as soon as possible and will provide information useful for management.
The cockle Cerastoderma edule fishery has traditionally been the most important shellfish species in terms of biomass in Galicia (NW Spain). In the course of a survey of the histopathological conditions affecting this species in the Ria of Arousa, a haplosporidan parasite that had not been observed in Galicia was detected in one of the most productive cockle beds of Galicia. Uni- and binucleate cells and multinucleate plasmodia were observed in the connective tissue mainly in the digestive area, gills and gonad. The parasite showed low prevalence, and it was not associated with abnormal cockle mortality. Molecular identification showed that this parasite was closely related to the haplosporidan Minchinia mercenariae that had been reported infecting hard clams Mercenaria mercenaria from the Atlantic coast of the United States. The molecular characterization of its SSU rDNA region allowed obtaining a fragment of 1,796 bp showing 98% homology with M. mercenariae parasite. Phylogenetic analysis supported this identification as this parasite was clustered in the same clade as M. mercenariae from the United States and other M. mercenariae-like sequences from the UK, with bootstrap value of 99%. The occurrence of M. mercenariae-like parasites infecting molluscs outside the United States is confirmed.