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The early stages of intertidal mussels, including the green-lipped mussel, Perna canaliculus, face both direct and indirect environmental threats. Stressors may influence physiological status and, ultimately, survival. An understanding of the nature of stress experienced is critical to inform conservation and aquaculture efforts. Here, we investigated oxidative stress dynamics in juvenile P. canaliculus in relation to emersion duration (1–20 h) and relative humidity (RH, 29–98%) by quantifying oxidative damage (protein carbonyls, lipid hydroperoxides, 8-hydroxydeoxyguanosine) and enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase and reductase). Mussels held in low RH during emersion experienced severe water loss (>70%), high mortality (>80%) and increased oxidative damage (35–45% increase compared to control conditions), while mussels held at high RH were not impacted, even after 20 h of air exposure. Following re-immersion, reoxygenation stress resulted in further increases in damage markers in mussels that had experienced dryer emersion conditions; protective action of antioxidants increased steadily during the 10 h re-immersion period, apparently supporting a reduction in damage markers after 1–5 h of immersion. Clearly, conditions during emersion, as well as duration, substantially influence physiological performance and recovery of juvenile mussels. Successful recruitment to intertidal beds or survival in commercial aquaculture operations may be mediated by the nature of emersion stress experienced by these vulnerable juveniles.
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Metabolites 2021, 11, 580. https://doi.org/10.3390/metabo11090580 www.mdpi.com/journal/metabolites
Article
Emersion and Relative Humidity Modulate Stress Response
and Recovery Dynamics in Juvenile Mussels
(Perna canaliculus)
Natalí J. Delorme 1,*, David J. Burritt 2, Norman L. C. Ragg 1 and Paul M. South 1
1 Cawthron Institute, Private Bag 2, Nelson 7042, New Zealand; norman.ragg@cawthron.org.nz (N.L.C.R.);
paul.south@cawthron.org.nz (P.M.S.)
2 Department of Botany, University of Otago, PO Box 56, Dunedin 9054, New Zealand;
david.burritt@otago.ac.nz
* Correspondence: natali.delorme@cawthron.org.nz; Tel.: +64-036-687-755
Abstract: The early stages of intertidal mussels, including the green-lipped mussel, Perna canaliculus,
face both direct and indirect environmental threats. Stressors may influence physiological status
and, ultimately, survival. An understanding of the nature of stress experienced is critical to inform
conservation and aquaculture efforts. Here, we investigated oxidative stress dynamics in juvenile
P. canaliculus in relation to emersion duration (1–20 h) and relative humidity (RH, 29–98%) by quan-
tifying oxidative damage (protein carbonyls, lipid hydroperoxides, 8-hydroxydeoxyguanosine) and
enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase and reductase).
Mussels held in low RH during emersion experienced severe water loss (>70%), high mortality
(>80%) and increased oxidative damage (35–45% increase compared to control conditions), while
mussels held at high RH were not impacted, even after 20 h of air exposure. Following re-immer-
sion, reoxygenation stress resulted in further increases in damage markers in mussels that had ex-
perienced dryer emersion conditions; protective action of antioxidants increased steadily during the
10 h re-immersion period, apparently supporting a reduction in damage markers after 1–5 h of im-
mersion. Clearly, conditions during emersion, as well as duration, substantially influence physio-
logical performance and recovery of juvenile mussels. Successful recruitment to intertidal beds or
survival in commercial aquaculture operations may be mediated by the nature of emersion stress
experienced by these vulnerable juveniles.
Keywords: green-lipped mussel; GreenshellTM mussel; Perna canaliculus; spat; emersion; oxidative
stress; reoxygenation stress; recovery; survival
1. Introduction
Emersion is a significant source of stress for marine organisms. During emersion,
organisms can be exposed to fluctuations in temperature, irradiance, and relative humid-
ity (RH), with many intertidal organisms having physiological and behavioural adaptions
that allow them to cope with such stressors [1,2]. For example, mussels can tolerate heat
exposure and prevent desiccation by modifying their gaping behaviour, metabolism, and
respiration [3–7]. The capacity to tolerate emersion in marine invertebrates closely relates
to their bathymetric distribution [8,9]. Some bivalve molluscs depress their metabolism
during emersion [10] or rely on anaerobic pathways to maintain ATP production for short
emersion periods [11,12]; air-gaping during long-term emersion exposure may subse-
quently assist in acid-base regulation [13]. These different strategies to deal with emer-
sion-related hypoxia are then likely to affect the organism’s responses following re-im-
mersion in seawater.
Citation:
Delorme, N.J.; Burritt, D.J.;
Ragg, N.L.C.; South, P.M. Emersion
and
Relative Humidity Modulate
Stress Response
and Recovery
Dynamics
in Juvenile Mussels
(
Perna canaliculus). Metabolites 2021,
11
, 580. https://doi.org/10.3390/
metabo11090580
Academic Editors
: Pierre Blier and
Hélène Lemieux
Received:
20 July 2021
Accepted
: 24 August 2021
Published:
27 August 2021
Publisher’s Note:
MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and institu-
tional affiliations.
C
opyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
diti
ons of the Creative Commons At-
tribution (CC BY) license (http://crea-
tivecommons.org/licenses/by/4.0/).
Metabolites 2021, 11, 580 2 of 17
Immersion in seawater after an emersion period is critical for the recovery and sur-
vival of the organism but is itself a stressor due to the oxidative damage caused to macro-
molecules (lipids, proteins and DNA) by rapid reoxygenation of the cells and the accumu-
lation of free radicals and reactive oxygen species (ROS) in the cells [14,15]. ROS are pro-
duced in the cell through normal metabolism, and in molluscs they represent around 1–
3% of the consumed oxygen [16]. ROS are neutralised by enzymatic and non-enzymatic
antioxidants [17]; however, under stressful conditions, levels/activities of cellular antiox-
idants can be too low to cope with the production of ROS, resulting in oxidative damage
and, ultimately, cell death [14,15,17]. Due to physical limitations to oxygen uptake during
periods of air exposure, emersion often does not result in an immediate increased in oxi-
dative damage [18]. However, the oxygenation of haemolymph in bivalves that had pre-
viously experienced emersion increased rapidly within the first hour of re-immersion in
seawater [19]. This reoxygenation can result in a significant increase in ROS formation in
bivalves, together with significant changes in oxidative damage and antioxidant levels/ac-
tivities [20–23]. To date, most studies of the effects of emersion and recovery in marine
bivalves have focused on adults [1823]. Such effects possibly vary among life stages due
to ontogenetic differences in metabolism, respiration, and behaviour, although few stud-
ies have reported emersion- and recovery-induced stress responses in juvenile marine in-
vertebrates, e.g., [24,25].
Juvenile mytilid mussels (‘spat’) may be particularly vulnerable to the repercussions
of emersion and reoxygenation stress. Settling juveniles typically colonise substrates free
of adult mussels [26], which may reflect an area that is particularly affected by the stress-
ors associated with emersion. Mussel spat also retain the capacity to resume pelagic drift-
ing by the production of a mucus ‘parachute’, facilitating relocation but elevating the risk
of predation [27]. It has been suggested that environmental stress may influence the re-
sumption of pelagic drifting [28], and hence indirectly affect subsequent survival. The
present study evaluated the stress response and recovery dynamics of juvenile, green-
lipped mussels, Perna canaliculus, which is also an important aquaculture species in New
Zealand [29,30]. The mussel industry routinely transfers juvenile mussels from their cap-
ture sites or from its single hatchery to marine farms around the country, a process that
can involve emersion of up to 72 h [29,31]. Previous studies have described the stress re-
sponse of P. canaliculus juveniles and adults to fasting, heat and simulated transport,
showing that fasted juveniles are less able to tolerate subsequent stress, and that transport
results in oxidative damage [3234]. Additionally, variations in RH during emersion can
affect the resettlement behaviour of juvenile P. canaliculus during recovery in seawater,
although the underlying dynamic of physiological responses of these stressed juveniles
remains unknown [28]. Therefore, the aim of this study was to assess the interactive effects
of variations in emersion duration and RH on the stress responses and recovery dynamics
of juvenile P. canaliculus during immersion in seawater. Stress responses were measured
by quantifying oxidative damage (protein carbonyls, lipid hydroperoxides, 8-hydroxyde-
oxyguanosine) and enzymatic antioxidants (superoxide dismutase, catalase, glutathione
peroxidase and reductase), as well as the water content and survival of the juvenile mus-
sels. These parameters were evaluated experimentally in the laboratory to test the hypoth-
esis that longer emersion times in a dryer environment would affect metabolism, stress
levels, condition and recovery dynamics of juvenile P. canaliculus.
2. Results
2.1. Emersion Conditions
During emersion, relative humidity (RH) was consistently above 90% for the high
RH treatment, with an average RH of 98 ± 2% (SD, n = 242; Figure 1A). In contrast, RH for
low and mid RH treatments was more variable. Low and mid RH treatments started at
approximately 15 and 60%, respectively, but average values over 20 h were, respectively,
29 ± 5% and 82 ± 11% (SD, n = 242 for each mean; Figure 1A). RH in the low RH treatment
Metabolites 2021, 11, 580 3 of 17
was 16 ± 2% during the first hour of emersion, increasing to 22 ± 4% after 5 h of emersion
and then to a maximum value of around 32% by the end of the 20 h of emersion (Figure
1B). RH in the mid RH treatment was 61 ± 1% during the first hour of emersion and in-
creased to 67 ± 4% after 5 h of emersion, before reaching a maximum value of around 94%
by the end of the 20 h emersion period (Figure 1B).
A
Relative Humidity Treatments
Relative H umidity (% )
0
20
40
60
80
100
120
Low Mid High
B
Time of emersion (h)
012345678910 11 12 13 14 15 16 17 18 19 20
Relative H umidity (% )
0
20
40
60
80
100
120
Low RH
Mid RH
High RH
Figure 1. Relative humidity (RH, %; 18 °C) during emersion in treatments. (A): Plot showing data
for each RH treatment (low, mid and high) and their respective means ± standard deviation (SD, n
= 242). (B): Time series for each of RH treatment during 20 h of emersion (average of two loggers
per treatment, 10 min sample interval). Short and long dashed lines show the end points of the 1 h
and 5 h emersion treatments, respectively.
2.2. Water Content
The water content of the juvenile mussels was reduced by increasing emersion time
and decreasing RH (Figure 2, Table 1). In the high RH treatment, water content was con-
sistently high at around 68–75% across emersion treatments (Figure 2). The water content
of mussels maintained at mid RH was high (~70%) and showed no change during the first
5 h of emersion, but then decreased to around 12% after 20 h of emersion (Figure 2). The
water content of the juveniles in the low RH treatment decreased over time and was lower
than the mid and high RH treatments after 1 and 5 h emersions (Figure 2). After 20 h of
emersion, the water content of juveniles in the low and mid RH treatments was similar,
with water content being between 8–12% (Figure 2).
Metabolites 2021, 11, 580 4 of 17
Figure 2. Water content (% live mass ± SE, n = 3) in juvenile Perna canaliculus exposed to different
relative humidity (RH; low, mid, high) and emersion (1, 5, 20 h) treatments. Control bar indicates
water content in juveniles that were not emersed (excluded from statistical analysis). Tukey pair-
wise comparisons show significant differences (p < 0.05) for the interaction between emersion time
and relative humidity treatments, which are denoted by different lower-case letters above bars.
Table 1. ANOVA results of water content, mortality and staining percentage data for Perna canalic-
ulus juveniles in different relative humidity (RH) treatments during emersion (E: 1, 5 and 20 h),
followed by recovery in seawater (R: 1, 5 and 10 h). Degrees of freedom (df), mean square (MS), F-
ratio and p-values are shown for each variable. Significant results (p < 0.05) are shown in bold.
Water Content
df
MS
F
p
Relative Humidity (RH)
2
0.565
32.785
<0.001
Emersion time (E)
2
0.276
16.023
<0.001
RH × E
4
0.077
4.515
0.011
Residual
18
0.017
Estimated Mortality
df
MS
F
p
Relative Humidity (RH)
2
4.436
422.941
<0.001
Emersion time (E)
2
7.987
761.58
<0.001
RH × E
6
1.663
158.529
<0.001
Residual (between-effects)
36
0.01
Recovery time (R)
2
0.776
159.186
<0.001
RH × R
4
1.662
30.832
<0.001
R × E
4
0.15
35.811
<0.001
RH × R × E
8
0.061
12.422
<0.001
Residual (within-effects)
72
0.005
Staining
df
MS
F
p
Relative Humidity (RH)
2
2.142
246.423
<0.001
Emersion time (E)
2
1.454
167.251
<0.001
RH × E
4
0.209
24.026
<0.001
Residual
36
0.009
2.3. Mortality Estimates: Observations and Staining
Observational live/dead assessments indicated interactive effects of emersion time,
RH and recovery time on estimates of mortality that increased with time of emersion, es-
pecially in the low and mid RH treatments (Figure 3, Table 1). The effect of recovery time
Metabolites 2021, 11, 580 5 of 17
varied among emersion and RH treatments with the percentage of estimated dead mus-
sels increasing over time in the low and mid RH treatments (Figure 3). Few juveniles that
were emersed for 1 h or held at high RH appeared to die during this experiment (Figure
3). At the end of the experiment, 0.5 ± 0.32% (SE, n = 5) of juvenile mussels were estimated
to be dead in control samples.
Figure 3. Estimates of mortality in juvenile Perna canaliculus exposed to different relative humidity
(RH; low, mid, high) and emersion (1, 5, 20 h) treatments, followed by recovery in seawater (1, 5, 10
h). Data represent mean percent of dead juveniles ± standard error (SE, n = 5).
Fast Green staining in control mussels was apparent in 11 ± 1.6% (SE, n = 5) of indi-
viduals. The percentage of stained mussels increased with emersion time, with the great-
est percentage occurring in mussels exposed to 20 h of emersion (Figure 4). A smaller
percentage of juveniles stained in high RH treatments at all emersion durations relative
to the mid and low RH treatments (Figure 4). Low and mid RH treatments had similar
effects on the percentage of stained juveniles within each of the 1 and 5 h emersion treat-
ments (Figure 4). After 20 h of emersion, there were fewer mussels stained in the mid RH
treatment compared to the low RH treatment, but the percentage stained in these treat-
ments was 84% greater than in the high RH treatment (Figure 4).
Figure 4. Fast Green staining of juvenile Perna canaliculus exposed to different relative humidity
(RH; low, mid, high) and emersion (1, 5, 20 h) treatments, followed by 10 h recovery in seawater.
Metabolites 2021, 11, 580 6 of 17
Control bar shows percentage of stained mussels that were continuously immersed in flowing sea-
water (excluded from statistical analysis). Data represent the mean percent of stained mussels ±
standard error (SE, n = 5). Tukey pair-wise comparisons show significant differences (p < 0.05) for
the interaction between emersion time and relative humidity treatments, which are denoted by dif-
ferent lower-case letters above bars.
2.4. Oxidative Damage
There were interactive effects of emersion time, RH treatment and recovery time on
levels of protein carbonyls (PCs), lipid hydroperoxides (LPs) and DNA damage, measured
as 8-OHdG, in juvenile mussels (Table 2).
There were strong effects of emersion and recovery duration on PCs levels in all but
the high RH treatment (Figure 5A, Table 2). After 1 h of emersion, levels of PCs were
higher for the low and mid RH treatments (relative to high RH) after 1 h of recovery,
which declined to similar levels to those observed in high RH after 5 and 10 h of recovery
(Figure 5A). After 5 h of emersion, levels of PCs in the low and mid RH treatments were
significantly elevated after 0, 1 and 5 h of recovery, but then declined in the mid RH treat-
ment to levels approaching baseline after 10 h of recovery (Figure 5A). An emersion time
of 20 h caused a more substantial increase in PCs levels in the low and mid RH treatments;
these were sustained over 10 h of recovery, while PCs levels in the high RH treatment
remained at basal levels (Figure 5A). The low RH treatment generally induced greater and
more variable PCs levels compared to mid and high RH (Figure 5A).
Levels of lipid hydroperoxides (LPs) were generally greater in mussels in the low
and mid RH treatments, rapidly rising during the first hour of re-immersion to maximum
levels that increased in correlation with emersion time (Figure 5B, Table 2). LP levels sub-
sequently decreased with increasing time of recovery, approaching baseline after 10 h,
except for mussels exposed to 20 h of emersion at low RH, where LP levels remained sig-
nificantly elevated (Figure 5B).
There were similar patterns for 8-OHdG levels with a general trend of increased 8-
OHdG levels in mussels after 1 h of recovery in seawater (Figure 5C, Table 2). An excep-
tion for this measure of DNA damage was that 8-OHdG continued to increase in the low
RH/20-h emersion treatment during recovery, in part driving a strong interaction among
the experimental factors (Figure 5C, Table 2).
Table 2. ANOVA results of oxidative damage biomarkers data for Perna canaliculus juveniles ex-
posed to different relative humidity (RH) treatments during emersion (E: 1, 5 and 20 h), followed
by recovery in seawater (R: 0, 1, 5 and 10 h). Significant results (p < 0.05) are shown in bold.
Protein Carbonyls (PCs)
df
MS
F
p
Relative Humidity (RH)
2
1.8
−2
86.5
<0.001
Emersion time (E)
2
1.4
−2
68.2
<0.001
Recovery time (R)
3
3.4
−3
16.6
<0.001
RH × E
4
1.9
−3
9.2
<0.001
RH × R
6
1.8
−4
0.8
0.541
E × R
6
3.5
−4
1.7
0.137
RH × E × R
12
7.3
−4
3.5
<0.001
Residual
72
2.1
−4
Lipid Hydroperoxides (LPs)
df
MS
F
p
Relative Humidity (RH)
2
2026.9
87.2
<0.001
Emersion time (E)
2
2281
98.1
<0.001
Recovery time (R)
3
1371.2
59
<0.001
RH × E
4
521.8
22.4
<0.001
RH × R
6
151.4
6.5
<0.001
E × R
6
175
7.5
<0.001
RH × E × R
12
62.4
2.7
0.005
Residual
72
23.3
DNA Damage (8-OHdG)
df
MS
F
p
Metabolites 2021, 11, 580 7 of 17
Relative Humidity (RH)
2
4206.1
125.8
<0.001
Emersion time (E)
2
6239.2
186.6
<0.001
Recovery time (R)
3
1055.1
31.6
<0.001
RH × E
4
2001.9
59.9
<0.001
RH × R
6
277.5
8.3
<0.001
E × R
6
80.1
2.4
0.036
RH × E × R
12
198.6
5.9
<0.001
Residual
72
33.5
Figure 5. Oxidative damage biomarkers in juvenile Perna canaliculus exposed to different relative
humidity (RH; low, mid, high) and emersion (1, 5, 20 h) treatments, followed by recovery in sea-
water (0, 1, 5, 10 h). Control bar (c) shows biomarker concentration in mussels that were continu-
ously immersed in flowing seawater (excluded from statistical analysis). (A): Protein carbonyls
(PCs); (B): Lipid hydroperoxides (LPs); (C): 8-hydroxydeoxyguanosine (8-OHdG). Data are mean
concentration ± standard error (SE, n = 3).
Metabolites 2021, 11, 580 8 of 17
2.5. Enzymatic Antioxidants
Enzymatic antioxidant activity was similar in all mussels sampled at the end of emer-
sion, regardless of duration and RH treatment, resembling levels in control animals (Fig-
ure 6 AD, Table 3). Activity consistently increased with recovery in seawater in low and
mid RH treatments after 1 and 5 h of emersion, and in mid RH treatment after 20 h of
emersion, driving RH × recovery interactions for all analyses (Figure 6 AD, Table 3). Ac-
tivity of enzymatic antioxidants for mussels from the high RH treatment remained at base-
line levels during recovery, regardless of emersion duration (Figure 6 AD).
Figure 6. Antioxidant biomarker activity in juvenile Perna canaliculus exposed to different relative humidity (RH; low, mid,
high) and emersion (1, 5, 20 h) treatments, followed by recovery in seawater (0, 1, 5, 10 h). Control bar (c) shows biomarker
activity in mussels that were continuously immersed in flowing seawater (excluded from statistical analysis). (A): Super-
oxide Dismutase (SOD); (B): Catalase (CAT); (C): Glutathione Peroxidase (GPx); (D): Glutathione Reductase (GR). Data
are mean concentrations ± standard error (SE, n = 3).
Table 3. ANOVA results of enzymatic antioxidant biomarkers data for Perna canaliculus juveniles
exposed to different relative humidity (RH) treatments during emersion (E: 1, 5 and 20 h), followed
by recovery in seawater (R: 0, 1, 5 and 10 h). Significant results (p < 0.05) are shown in bold.
Superoxide Dismutase (SOD)
df
MS
F
p
Relative Humidity (RH)
2
1260.5
62.2
<0.001
Emersion time (E)
2
262.4
12.9
<0.001
Recovery time (R)
3
873.7
43.1
<0.001
RH × E
4
161.5
8
<0.001
RH × R
6
232.8
11.5
<0.001
E × R
6
36.2
1.8
0.115
RH × E × R
12
31.7
1.6
0.123
Residual
72
20.3
Catalase (CAT)
df
MS
F
p
Relative Humidity (RH)
2
243.5
72.7
<0.001
Emersion time (E)
2
41
12.2
<0.001
Recovery time (R)
3
223.8
66.8
<0.001
RH × E
4
21.6
6.4
<0.001
RH × R
6
49.6
14.8
<0.001
Metabolites 2021, 11, 580 9 of 17
E × R
6
4.5
1.4
0.248
RH × E × R
12
3.2
1
0.487
Residual
72
3.4
Glutathione Peroxidase (GPx)
df
MS
F
p
Relative Humidity (RH)
2
521
66.4
<0.001
Emersion time (E)
2
61.6
7.9
<0.001
Recovery time (R)
3
308.2
39.3
<0.001
RH × E
4
21.9
2.8
0.033
RH × R
6
62.2
8
<0.001
E × R
6
10.7
1.4
0.239
RH F E × R
12
13.1
1.7
0.092
Residual
72
7.8
Glutathione Reductase (GR)
df
MS
F
p
Relative Humidity (RH)
2
18.9
42.5
<0.001
Emersion time (E)
2
2
4.5
0.015
Recovery time (R)
3
13.2
29.8
<0.001
RH × E
4
1
2.3
0.067
RH × R
6
4.4
9.8
<0.001
E × R
6
0.2
0.5
0.811
RH × E × R
12
0.1
0.1
1
Residual
72
0.5
3. Discussion
This study showed that the effects of emersion and the re-immersion dynamics of
juvenile Perna canaliculus are complex and mainly depend on the conditions that the mus-
sels experience during emersion. Juvenile mussels that experienced longer emersion at
low and mid relative humidity (~1560% RH) had increased water loss, increased oxida-
tive damage and antioxidant enzymatic activity. These elevated levels, however, only
tended to become apparent following re-immersion. The accumulation of oxidative dam-
age in juvenile P. canaliculus, despite a corresponding increase in antioxidant activity, was
correlated to increasing mortality rates during the 10 h re-immersion monitoring period
(up to ~95% following 20 h emersion at low-mid RH). This correlation between emersion
time, oxidative damage, and mortality was tested for all oxidative stress markers at the
different RH levels at the end of the recovery time (see supplementary material). The
strongest correlation was observed for oxidative damage in the form of PCs at low and
mid RH, where mortality increased steadily with PC levels, as time of emersion increased
(r2 = 0.9991 at low RH and r2 = 0.7273 at mid RH). LPs and DNA damage also correlated
to mortality observations only at low RH, but these correlations were weaker (r2 = 0.8630
and r2 = 0.7844 for LPs and DNA damage, respectively). This suggests that mussels that
experience emersion at low RH are most likely to die due to oxidative stress, whereas
oxidative damage is reduced at mid RH probably due to the specific action of antioxi-
dants. At mid RH, it is likely that mussels were not dead but compromised, widely gaping
and unable to close their valves after emersion and RH stress and are therefore classified
as “dead”. Surviving mussels could also be physiologically impaired due to the high oxi-
dative damage, potentially resulting in altered biological functions or even further mor-
tality with increasing immersion time.
Green-lipped mussels naturally colonise rocks of the lower littoral and sub-tidal
zones of New Zealand [35]. The emersion times used in this study are representative of a
typical exposure period for an intertidal P. canaliculus (1 h), an exceptional low tide event
(5 h), and an artificial emersion time which would represent a common transport time for
mussel spat from hatchery to the grow-out farms (20 h). It should be noted that the juve-
nile mussels used in this study are hatchery produced from subtidal mussel populations
(farmed); the results shown here might vary from the potential stress response of wild
spat which may have previously experienced emersion [36] and associated stressors, with
the potential to either increase [37] or reduce [32] subsequent stress tolerance.
Metabolites 2021, 11, 580 10 of 17
Emersion of juvenile P. canaliculus caused severe water loss when RH was lower dur-
ing emersion, and with increasing exposure times. Mussels isolate their soft tissues from
the external environment by closing their valves during emersion, using periodic gaping
behaviour to decrease their body temperature and facilitate gas exchange, allowing adult
mussels to withstand long periods of emersion [4,6,38]. However, few studies have as-
sessed the role of gaping in juvenile mussels. In the present study, there were two lines of
evidence to suggest that juvenile P. canaliculus used gaping behaviour as a mechanism to
reduce stress during emersion. First, there was a significant decrease in the water content
of emersed juveniles that was exacerbated as emersion duration increased. Second, the
mid humidity treatment showed increasing RH with the time of emersion, indicating that
moisture from the mussels was released into the container during incubation. Even
though RH increased in the mid RH treatment to levels ca. 80%, mussels experienced in-
creased oxidative damage and antioxidant activity during the subsequent re-immersion
period, compared to mussels held in high RH during emersion. This suggests that desic-
cation stress could also play a crucial role in the recovery dynamics of P. canaliculus.
It can be challenging to determine whether juvenile mussels are dead, moribund or
alive using visual observations [28,39]. Here, our estimates suggested that mortality in-
creased with recovery time, especially in the more severe RH treatments, with no differ-
ences between mid and low RH treatments at the end of the 10 h recovery period. Follow-
ing 20 h emersion and low or mid RH, for example, >90% of individuals subsequently
appeared unresponsive in water and took up Fast Green stain. However, measures of an-
tioxidant enzyme activities suggest that these mussels were still metabolically active in
the mid treatment. Indeed, given the substantial increases in antioxidant enzyme activities
over the 10 h immersion period, it seems likely that many of these mussels were alive, but
moribund and unable to respond to tactile stimulus or an osmotic shock (i.e., valve closure
when immersed in freshwater and stain). This hypothesis could be tested with extended
immersion periods to determine whether these mussels completely recover. By contrast,
the high RH appears relatively benign, even when spat are emersed for 20 h, with most
mussels showing signs of life or the ability to respond to tactile or osmotic stimulus and
are therefore more likely to remain viable [39].
Marine littoral organisms experiencing natural emersion due to tidal cycles can ac-
cumulate modest levels of ROS during air exposure as they shift to anaerobic metabolism
[17,40]. Despite demonstrating net metabolic depression [41], many enzymatic antioxi-
dants are activated during the emersion period as a preparation for the reoxygenation
stress (“preparation for oxidative stress”, POS) [42–44]. When organisms are immersed in
the water for recovery, reoxygenation of haemolymph occurs rapidly (within one hour)
[19], causing an oxidative burst, and the generation of large amounts of ROS [43]. In in-
vertebrates, the oxidative burst is less intense and happens more slowly than in verte-
brates; nonetheless, the excessive production of ROS can still result in oxidative damage
[45]. In this study, juvenile P. canaliculus exposed to low and mid humidity air showed
increased oxidative damage after 20 h of emersion compared to control mussels that re-
mained submersed in seawater. Here, P. canaliculus experienced a more extreme emersion
stress than most intertidal species as they moved from a completely subtidal environment
to an extreme emersion period (20 h). However, oxidative stress was minimised if mussels
were maintained in high humidity during emersion, where damage levels were similar to
non-emersed control mussels. Reoxygenation has been shown to increase oxidative dam-
age in the mussel Mytilus edulis [20], the gastropod Crepipatella dilatata [46], and the oyster
Crassostrea virginica [47]. In the present study, reoxygenation stress resulted in a rapid in-
crease in oxidative damage markers in all the humidity treatments, but levels were signif-
icantly higher following low and mid humidity exposure during emersion. Oxidative
damage in the mussels that were exposed to high humidity during a 20 h emersion
showed a decrease in damage to baseline levels after the mussels had been in seawater for
10 h.
Metabolites 2021, 11, 580 11 of 17
Antioxidant enzyme activity in juvenile P. canaliculus did not increase during the
emersion period at any humidity level. This suggests that juvenile P. canaliculus may have
a reduced POS (i.e., preparation for oxidative stress) capacity to cope with reoxygenation
stress during recovery, as seen in other invertebrate species [43]. In adults of the brown
mussel, Perna perna, emersion stress for 48 h lowered the activity of enzymatic antioxi-
dants; however, the levels of the non-enzymatic antioxidant glutathione (GSH) showed a
rapid and persistent increase during emersion [48]. In the mussel Mytilus edulis, 48 and 72
h anoxia in seawater had little effect on the activity of antioxidants; however, antioxidant
activity was suppressed after 72 h anoxia followed by 24 h of reoxygenation [20]. In the
present study, there was an increase in enzymatic antioxidant activity of juvenile P. cana-
liculus in all treatments following re-immersion, which agrees with similar findings in
other invertebrate species [46,49–51]. Levels of antioxidant activity in mussels held in high
humidity conditions during emersion returned to baseline levels after 10 h of recovery. In
contrast, mussels held at mid humidity during emersion showed no indication of declin-
ing after 10 h in seawater. It should be noted that mussels held at low humidity during
emersion typically displayed low levels of antioxidant activity, similar to mussels that
were held at high humidity. However, this result is likely to be an artifact of the high
mortality of the mussels in the low humidity treatment, associated with high oxidative
damage in the mussels sampled, but low antioxidant activity which is likely to have come
from the small proportion of live mussels.
In this study, the increased action of antioxidants observed after short emersion pe-
riods (1 and 5 h) helped maintain relatively low levels of oxidative damage at all RH levels
after re-immersion. However, at longer emersion (20 h), the activity of some antioxidants
becomes compromised, resulting in an accumulation of oxidative damage in the tissues.
Emersion can be an occasional or regular event for juvenile P. canaliculus. For exam-
ple, P. canaliculus can be an intertidal organism that experiences semidiurnal low tides, or
they can be occasionally cast ashore while attached to drift algae. Juvenile P. canaliculus
are also routinely emersed to transfer them to sea-based nursery farms for aquaculture, a
process that can take as long as 3 days and be highly variable in terms of environmental
conditions [52]. Losses of juvenile P. canaliculus after they are seeded onto a marine farm
are a common problem for the mussel industry in New Zealand, where most of the juve-
niles are lost during the first few months of aquaculture [53–56]. Based on the results of
the present study, it is possible that conditions in which the juvenile mussels are trans-
ported trigger a series of molecular, biochemical and physiological responses in the mus-
sels that could have carry-over effects for the mussels after seeding, potentially affecting
retention and survival of the juveniles. For example, juvenile resettlement behaviour was
slowed and reduced by lower RH conditions during emersion [28]. The data presented
here suggest that such impacts on behaviour could correspond to the physiological con-
dition of the juveniles which, in the case of juveniles emersed in drier conditions, reflect
increased ROS damage and antioxidant activity.
Complex, multi-species mussel guilds occupy the New Zealand rocky shore, with
lower-littoral Perna canaliculus giving way to Mytilus, Aulacomya and Xenostrobus species
on the higher shore [35,57]. Thermotolerance has been demonstrated to be a key determi-
nant of this vertical zonation [57], which is turn likely to be influenced by region, latitude
and genetic structure [58]. Based on the findings presented here, it would be valuable to
consider the role of juvenile emersion and, in particular, reoxygenation stress as factors
influencing natural distribution in an increasingly marine heatwave-prone region [59].
Overall, the present study showed that juvenile P. canaliculus are extremely sensitive
to low humidity and prolonged emersion times, with mussels held in low RH during
emersion experiencing severe water loss, high oxidative damage and high mortality,
while mussels held at high RH were not impacted, even after 20 h of air exposure. These
findings have significant implications for natural shoreline distribution and the aquacul-
ture industry where juvenile mussels are routinely emersed during the production cycle.
Metabolites 2021, 11, 580 12 of 17
For aquaculturists, transportation methods should aim to maintain mussels in a high hu-
midity environment at a constant temperature for the shortest possible time to mitigate
the deleterious effects described in this study. When the time of emersion cannot be short-
ened, mussels should be held in a high relative humidity environment to minimise the
stress responses elicited in the juvenile mussels, including increased oxidative damage
and subsequent mortality. Further research is required to unravel the complex factors in-
fluencing resilience and environmental stress in the juveniles that appear to represent a
major life-stage bottleneck for both cultured and wild P. canaliculus populations.
4. Materials and Methods
4.1. Experimental Design
Green-lipped mussel juveniles (~1 mm) were collected from a commercial hatchery
(SPATnz) and transported to the laboratory of the adjacent Cawthron Aquaculture Park
(Nelson, New Zealand). Mussels were weighed and separated into 178 circular sieves (8
cm diameter, 200 µm mesh size). Separate sets of experimental sieves were allocated for
the determination of water content (27 sieves; ~120 mg of mussels in each sieve), assess-
ments of survival (50 sieves; ~10 mg of mussels in each sieve) and oxidative biomarker
analysis (111 sieves; ~1 g of mussels in each sieve). The sieves were then placed in a shal-
low acclimation tank with flowing seawater at 18 °C containing a mixture of axenically
cultured microalgae (Chaetoceros calcitrans, C. muelleri and Pavlova lutheri). All sieves were
supplied with food ad libitum during the first 24 h after collection before experimentation.
Experimental treatments consisted of three relative humidity (RH) treatments (low =
~15%, mid = ~60%, and high = ~90%), four emersion times (0, 1, 5 and 20 h) and three
recovery times (1, 5 and 10 h) following re-immersion in seawater. Control mussels were
not emersed during the experiment. Replication consisted of three replicated sieves per
treatment for oxidative damage and antioxidant biomarker analyses, three replicated
sieves for water content analysis, and five replicated sieves for survival. Lowered RH lev-
els were achieved by adding different amounts of desiccant (silica gel) to a circular, 750
mL air-tight plastic container; for the high RH treatment, a seawater-saturated cotton cloth
was added to the containers. Following acclimation, the experimental sieves with mussels
were blot-dried and randomly allocated to the different RH treatments. All containers
were tightly closed immediately after addition of the experimental sieves and placed in
an incubator at 18 °C. RH loggers (Hygrochrons, iButtonLink Technology, Whitewater,
WI, USA) sampling at 10 min intervals were added to 6 of the containers allocated to 20 h
of emersion (two per RH treatment). After each of the emersion treatments, the mussels
in the sieves were either sampled, assessed as described below, or randomly assigned to
re-immersion treatments.
4.2. Water Content and Mortality Estimates
Water content in juvenile mussels was determined as percent mass loss by weighing
(wet), drying (at 100 °C for 24 h), and re-weighing.
Sieves allocated to survival monitoring were surveyed repeatedly throughout the re-
immersion recovery period. A stereomicroscope was used to observe the immersed mus-
sels; wide-open individuals that did not respond to a tactile stimulus (prodding with for-
ceps) were considered to be dead [25].
After the last recovery time point (10 h), mussels were stained using the Fast Green
method [39], with minor modifications, to provide an indication of viability. In brief, mus-
sels were transferred from the experimental sieves into freshwater (~20 mL) in 35 mL plas-
tic containers to induce valve closure. One drop of concentrated Fast Green dye was added
to each container, and the mussels were left in the stain for 1 h. Individuals that were
incapable of sustained valve closure throughout this period were considered inviable
(dead or moribund) and took up dye; the green-stained tissues of these spat could subse-
quently be discerned through the translucent valves. The mussels were subsequently
Metabolites 2021, 11, 580 13 of 17
rinsed and frozen at −20 °C. Samples were thawed and mussels were counted; the per-
centage of stained individuals per sample was calculated and used as the response varia-
ble in analyses.
4.3. Oxidative Damage
Juvenile mussels were sampled for oxidative biomarker analyses before emersion
(control), directly after the completion of the emersion periods (0 h recovery) or after 1, 5
or 10 h of recovery in seawater. Three sub-samples of ~130–140 mg of juvenile mussels
(fresh weight) were taken from each of the replicate sieves for each assay. Each sub-sample
was placed into 2 mL cryo-vials, flash frozen in liquid nitrogen and stored at −80 °C until
analyses. The sub-samples were used to determine oxidative damage (protein carbonyls
(PCs), lipid hydroperoxides (LPs) and 8-hydroxydeoxyguanosine (8-OHdG)) and antiox-
idant enzyme activity (superoxide dismutase (SOD), catalase (CAT), glutathione peroxi-
dase (GPx) and glutathione reductase (GR)).
4.3.1. Macromolecule Extraction
Macromolecule extractions (protein, lipid and DNA) for determination of oxidative
damage in juvenile P. canaliculus were performed according to Delorme et al. [32] In brief,
total protein was extracted on ice by adding 900 µL of ice-cold enzyme extraction buffer
(100 mM potassium phosphate [pH 7.5] containing 50 mM NaCl, 0.1 mM Na2EDTA, 1%
polyvinylpyrrolidone−40, 2 mM phenylmethylsulfonyl fluoride and 0.1% TritonX−100)
and homogenising for 30 s at 1500 rpm (1600 MiniG®, SPEX®) using zirconia/silica beads
and a pre-chilled cryo-block (SPEX®). The samples were then centrifuged for 15 min at
17,000× g at 4 °C and the supernatant (i.e., protein extract) purified using ultrafiltration
and purification columns (AMICON). The purified protein extract was then washed and
reconstituted with 250 µL of 50 mM potassium phosphate buffer (pH 7.2), placed in a 1.5
mL microcentrifuge tube, blown with oxygen-free nitrogen and stored at −80 °C. Protein
content was determined by the Lowry protein assay [60]. Samples were diluted with po-
tassium phosphate as required before analysis. The levels of protein carbonyls (PCs) were
determined via reaction with 2.4-dinitrophenylhydrazine (DNPH) as described by Rez-
nick and Packer [61] and expressed as nmols of carbonyls mg of protein−1.
Total lipids were extracted by adding 600 µL of methanol:chloroform (2:1 v/v) and
homogenised as described above. The homogenised sample was left to stand for 5 min
and an extra 400 µL of chloroform were added and vortexed vigorously for 30 s. Then,
400 µL of MilliQ water were added and the sample vortexed again for 30 s. The samples
were then centrifuged at an ambient temperature for 30 s at 17,000× g. Finally, the chloro-
form phase (bottom layer) was removed and transferred to a clean 1.5 mL microcentrifuge
tube, blown with oxygen-free nitrogen and stored at −80 °C until analysis. The level of
lipid hydroperoxides (LPs) in the samples was determined by absorbance at 500 nm using
the ferric thiocyanate method described by Mihaljevic et al. [62], adapted for measurement
in a microtitre plate reader. A calibration curve with t-butyl hydroperoxide was used and
the LP content calculated as nmol of lipid hydroperoxide per mg of fresh (wet) mussel
weight.
PC and LP assays were carried out using a Victor 1420 Multilabel plate reader (Perkin
Elmer Wallac, Waltham, MA, USA) fitted with a temperature control cell (set to 25 °C)
and an auto-dispenser. Data were acquired and processed using the WorkOut 2.0 software
package (Perkin Elmer, Waltham, MA, USA).
DNA extraction was performed using an ISOLATE II Genomic DNA Kit (Bioline,
Memphis, TN, USA), with one minor modificationthe samples were crushed and ho-
mogenised using a tube pestle after the addition of the pre-lysis buffer. The final DNA
extracts were placed in a 1.5 mL microcentrifuge tube, blown with oxygen-free nitrogen
and stored at −80 °C until analyses. The level of oxidised DNA was calculated by quanti-
fying the amount of 8-hydroxydeoxyguanosine (8-OHdG) present using high-perfor-
Metabolites 2021, 11, 580 14 of 17
mance liquid chromatography (HPLC) followed by UV detection of guanine and electro-
chemical detection (coulometric) of 8-OHdG as described previously for P. canaliculus ju-
veniles [32].
4.3.2. Enzymatic Antioxidants
The remaining protein extract was used to perform antioxidant enzyme assays: su-
peroxide dismutase activity (SOD), catalase (CAT), glutathione peroxidase (GPx) and glu-
tathione reductase (GR) as described by Delorme et al. [32] In brief, SOD was determined
using a Cayman Chemicals Superoxide Dismutase Assay Kit and the activity expressed
as units of SOD mg of protein1. CAT was assayed using the chemiluminescent method of
Maral et al. [63], as adapted by Janssens et al. [64] for 96-well microplates, and the activity
expressed as µmol min−1 mg protein−1. GPx activity was measured according to the spec-
trophotometric method described by Paglia and Valentine [65] and expressed as nmol
min−1 mg of protein−1. GR was assayed using the method of Cribb et al. [66], with minor
modifications and activity expressed as nmol min−1 mg of protein−1. All enzymatic assays
were carried out using a Perkin Elmer Wallac Victor 1420 multilabel counter (Perkin
Elmer, Waltham, MA, USA) as detailed above.
4.4. Statistical Analyses
All analyses were carried out using analysis of variance (ANOVA) with α = 0.05 un-
less otherwise stated. Assumptions of ANOVA were checked using appropriate tests
(ShapiroWilk, Brown Forsythe, Mauchly) and graphical observations. Water content,
mortality and staining percentage data were arcsine-square root transformed prior to
analysis. Water content data were not normally distributed but met the assumption of
homoscedasticity and were analysed using a two-way ANOVA with RH level and emer-
sion time as factors since balanced ANOVA is robust to deviations from normality [67].
Estimates of mortality data were analysed using a repeated measures ANOVA with RH
and emersion time as the between-subjects effects, and re-immersion time as the within-
subjects effect to account for repeated observations of individual sieves. Estimated mor-
tality data were non-normal and variances were heterogeneous but met the assumption
of sphericity and were analysed with α = 0.01 [68]. Staining data were analysed using a
two-way ANOVA (α = 0.01), with RH level and emersion time as fixed factors. All oxida-
tive damage (PCs, LPs, 8-OHdG) and enzymatic antioxidants (SOD, CAT, GPx, GR) data
were analysed with three-way ANOVA using RH, emersion time, and recovery time as
fixed factors. PC data did not meet parametric assumptions and were transformed to the
reciprocal before analysis. Differences among treatments were identified using Tukey
pair-wise tests with α = 0.05. Analyses were carried out using Sigma Plot 14.0 (SYSTAT
Software, Inc., Chicago, IL, USA) or Statistica 12 (Statsoft) software.
Supplementary Materials: The following are available online at www.mdpi.com/arti-
cle/10.3390/metabo11090580/s1, Figure S1: Correlations between oxidative damage and mortality
observations after 10 h of recovery in seawater. A: Protein carbonyls (PCs); B: Lipid hydroperoxides
(LPs); C: 8-hydroxydeoxyguanosine (8-OHdG; DNA damage). Data from different emersion times
were combined to plot oxidative damage versus mortality across emersion time for each relative
humidity (RH) treatment, Figure S2: Correlations between oxidative damage and mussel staining
after 10 h of recovery in seawater. A: Protein carbonyls (PCs); B: Lipid hydroperoxides (LPs); C: 8-
hydroxydeoxyguanosine (8-OHdG; DNA damage). Data from different emersion times were com-
bined to plot oxidative damage versus staining across emersion time for each relative humidity (RH)
treatment.
Author Contributions: Conceptualization, N.J.D. and P.M.S.; methodology, N.J.D. and P.M.S.; for-
mal analysis, N.J.D. and P.M.S.; investigation, N.J.D. and P.M.S.; resources, D.J.B.; writingoriginal
draft preparation, N.J.D.; writingreview and editing, N.J.D., D.J.B., N.L.C.R. and P.M.S.; visuali-
zation, N.J.D. and P.M.S.; project administration, N.L.C.R. All authors have read and agreed to the
published version of the manuscript.
Metabolites 2021, 11, 580 15 of 17
Funding: This research was funded by the New Zealand Ministry for Business, Innovation and Em-
ployment, through the Cawthron Shellfish Aquaculture Platform, Contract No. CAWX1801.
Institutional Review Board Statement: No ethical approval was required for this study. According
to the New Zealand Animal Welfare Act, ethical approval for work using molluscs is not needed
(except for cephalopods).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to the value of researchers’ interaction
when/if the data are requested.
Acknowledgments: We thank SPATnz for supplying mussel spat, and to Leonardo Zamora, Jessica
Ericson, Karthiga Kumanan, Jolene Berry, Joanna Copedo and Bridget Finnie for their technical sup-
port and advice. Special thanks to Leonardo Zamora and Joanna Copedo for helping with designing
and producing the graphic abstract.
Conflicts of Interest: The authors declare no conflict of interest.
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... Juvenile P. canaliculus can be stranded by the receding tide or exposed to air during transfer from the hatchery to grow-out facilities in aquaculture (Delorme et al., 2020;Jeffs et al., 2018). Additionally, emersion in juvenile P. canaliculus has shown to result in high oxidative damage, particularly after 1 h of rehydration after emersion (Delorme et al., 2021a). Hence, the present study applied emersion and relative humidity treatments as relevant stressors likely to induce a range of ROS levels in P. canaliculus juveniles (Figs 1 and 2). ...
... Since only living, apparently vigorous juvenile mussels were selected for staining after treatment, it is possible that under prolonged emersion conditions the antioxidant mechanisms in the selected mussels overcompensated for the large accumulation of ROS, resulting in a relatively low ROS signal (mean of 16% ROS) compared with the other treatments (mean between 26-38% ROS), but still higher than control mussels (mean of 5% ROS). Previous research has shown that the antioxidant activity increases greatly in juvenile P. canaliculus after exposure to different emersion times and relative humidity levels, especially when mussels are exposed to mid humidity conditions (Delorme et al., 2021a). This result suggests that selected (living) mussels were able to cope with the (E) 20 h emersion at moderate RH (20 h-M); (F) Dead mussels that were killed by freezing at −20°C prior to the staining and fixing. ...
... prolonged stress caused by a 20 h emersion and 1 h of recovery in seawater, but their subsequent capacity to completely recover and survive in the long term remains unknown. Recent studies in juvenile P. canaliculus have shown that survival during recovery (up to 10 h) is greatly compromised as relative humidity decreases (Delorme et al., 2021a). It is also worth noting that the strength of the ROS signal observed in dead mussels was comparable to that of stressed mussels. ...
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Stress and survival of the juvenile New Zealand green-lipped mussel, Perna canaliculus, is a poorly understood bottleneck in the ecological and economic performance of a significant aquaculture crop. This species was therefore selected as a model organism for the development of a new method to quantify oxidative stress in whole individuals. An in vivo ROS-activated stain (CellROX™) was administered to anaesthetised, translucent juveniles that were subsequently formaldehyde fixed and then visualised using confocal microscopy. Subsequent application of image analysis to quantifying ROS-positive tissue areas was successfully used to detect stress differences in juvenile mussels exposed to varying levels of emersion. This integrated method can be used to localise and quantify ROS production in individual translucent bivalve life stages (larval and juvenile), while relative stability following fixation greatly expands potential practical field applications. This article has an associated First Person interview with the first and third authors of the paper.
... Treating yellowtail kingfish (Seriola lalandi) adults with O. ficus indica extracts reduced stress biomarkers associated with the complex stressors associated with three hours of live transport (Boerrigter et al., 2014). For mussels, transport of spat from hatchery or wild collection site to farm exposes these vulnerable life stages to a multitude of stressors (temperature fluctuations, starvation, desiccation) that often result in reduced survival of spat post-transport and high oxidative stress (Carton et al., 2007;Delorme et al., 2021;South et al., 2020). Treating P. canaliculus spat with O. ficus indica before transport would temporarily stimulate protective mechanisms within the mussels and potentially increase survivorship during relaying. ...
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Cultured Greenshell™ mussels (Perna canaliculus) are exposed to biotic and abiotic stresses, all of which can negatively impact growth, fecundity, and survival. These impacts are especially pronounced in sensitive larval and spat stages and may result in substantial losses when spat are transferred from hatchery to grow-out farms. Immersion in solutions of plant extracts have been shown to improve stress tolerance in certain aquatic species and may offer new husbandry and market access opportunities, e.g., more amenable to organic farming practices. Here the efficacy of immersing P. canaliculus spat in ‘high’ or ‘low’ concentrations of prickly pear cactus (Opuntia ficus indica) solutions was examined. Solutions were made from either dry cactus leaf powder i.e., 4.5 (low) or 45 (high) g L⁻¹ or cactus fruit extract (4 μl or 40 μl L⁻¹) and mussels immersed for 1 h to stimulate potential defence mechanisms within mussels prior to acute thermal challenge. Following a subsequent 1 h thermal shock, the predicted 50% survival temperature (LT50) was raised following pre-treatment with O. ficus indica solutions. However, higher resilience to thermal stress was evident in P. canaliculus spat immersed in low concentration solutions of O. ficus indica powder (LT50 = 32.3 °C) and extracts (LT50 = 32.4 °C) compared to spat in immersed in higher concentrations (i.e., LT50 = 31.8 °C ‘Powder’ and 31.4 °C ‘Extract’ respectively) and to naïve controls (LT50 = 30.7 °C). The results provide evidence for the potential for bioactive plant extracts to augment physiological resilience in a shellfish species. While internal biochemical validation is required, the straightforward exposure protocols applied here would be readily transferrable into commercial hatchery culture methods for P. canaliculus.
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