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Marine Biology Research
ISSN: 1745-1000 (Print) 1745-1019 (Online) Journal homepage: http://www.tandfonline.com/loi/smar20
Impact of the invasive parasitic copepod Mytilicola
orientalis on native blue mussels Mytilus edulis in
the western European Wadden Sea
M. Anouk Goedknegt, Sarah Bedolfe, Jan Drent, Jaap van der Meer & David
W. Thieltges
To cite this article: M. Anouk Goedknegt, Sarah Bedolfe, Jan Drent, Jaap van der Meer & David
W. Thieltges (2018): Impact of the invasive parasitic copepod Mytilicola orientalis on native blue
mussels Mytilus edulis in the western European Wadden Sea, Marine Biology Research, DOI:
10.1080/17451000.2018.1442579
To link to this article: https://doi.org/10.1080/17451000.2018.1442579
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Published online: 29 Mar 2018.
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ORIGINAL ARTICLE
Impact of the invasive parasitic copepod Mytilicola orientalis on native blue
mussels Mytilus edulis in the western European Wadden Sea
M. Anouk Goedknegt , Sarah Bedolfe, Jan Drent , Jaap van der Meer and David W. Thieltges
Department of Coastal Systems, NIOZ Royal Netherlands Institute for Sea Research, and Utrecht University, P.O. Box 59, 1790 AB Den Burg,
The Netherlands
ABSTRACT
Invasive species can indirectly affect native species by modifying parasite–host dynamics and
disease occurrence. This scenario applies to European coastal waters where the invasive
Pacific oyster (Magallana gigas) co-introduced the parasitic copepod Mytilicola orientalis that
spills over to native blue mussels (Mytilus edulis) and other native bivalves. In this study, we
investigated the impact of M. orientalis infections on blue mussels by conducting laboratory
experiments using controlled infections with larval stages of the parasitic copepod. As the
impact of infections is likely to depend on the mussels’food availability, we also tested
whether potential adverse effects of infection on mussels intensify under low food
conditions. Blue mussels that were experimentally infected with juvenile M. orientalis had a
significantly lower body condition (11–13%) compared with uninfected mussels after nine
weeks of infection. However, naturally infected mussels from a mixed mussel and oyster bed
did not significantly differ in body condition compared with uninfected mussels. Contrary to
effects on mussel condition, we did not find an effect of experimental infections on
clearance rates, shell growth or survival of blue mussels and no clear sign of exacerbating
effects of food limitation. Our study illustrates that invasive species can indirectly affect
native species via parasite co-introductions and parasite spillover. The results of this study
call for the integration of such parasite-mediated indirect effects of invasions in impact
assessments of invasive species.
ARTICLE HISTORY
Received 18 December 2016
Accepted 14 February 2018
SUBJECT EDITOR
Roy Kropp
KEYWORDS
Invasive species; parasite co-
introduction; parasite
spillover; controlled
infections; Pacific oyster;
Magallana gigas
Introduction
Invasive species affect native species, communities and
ecosystems worldwide (Davies 2009; McGeoch et al.
2010; Lockwood et al. 2013), often directly via preda-
tion and competition (Parker et al. 1999; Simberloff
et al. 2013). However, invasive species can also exert
indirect effects, for example by changing habitat struc-
ture or modifying parasite–host dynamics. Parasite-
mediated indirect effects of invasive species can take
place via several mechanisms, including the co-intro-
duction of a parasite with an invasive host species
(parasite co-introduction; Lymbery et al. 2014). Conse-
quently, in the invaded range, a co-introduced parasite
might spill over from its invasive host to novel native
host species (parasite spillover; Prenter et al. 2004;
Kelly et al. 2009). Such parasite spillover events also
occur in marine ecosystems and can lead to emerging
diseases and subsequent mass mortalities of native
hosts with, in several cases, knock-on effects on
native communities and ecosystems (Goedknegt et al.
2016).
Parasite co-introduction and spillover have also
occurred with the introduction of the Pacific oyster,
Magallana (previously Crassostrea)gigas (Thunberg,
1793) to European coastal waters for aquaculture pur-
poses (Troost 2010). With the initial oyster imports in
the 1960s and 70s, the intestinal parasitic copepod
Mytilicola orientalis Mori, 1935 was co-introduced to
Europe (His 1977). The parasite’s native range is in Japa-
nese waters, and it has a direct life cycle with a free-
living larval dispersal stage, after which it resides in
the digestive tract of molluscs (Mori 1935). After its
co-introduction to Europe, the parasite spread first via
its principal host, the Pacific oyster, but was later
additionally found in wild native blue mussels (Mytilus
edulis Linnaeus, 1758), common cockles (Cerastoderma
edule (Linnaeus, 1758)), Baltic tellins (Limecola (formerly
Macoma)balthica (Linnaeus, 1758)) and European flat
oysters (Ostrea edulis Linnaeus, 1758), indicating
© 2018 Informa UK Limited, trading as Taylor & Francis Group
CONTACT M. Anouk Goedknegt Anouk.Goedknegt@nioz.nl Department of Coastal Systems, NIOZ Royal Netherlands Institute for Sea Research, and
Utrecht University, P.O. Box 59, Den Burg Texel 1790 AB, The Netherlands
Supplemental data for this article can be accessed https://doi.org/10.1080/17451000.2018.1442579. The underlying research materials for this article can
be accessed at https://doi.org/10.4121/uuid:7a4d46a5-048d-4ecc-a8c7-2de3b78d122e
MARINE BIOLOGY RESEARCH, 2018
https://doi.org/10.1080/17451000.2018.1442579
Published online 29 Mar 2018
several spillover events (His 1977; Stock 1993; Pogoda
et al. 2012; Goedknegt et al. 2017a). In particular,
native blue mussels are increasingly serving as new
hosts, with infection prevalences being similar to or
even exceeding those in Pacific oysters in some areas
(Pogoda et al. 2012; Goedknegt et al. 2017a). Native
blue mussels are also infected by Mytilicola intestinalis
Steuer, 1902, a related parasite species described
from the Mediterranean Sea (Steuer 1902), which has
a similar direct life cycle to M. orientalis (Caspers
1939; Grainger 1951; Dethlefsen 1985; Gee and Davey
1986), and was first found in the Wadden Sea in 1938
(Caspers 1939). Mytilicola intestinalis became infamous
as the ‘red worm disease’, because it was thought to
be the causative agent of mass mortalities of blue
mussels in the North Sea in the 1950s and 60s (Korringa
1968; Blateau et al. 1992). However, so far experimental
evidence to support this hypothesis is scarce. Histori-
cally, especially juvenile stages of M. intestinalis were
held responsible for mortalities of mussels (Korringa
1950; Dethlefsen 1985) because of their presence in
the ramifications of the digestive gland (Campbell
1970). In addition, the energy demand of young infec-
tive stages is expected to be high after exploiting their
egg yolk, the only food source available during the
pelagic larval phase (Grainger 1951). This increases
the chance for mussels to be negatively affected by
juvenile parasites. For mature M. intestinalis, lethal and
sublethal effects on important host fitness components
such as body condition, filtration rate and reproduction
do not seem to be unidirectional and have been contro-
versially discussed (Lauckner 1983). Only recently, con-
trolled infection experiments have been conducted
with M. intestinalis and blue mussels, in which a
reduction in blue mussel dry weight was found as a
result of infection by the parasite in sympatric para-
site–host populations (Feis et al. 2016). Similarly,
although M. orientalis is generally considered a serious
pest (Holmes and Minchin 1995) and is registered on
the list of 100 worst invaders of the Mediterranean
Sea (Streftaris and Zenetos 2012), studies on its lethal
and sublethal effects on M. gigas (Katkansky et al.
1967; Deslous-Paoli 1981; De Grave et al. 1995;Steele
and Mulcahy 2001)andOstrea lurida Carpenter, 1864
(Odlaug 1946) have been inconclusive and they have
generally lacked an experimental approach. The
effects of M. orientalis on its new host, the blue
mussel, have not been studied to date. A potential
effect of M. orientalis on native blue mussels is likely to
be modified by environmental factors and species inter-
actions (Hepper 1953; Campbell 1970; Lauckner 1983;
Troost 2010). For example, seasonal cycles, extreme
temperatures and inter- and intraspecific competition
may lead to food limiting conditions that can either alle-
viate or intensify the adverse effects of infection by the
parasitic copepod. Along these lines, Moore et al. (1977)
postulated that M. intestinalis actually lives in a com-
mensal relationship with their host, but that this
relationship can turn into a negative interaction in
times of serious food limitation. However, experimental
evidence for an augmented effect of Mytilicola spp.
infections on hosts at low food levels is lacking.
In this study, we investigated the effects of
M. orientalis infection on the native blue mussel
M. edulis by conducting two laboratory experiments
that used controlled infections of mussels with larval
stages of the copepod.Following the hypothesis of
Moore et al. (1977), we also tested whether the poten-
tial adverse effects of infection on survival, clearance
rate, body condition and shell growth of blue mussels
intensified under low food conditions. In addition, we
collected mussels in the field to determine natural
infection levels and to investigate the effect of Mytili-
cola infections on mussel body condition in a natural
environment. This combination of lab and field investi-
gations allowed us to assess the impact of the spillover
of the invasive parasite M. orientalis from invasive
Pacific oysters on native blue mussels.
Materials and methods
Field survey
To determine the natural prevalence (the proportion of
infected individuals) and mean infection intensity
(mean number of parasites in infected mussels) of Myti-
licola orientalis in blue mussels (Mytilus edulis) in the
wild, we collected 30 mussels of 30–50 mm length
from a mixed bed of Pacific oysters (Magallana gigas)
and blue mussels located on the Vlakte van Kerken, a
tidal flat on the east coast of the island of Texel
(Figure 1) in the western European Wadden Sea
(southern North Sea) on 11 June 2014. Additionally,
we collected from this bed another 39 mussels (34–
54 mm) to analyse the effect of infection status
(infected/uninfected) on mussel body condition.
Experimental infections
Uninfected mussels (30–35 mm; n= 150) for the exper-
iments were haphazardly collected from basalt groynes
on the north-west shore of the Dutch mainland (Julia-
nadorp, Figure 1) on 11 September 2014. Previous
explorations by M. A. Goedknegt had shown that Myti-
licola spp. does not occur at this location, which was
verified by haphazardly sampling and screening 30
2M. A. GOEDKNEGT ET AL.
additional mussels, which were all found to be free of
infection. Any epifauna (mostly barnacles) on the
mussels was carefully removed from the shells to
ensure that copepod larvae could infect mussels
without being eaten or physically obstructed during
experimental infections (Johnson and Thieltges 2010).
Until the infection procedure, collected mussels were
maintained in 75-l flow-through tanks at 18°C under a
24-hour light cycle (12 h light and 12 h dark) and fed
three times per week with fresh Isochrysis galbana
Parke, 1949 culture, or alternatively with Phyto-Feast®
when fresh culture was unavailable (on average once
a week).
To acquire a source for M. orientalis larvae, mussels (n
= 140) were haphazardly collected from a mixed bed
with known Mytilicola infections located on a tidal flat
on the east coast of the island of Texel (Figure 1)on
22 August 2014. Within two days of collection, mussels
were dissected and gravid M. orientalis females
extracted, which were identified and distinguished
from M. intestinalis based on descriptions of Mori
(1935) and Elsner et al. (2011). The egg sacs were separ-
ated from the female and placed in individual petri
dishes (Ø 60 mm) filled with seawater. They were incu-
bated at approximately 30°C to expedite the larval
development time (based on results of a pilot study)
and were monitored daily. Larval stages were identified
based on descriptions of M. intestinalis larvae by Gee and
Davey (1986). When larvae had developed into the infec-
tive copepod I stage (Gee and Davey 1986), uninfected
mussels (n= 74) were exposed to the larvae.
Before exposure to infective larvae, the shell length
(maximum anteroposterior axis) of each mussel was
measured with callipers to the nearest mm. Because
of variation in larval hatching and development time,
insufficient larvae were available to infect all mussels
at once, ultimately resulting in two temporal exper-
iments with infected mussels. Individual mussels in
the first experiment (n= 34 mussels) were exposed to
parasites in a 100-ml cup and after 24 h, mussels and
filtered seawater were transferred to a 1000-ml con-
tainer for the following eight weeks of the experiment.
For individual mussels in the second experiment (n=
30), exposure was carried out directly in the 1000-ml
container, where they remained for the entire nine
weeks of this study.
Exposure of mussels to infective larvae was done by
carefully pipetting (200-μl pipette) 25 larvae from the
Petri dish (by the use of a stereo microscope) and
depositing them into the container with an uninfected
mussel and filtered seawater. To promote filtration and
uptake of infective larvae, small amounts of algal
culture (I. galbana) were added to the seawater
during exposure of mussels of the second experiment.
Five extra mussels for each of the two experiments
were artificially infected and sacrificed for examination
of larval development at mid-way points during the
experiments. Control (uninfected) mussels (Experiment
1: n= 34, Experiment 2: n= 30) were treated identically
to exposed mussels within each experiment (including
the transfer of filtered seawater and addition of small
amounts of algal culture) but without the addition of
copepodites.
Experimental set-up
The experiment was set up immediately after exposing
the mussels to the parasite larvae, which was for the
first experiment on 10 September 2014 and the
second experiment on 7 October 2014. The experiment
was run in a two-factorial design with M. orientalis
infection (infected/uninfected) and food level (high/
low) as fixed factors and set up in blocks of four
1000-ml containers each containing one individual
mussel. Thus, each block contained a replicate of the
following treatments: uninfected mussel –low food
level, infected mussel –low food level, uninfected
mussel –high food level, and infected mussel –high
food level. The first experiment contained 17 of these
replicated blocks (n= 68 mussels in total), while the
second experiment contained 15 replicated blocks (n
= 60 mussels in total). The containers were kept in a
climate-controlled room at 18°C and seawater was
replaced weekly. All mussels were fed three times per
week, with mussels in the high food treatment receiv-
ing 50 ml algae mixture and mussels in the low food
Figure 1. Sampling locations of wild blue mussels (Mytilus
edulis) (squares; n= 30 and n= 39), blue mussels as a source
for gravid Mytilicola orientalis females (circles; n= 140) and
naturally uninfected blue mussels (triangles; n= 150).
MARINE BIOLOGY RESEARCH 3
treatment receiving half that quantity. When fresh
algae culture was unavailable, on average once per
week, 0.1 ml of PhytoFeast® per mussel was provided
in the high food treatment, while 0.05 ml was provided
to mussels in the low food treatment. Only fresh algal
culture was provided on days when clearance rates
were measured.
After dissections at the end of the experiment, we
found that some experimental blocks contained
mussels with failed infections (exposed to larvae but
not found to be infected at the end of the experiment),
or unanticipated infections (found to be infected
despite not having been exposed to larvae). In these
cases, affected blocks were excluded from the analysis
to preserve a balanced design with a complete dataset.
In the first experiment, four mussels (out of 34 mussels
exposed to larvae) remained uninfected and one
mussel was unintentionally infected, while in the
second experiment infection success was lower and
seven (out of 30) mussels were uninfected. After
removing all blocks with failed and unanticipated infec-
tions, 12 blocks were left for the first experiment (n=48
mussels) and 10 blocks for the second experiment (n=
40 mussels).
Measurement of clearance rate, body condition
and shell growth
Clearance rate
We conducted the first clearance rate measurements of
the mussels of the first experiment one week after
exposure to M. orientalis larvae, while the clearance
rate of mussels of the second experiment was measured
immediately (one day) after exposure. We continued to
measure the clearance rate of each mussel once per
week, to assess if and when larval maturation affected
the clearance rate of mussels.
Clearance rate was assessed by means of the indirect
clearance method (Riisgård 2001;Petersenetal.2004).
One day prior to the measurement, all containers were
refreshed with filtered seawater and mussels were
checked for survival (severely gaping mussels in smelly
water were assigned as dead). In the morning before
the test, we made dilutions of live algal (I. galbana)
culture and analysed its density using a CASY® Cell
Counter and Analyser System Model TT (Schärfe
System GmbH). The algal dilutions were calibrated to
Relative Fluorescence Units (RFU) using a Trilogy® Lab-
oratory Fluorometer (Turner Designs), which allowed
us to measure a large number of samples in a short
period of time. Fluorometer measurements required
1.5 ml of water that we obtained from each experimen-
tal container with a 2-ml pipette.
We measured background fluorescence levels (RFU)
immediately before the test and created a calibration
curve to calculate the amount of algal culture needed
to create an initial starting concentration of 13 × 10
3
–
14 × 10
3
algal cells per ml in each experimental con-
tainer. This level was chosen to avoid very high or
very low algal densities, which are known to hamper fil-
tration by mussels (Clausen and Riisgård 1996) and
because it falls in the middle of the range in which
mussel filtration rate is independent of food density
(Riisgård and Randløv 1981). Algal culture of the calcu-
lated quantity was added once to all experimental con-
tainers and fluorescence was measured immediately
after addition (t
0
) and again after one (t
1
) and two
hours (t
2
). The measured RFU values at these measure-
ment intervals were corrected for background fluor-
escence after which these values were transformed to
number of algal cells by the use of the calibration
curve. Subsequently, the decrease in algal cells over
two hours was estimated by calculating the slope of
the regression line of the ln-transformed cell numbers
as a function of time (in min; after Stier et al. 2015).
Finally, to assess the clearance rate in ml min
−1
we mul-
tiplied the slope of each individual regression by
−1000, to account for the 1000 ml volume of water in
which the mussels were kept during the measure-
ments. Some mussels did not filter at all during the
measurements and therefore a separate category (suc-
cessful filtration: yes/no) was used as a random effect in
the clearance rate mixed model to take this variation
into account.
Body condition and shell growth
The experiments ran for eight (Experiment 1) and nine
weeks (Experiment 2) and immediately after termin-
ation of the experiments all mussels were measured,
screened for presence of M. orientalis larvae, frozen,
freeze-dried and weighed to assess body condition.
We separated the shells from the mussel tissue and
extracted adult copepods from the intestines. Larvae
were left in the tissue, as these were too small to
handle without disturbing the mussel flesh. Mussel
tissue was compressed between glass slides and exam-
ined under a stereo microscope (magnification 10–
80×) to account for all parasitic copepods, including
larvae and juveniles (Gee and Davey 1986). We then
carefully removed the mussel tissue from the plates,
deposited it in glass vials, froze (−20°C for at least 24
hours) and freeze-dried it (48 hours) to ultimately
measure the dry weight of the tissue. Condition index
was determined as CI = DW L
−3
, where DW is the dry
weight (mg) of the tissue and L is the final shell
length (cm, after Petersen et al. 2004). At termination
4M. A. GOEDKNEGT ET AL.
of the experiment, the length of each individual mussel
shell was measured to the nearest mm with callipers.
Shell growth was then calculated by extracting initial
length from the final length of each mussel.
Statistical analysis
All statistical analyses were performed using the stat-
istical software environment R (R Development Core
Team 2015) and model assumptions were confirmed
using qq plots and histograms (Zuur et al. 2010).
When data were not normally distributed, we applied
appropriate transformations. Pvalues of < 0.05 were
considered significant.
The condition index of wild mussels was log
10
-trans-
formed, before the difference in naturally infected and
uninfected mussels was analysed with a Student’st-
test. The results of the two laboratory experiments
were analysed separately, but using similar models.
To test for effects of the parasite on clearance rate of
the mussels, we applied a square-root transformation
and used a linear mixed model (lmm; lmer function
from the package lme4; Bates et al. 2015) with infection
status, food level, time, the interaction between these
three variables and experimental block as fixed
factors. Individual mussels and successful filtration
were included as random effects. We used a similar
model to investigate the effect of infection intensity
(number of M. orientalis individuals per infected
mussel) on clearance rate of infected mussels, with
the number of M. orientalis individuals as predictor vari-
able in the model. For all these mixed models, Pvalues
were obtained by comparing the full model (with all
fixed effects) against a reduced model (without the
fixed effect in question) with a likelihood ratio test.
Condition index was log
10
-transformed to improve
normality of the data. To test for effects of the parasites
on the condition of blue mussels, we applied a general
linear model (glm) with infection status, food level,
their interaction and the blocking factor as explanatory
variables. Again, we used a similar model to investigate
the effect of infection intensity on the condition index
of infected mussels, but we replaced infection status
with M. orientalis intensity.
Finally, to test for the effects of the parasite on mussel
shell growth, we first modelled shell growth against
mussel shell length at the start of the experiment with
a linear model. We took the residuals from this model
as a proxy for shell growth (corrected for initial length)
and subsequently used a general linear model to test
for the effects of infection status, food level, their inter-
action, and experimental blocking as explanatory vari-
ables. Finally, we tested the effect of intensity of
infection on (corrected) shell growth of infected
mussels, by using a similar linear model where we
replaced the infection category by numbers of
M. orientalis.
Results
Natural infections
Mytilicola orientalis prevalence in blue mussels (Mytilus
edulis,n= 30, 30–50 mm) in a mixed mussel (M. edulis)
and Pacific oyster (Magallana gigas) bed on a tidal flat
on the east coast of Texel was 53% with a mean (± SE)
intensity of 1.8 (± 0.3) individuals per infected host.
From this sample batch, 10 mussels had the same
size class as the mussels used in our experiment (30–
35 mm) and this group had a prevalence of 50% and
mean intensity (± SE) of 3.0 ± 0.7. Naturally infected
mussels (n= 18) tended to have 20% lower condition
indices than uninfected mussels (n= 21), however the
difference was not statistically significant (t= 8.880, P
= 0.068; Figure 2; for raw data see Goedknegt et al.
2018)).
Figure 2. Condition index (log
10
-transformed) of uninfected
blue mussels (Mytilus edulis)(n= 21) and mussels infected
with Mytilicola orientalis (n= 18) collected on a mixed blue
mussel (M. edulis) and Pacific oyster (Magallana gigas) bed
on a tidal flat on the east coast of Texel (Vlakte van Kerken,
Texel, The Netherlands; see Figure 1). The boxes represent
the interquartile range, the whiskers denote the lowest and
highest values within the 1.5 interquartile range, the black
line in each box denotes the median and the black dots rep-
resent the mean condition indices of each group. Note the
truncated y-axis.
MARINE BIOLOGY RESEARCH 5
Success of controlled infections
Hatching success
Dissection of 140 source mussels from a mixed blue
mussel and Pacific oyster bed on the east coast of
Texel (Vlakte van Kerken) produced 60 egg sacs (preva-
lence of gravid females was 43%). The time from egg
sac extraction to hatching of copepod larvae was
highly variable and ranged from immediate hatching
to 8 d after extraction, with an average of 4.4 d. At
early phases the eggs were opaque (Figure 3a), but
when close to hatching, the eggs became transparent
and the red eye spots of the larvae became visible
through the egg case (Figure 3b). All eggs within an
individual egg sac developed at similar rates. The nau-
plius phase (Figure 3c) lasted a maximum of 1 d, and
infective copepodite I larvae (Figure 3d) appeared on
average 4.8 d after egg extraction, though the earliest
larvae metamorphosed within two days. After eight
days, larval survival declined, and the collection
period was terminated. The nauplii were 200–220 μm
in length and the copepodite I (infective) stages were
240–290 μm long. An average of 50 copepodite
larvae successfully emerged from a single female’s
egg sac pair, although the maximum recorded was
more than 200 copepodites in a pair. Hatching
success ranged from 0 to 100%, and overall, 26.1% of
the eggs failed to hatch.
Infection success
In the first experiment, mussels had a higher infection
success rate (88%) than in the second experiment
(76%). The maximum number of individual
M. orientalis found in a single mussel was 12. Average
intensities of controlled infections in both experiments
were comparable to those of similar-sized mussels in
the field (mean ± standard deviation, SD; Experiment
1: 3.0 ± 2.4, Experiment 2: 3.5 ± 3.2). Like adults, juvenile
copepods were found in the digestive tract near the
stomach of blue mussels and were approximately
2 mm long at termination of the experiment (Figure
4). Therefore, the copepodites had increased about
10 times in size, growing at a rate of about 30 μm
day
−1
after infecting their hosts. As the copepods
were not yet grown to mature size, it was impossible
to determine their sex. The uninfected control
mussels were confirmed to be free of infection,
except for one mussel in the first experiment that
was infected with two adult female M. orientalis.
Effects on mussel clearance rate
No mortality occurred in either infected or uninfected
mussels in either of the two experiments. In both
experiments, 13% of the mussels did not filter during
the experimental period, and non-filtering mussels
occurred across all treatments. We did not find signifi-
cant overall effects of infection status or food limitation
on mussel clearance rates (Supplementary Table SI),
but clearance rate significantly differed over time in
the second (lmm; P= 0.150 * 10
−13
;Figure 5b) but
not in the first experiment (P= 0.722; Figure 5a; for
raw data see Goedknegt et al. 2018). This difference
probably results from the relatively high clearance
rates in the first week and relatively lower clearance
rates in week 7 of Experiment 2 in comparison to Exper-
iment 1 (Figure 5). When testing for the effects of
M. orientalis intensity upon infected mussels, we
found no significant results for any of the factors in
the first experiment and no significant interaction
between food level and infection intensity, but a sig-
nificant effect of time (P= 0.261 * 10
−6
; Table SI) in
the second experiment.
Effects on mussel body condition
Experimentally infected mussels had significantly lower
body condition indices compared with uninfected
Figure 3. Developmental phases of Mytilicola orientalis: (a) a pair of egg sacs, (b) eggs about to hatch (note the red eye spots), (c)
nauplius and (d) infective copepodite I larva. The white scale bars denote 200 μm.
6M. A. GOEDKNEGT ET AL.
mussels (mean ± standard error, SE: Experiment 1:
infected mussels 0.74 ± 0.03, uninfected mussels 0.81
± 0.01; Experiment 2: infected mussels 0.68 ± 0.02,
uninfected mussels 0.75 ± 0.02; Figure 6; Supplemen-
tary Table SII; for raw data see Goedknegt et al. 2018).
Furthermore, infected mussels kept under low food
levels had the tendency to have lower condition
indices, whereas uninfected mussels had slightly
increased condition indices (Figure 6). However, in
both experiments, the effect of food level and the inter-
action between infection status and food level was not
significant (Table SII).
In additional analyses, where we tested for an effect
of M. orientalis intensity and food limitation on the con-
dition index of infected mussels, we found different
results for both experiments. In the first experiment,
we found a positive relationship between M. orientalis
intensity and body condition of mussels (P= 0.046),
but this result was not significant in the second exper-
iment (Table SII). Additionally, the block factor was sig-
nificant in the first experiment (P= 0.013), but not
Figure 4. Developmental status of Mytilicola orientalis infections in blue mussels (Mytilus edulis) after approximately (a) 5.5 weeks
(scale bar denotes 1000 μm) and (b) 8 weeks (scale bar denotes 500 μm) after exposure to infective larvae.
Figure 5. Mean clearance rate (± SE) of uninfected blue
mussels (Mytilus edulis) (grey) and mussels infected with Myti-
licola orientalis (black) fed under high (circles) and low (tri-
angles) food levels. Clearance rates were measured weekly
after exposure to infective larvae for each of the four treatment
groups in the first (a) and second (b) experiment.
Figure 6. Boxplots of the condition index (log
10
-transformed)
of blue mussels (Mytilus edulis) in the first (a) and second (b)
experiment that were either infected (grey) or uninfected
(white) and kept under high or low food level conditions
during the nine weeks of the experiment. The boxes represent
the interquartile range, the whiskers denote the lowest and
highest values within the 1.5 interquartile range, the black
line in each box denotes the median, the large black dots rep-
resent the mean condition indices of each group and the
smaller dots outside the boxes are outliers. Note the truncated
y-axis.
MARINE BIOLOGY RESEARCH 7
significant in the second experiment (Table SII). Finally,
we did not find any significant effects of food level or
an interaction between intensity and food level in
both experiments (Table SII).
Effects on mussel shell growth
Mean mussel shell growth ± SE was 0.56 ± 0.05 mm in
the first experiment and 0.49 ± 0.07 mm in the second
experiment, which is an average of about 0.01 mm
day
−1
. In both experiments, mussel shell growth (cor-
rected for initial shell length) was not significantly
affected by M. orientalis infection status, food level,
the interaction between those variables or the blocking
factor (Supplementary Table SIII; for raw data see Goed-
knegt et al. 2018). Furthermore, among only the
infected mussels we did not detect any significant
effect of infection intensity, food level, an interaction
between those terms or an effect of experimental
blocking on the shell growth of infected mussels in
either experiment (Table SIII).
Discussion
This study experimentally tested for the effects of the
invasive parasitic copepod Mytilicola orientalis (which
has recently spilled over from invasive Pacific oysters
(Magallana gigas)) on native blue mussels (Mytilus
edulis).In laboratory experiments, we found significant
negative effects of infection with (juvenile stages of)
the invasive parasite on the body condition of
mussels, although the feeding, growth and survival
were not affected.
The detrimental effect of early developmental
stages of Mytilicola infection has been previously
suggested for the congeneric M. intestinalis (Korringa
1950; Dethlefsen 1985) because of their presence in
the ramifications of the digestive gland (Campbell
1970). This is the digestive organ in molluscs, and infec-
tions may compromise its functioning. As stable
isotope analyses suggest that Mytilicola feeds on host
tissue (Gresty and Quarmby 1991; Goedknegt et al.
2017b), the energy demand of the growing copepods
can be expected to lead to a significant loss of tissue,
ultimately resulting in a potential lower host condition
(11–13% reduction in our experiments). Moreover,
when Mytilicola feeds on host tissue, the resulting
metaplasia of the host gut epithelium (Moore et al.
1977) needs to be repaired, which is an energetically
demanding process for the host and likely to reduce
host condition. When Mytilicola matures, the effects
of the parasite may become less severe as the cope-
pods move away from the digestive gland and
migrate further down the digestive tract (Grainger
1951; Gee and Davey 1986). A decrease in harmfulness
with parasite age may also explain why we did not find
significant difference in condition between infected
and uninfected mussels in the field, as those infections
consisted of a mix of juvenile and adult stages of
M. orientalis. Generally, adverse effects of M. orientalis
on host condition have also been reported for
oysters, Ostrea lurida (Odlaug 1946) and M. gigas (Kat-
kansky et al. 1967) and are known to increase with
infection intensity (Katkansky et al. 1967). Similarly,
for the congeneric species M. intestinalis reductions in
dry weight of its blue mussel host are more severe
when the parasite occurs in higher numbers in sympa-
tric parasite–host populations (Feis et al. 2016).
However, in our experiments we could not find a
general trend of declining mussel condition with
M. orientalis intensity, as the two experiments gave
contrary results in this respect. Nevertheless, the
general negative effect of infections with early stages
of M. orientalis on mussel body condition suggests
that native mussels may experience negative effects
from the spillover of this invasive parasite. The exact
mechanism behind the loss in body condition and
the effect of the parasite on the energy budgets
(lipids/glycogen content) of mussels, are a topic for
future studies.
In contrast to the adverse effects of juvenile
M. orientalis on mussel condition, we found no evi-
dence that M. orientalis infection impacted the clear-
ance rates of mussels. Previous work has observed a
reduced filtration capacity in mussels infected with tre-
matode metacercariae, which encyst in mussel gills and
palps and interfere with filtration (Stier et al. 2015).
However, as Mytilicola resides in the mussels’intestines
it may not directly affect gill function in the same way
as trematode metacercariae. Instead, Mytilicola infec-
tions may only indirectly affect filtration by influencing
host energy requirements and expenditure. Alterna-
tively, mussels could intensify their filter activity when
infected with M. orientalis to counterbalance the
higher energy demand caused by the parasite.
However, we did not observe any significant effects
of parasite infection on mussel clearance rates. We
acknowledge that our inference in this respect might
have been hampered due to the variation in clearance
rates we observed over time, especially in the second
experiment. Part of this variation is explained by the
mussels that did not filter during our experiments,
which we therefore included as a random effect in
the model. Overall, observed clearance rates were rela-
tively low and in many cases dropped to less than
10 ml min
−1
, which is lower than filtration rates
8M. A. GOEDKNEGT ET AL.
previously reported for mussels under comparable
algal concentrations (Clausen and Riisgård 1996; Stier
et al. 2015). The underlying reasons for these low
values are not known, but may explain the limited
shell growth of all mussels (on average 0.01 mm
day
−1
). The absence of any effect of M. orientalis on
mussel shell growth may also be related to the low
growth, but this result also corresponds with observa-
tional studies of Pacific oysters which did not detect
negative effects of infections on growth (Katkansky
et al. 1967; Steele and Mulcahy 2001). Finally, a nega-
tive effect of the parasite on mussel survival could
have been expected given that its congeneric
M. intestinalis has been considered to be the causative
agent of mussel mass mortalities in the past (Korringa
1968; Blateau et al. 1992). However, in our experiments
there was no mortality of mussels among treatments,
illustrating the sub-lethality of the parasite that has
also previously been shown for Pacific oysters (Kat-
kansky et al. 1967).
In contrast to our expectation, we did not find clear
evidence that food shortage exacerbated the effects of
Mytilicola infections at the food levels applied, which
were chosen to lie within the range of concentration
where mussels actually filter. It may well be that
adverse effects of infections under even more
extreme scenarios, such as severe starvation, may
occur and future studies could investigate the effects
of the parasite under more extreme food conditions.
In general, controlled infections of hosts with Mytili-
cola infective larval stages proved to be an effective
method to study effects of the parasite on blue
mussel hosts, which can be applied in subsequent
studies with other host species as they would help to
overcome the lack of strong inference in earlier corre-
lative studies on both Mytilicola species. Here, we
have developed a successful technique to harvest the
invasive parasite M. orientalis and to infect its new
blue mussel host under laboratory conditions. The
lack of mortality of mussels among treatments during
the experiments, suggests that only sublethal effects
of the parasite occurred, and that our experimental
procedures were non-lethal. Furthermore, we have
documented the maturation of M. orientalis larval
stages from the moment of hatching to the develop-
ment of the infective stage (see also Pogoda et al.
2012), which typically took less than one week under
the relatively high temperatures used to increase
development speed (i.e., the planktonic phase is likely
to be longer in natural populations). Our infection
methods were successful (success rate 71–88%), and
we achieved mean intensities (about 3 copepods per
infected host) that were similar to intensities observed
in natural populations of similar-sized infected mussels.
As the results of our varying infection techniques only
marginally differed between experiments, the addition
of food during parasite exposure and the size of the
infection containers do not appear to drastically
affect the outcome of laboratory infections. Given
that Gee and Davey (1986) estimated a maturation
period of 70.8 (± 16.6, 95% confidence interval) days
at 14–18°C and just 8.3 (± 4.1) days at 18–22°C for
M. intestinalis, we expected our experiments to
provide ample time for the parasites to achieve matur-
ity. However, our screenings unexpectedly revealed
almost exclusively juvenile M. orientalis after nine
weeks in the experiment, carried out at 18°C. This
may indicate that M. orientalis maturation times are sig-
nificantly longer than M. intestinalis because of a lower
tolerance for cool temperatures and further studies will
be needed to determine developmental times of the
parasite at various temperatures.
In conclusion, this is the first study in which con-
trolled laboratory infections with the invasive
copepod M. orientalis were performed on its new
native blue mussel host. We discovered that infections
with early stages of the copepod (up to nine weeks)
lead to lower condition of infected mussels. As our
study was performed with juvenile stages of the para-
sitic copepod, potential impacts of adult parasites
remain to be investigated. In our experimental study,
we challenged the mussels with only two stressors
(infection with M. orientalis and limiting food con-
ditions). However, for mussels living on natural
mussel beds, stressors may be more diverse and
severe (e.g., extreme temperatures, infections with
multiple parasite species, resource competition with
other species), opening perspectives for future
studies. Such studies will be important to identify the
full range of indirect effects of invasive oysters and
other invasive species on native biota via parasite co-
introductions and subsequent indirect parasite-
mediated effects via parasite spillover.
Acknowledgements
We are grateful to Jarco Havermans for help in the field and
laboratory, Josje Snoek and Kirsten Scholten for maintaining
the algae cultures and Rob Dekker for pointing out unin-
fected mussel populations on basalt groins near Julianadorp.
We also thank three anonymous reviewers and Subject Editor
Dr Roy Kropp for their useful comments on earlier drafts of
this manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
MARINE BIOLOGY RESEARCH 9
Funding
This work was supported by the Nederlandse Organisatie
voor Wetenschappelijk Onderzoek (NWO) and the German
Bundesministerium für Bildung und Forschung (BMBF)
[grant number 839.11.002].
ORCID
M. Anouk Goedknegt http://orcid.org/0000-0002-8637-0779
Jan Drent http://orcid.org/0000-0003-1277-5957
Jaap van der Meer http://orcid.org/0000-0003-4818-2408
David W. Thieltges http://orcid.org/0000-0003-0602-0101
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