Pre‑hatching development in the intertidal zone negatively affects juvenile survival and physiology in the muricid gastropod Acanthina monodon

Abstract

Encapsulated development in the intertidal environment can potentially expose developing embryos to environmental stresses, particularly during low tides. Such stresses can affect juvenile performance after hatching. Capsules-containing advanced pre-hatching stages of the snail Acanthina monodon were collected during July–August 2017 from rocks in the intertidal and subtidal environments along the coast of Valdivia, Chile (Calfuco beach, 39°79′27″S; 73°39′27″W) and brought to the laboratory, where hatching of the juveniles took place. The number of embryos per capsule in relationship to capsule size was determined for capsules from the two environments, as were the juvenile hatching size and the number of juveniles hatching from each capsule. Survival and respiratory performance were also monitored for juveniles from the two locations. Neither embryonic packaging nor the number of juveniles hatched per capsule, nor the hatching size of the juveniles evidenced any differences for capsules that were collected in the two different environments. In general, juvenile survival was low (< 10% at 4 week post-hatching) regardless of capsule origin. However, survival and standardized rates of oxygen consumption were substantially higher for juveniles from subtidal capsules. This suggests that environmental stressors had a detrimental effect on embryos from intertidal capsules.

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Vol.:(0123456789)
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Marine Biology (2018) 165:155
https://doi.org/10.1007/s00227-018-3412-1
ORIGINAL PAPER
Pre‑hatching development intheintertidal zone negatively aects
juvenile survival andphysiology inthemuricid gastropod Acanthina
monodon
O.R.Chaparro1· L.P.Salas‑Yanquin1· A.S.Matos2· J.A.Bűchner‑Miranda1· M.W.Gray3· V.M.Cubillos1·
J.A.Pechenik4
Received: 11 May 2018 / Accepted: 1 September 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
Encapsulated development in the intertidal environment can potentially expose developing embryos to environmental stresses,
particularly during low tides. Such stresses can affect juvenile performance after hatching. Capsules-containing advanced
pre-hatching stages of the snail Acanthina monodon were collected during July–August 2017 from rocks in the intertidal
and subtidal environments along the coast of Valdivia, Chile (Calfuco beach, 39°79′27″S; 73°39′27″W) and brought to the
laboratory, where hatching of the juveniles took place. The number of embryos per capsule in relationship to capsule size was
determined for capsules from the two environments, as were the juvenile hatching size and the number of juveniles hatching
from each capsule. Survival and respiratory performance were also monitored for juveniles from the two locations. Neither
embryonic packaging nor the number of juveniles hatched per capsule, nor the hatching size of the juveniles evidenced any
differences for capsules that were collected in the two different environments. In general, juvenile survival was low (< 10%
at 4week post-hatching) regardless of capsule origin. However, survival and standardized rates of oxygen consumption were
substantially higher for juveniles from subtidal capsules. This suggests that environmental stressors had a detrimental effect
on embryos from intertidal capsules.
Introduction
Intertidal invertebrates are routinely subjected to severe,
periodic stresses, including desiccation and UV irradia-
tion, and substantive changes in salinity, pH, and oxygen
concentrations (Pechenik etal. 2001, 2016; Thiyagarajan
etal. 2007; Segura etal. 2014). Some intertidal invertebrates
deposit their embryos in physically complex “egg capsules,”
which they then abandon (Garrido and Gallardo 1993;
Rawlings 1999; Przeslawski etal. 2004); the encapsulated
embryos, therefore, experience the same sorts of environ-
mental stresses until they hatch. In benthic marine inver-
tebrates, abiotic stressors are more severe in the intertidal
than in the subtidal (Moran 1999; Jenewein and Gosselin
2013; Bashevkin etal. 2017). Although the direct impact of
such stresses on intertidal development has been examined
for some invertebrate species (e.g., Pechenik 1982; Rawl-
ings 1996; Przeslawski 2005), the possibility that non-lethal
stresses experienced by encapsulated intertidal embryos
might impact post-hatching development has not previously
been considered.
Responsible Editor: J. Grassle.
Reviewed by G. Pastorino and an undisclosed expert.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s0022 7-018-3412-1) contains
supplementary material, which is available to authorized users.
* O. R. Chaparro
ochaparr@uach.cl
1 Instituto de Ciencias Marinas y Limnológicas, Universidad
Austral de Chile, Valdivia, Chile
2 Laboratório de Invertebrados Marinhos, Departamento de
Biologia, Centro de Ciências, Universidade Federal doCeará,
Fortaleza, Brazil
3 Center forEnvironmental Science, Horn Point Laboratory,
University ofMaryland, Cambridge, MD, USA
4 Biology Department, Tufts University, Medford, MA02155,
USA

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The previous studies have shown that some stresses (but
not all, e.g., Diederich etal. 2011) experienced during lar-
val development can alter post-metamorphic performance
(reviewed by Pechenik 2018). For example, Hettinger etal.
(2012) showed that exposing oyster larvae to high PCO2
levels caused carry-over effects expressed as reduced juve-
nile growth rates. Stresses experienced during encapsulated
development might have similar effects on later develop-
ment. Such effects might be especially common in species
with direct development, in which individuals hatch from
their egg capsules only after metamorphosis; in species
with direct development, the embryos will spend a longer
time in their egg capsules than will related species exhibit-
ing “mixed” development, in which free-living larvae are
released after briefer encapsulation (Pechenik 1979).
In many cases, the capsule wallsprovide some protection
from environmental stresses for the encapsulated embryos
(Pechenik 1982; Rawlings 1996; Chaparro etal. 2008). On
the other hand, the capsular wall thickness could also be a
handicap, since the walls will restrict the inward diffusion of
oxygen (Lardiés and Fernández 2002; Brante 2006; Brante
etal. 2008; Segura etal. 2010) and the elimination of waste
products.
The Caenogastropoda Acanthina monodon (Muricidae) is
an encapsulating marine gastropod that inhabits a large part
of the Chilean coast (Dye 1991; Reid and Osorio 2000). It
can be found in rocky habitats in both shallow subtidal and
intertidal areas (Osorio etal. 1979). This species reproduces
during much of the year by producing egg capsules that con-
tain developing embryos and nurse eggs, which typically
account for ~ 7% of the eggs in the capsules. Females aban-
don their egg capsules soon after they have been deposited
(Gallardo 1979) in other caenogastropods. The encapsulated
embryos develop in the capsules for 55–80 days (Gallardo
1979), until crawling juveniles finally emerge. While the
subtidally placed egg capsules remain submerged during
development, intertidal capsules are exposed to air during
extreme low tides for 3–4h (L. Salas, pers. obs.). In the
summer, the intertidal capsules are exposed to higher tem-
perature than those in the nearby seawater (average summer
water temperature, approx. 13°C; average summer air tem-
perature, 18°C, Windfinder 2018), and to UV irradiation
and water loss, through evaporation. Encapsulated embryos
at low tide can also be exposed to relatively low oxygen con-
ditions, due to the potential difficulties in the inward diffu-
sion of oxygen in the air. Capsule walls could limit diffusion
of oxygen into the interior of the egg capsules, as recorded
by Segura etal. (2010) for capsules of the brooding gastro-
pod Crepipatella dilatata.
In consideration of the above, encapsulated embryos that
develop intertidally are likely to experience greater levels
of stress than those developing subtidally. Sublethal stresses
may influence juvenile performance, either at the time of
hatching or later in development, affecting growth, survival,
and physiological responses, particularly in the consumption
of oxygen as an indicator of metabolic activity.
The present study compares sizes at hatching, post-
hatching mortality, and respiratory performance for juve-
niles emerging from subtidal and intertidal egg capsules;
only the intertidally collected egg capsules will have been
exposed to the stressors associated with the periodic emer-
sion associated with tidal cycles. We postulated that juve-
niles emerging from intertidally collected capsules would
be smaller at hatching, have higher juvenile mortality, and
have lower rates of oxygen consumption relative to those
from subtidal habitats.
Materials andmethods
Capsules of the muricid gastropod Acanthina mono-
don (Sánchez etal. 2011) were collected in July–August
2017 from intertidal and subtidal rocky areas in Calfuco
(39°79′27″S; 73°39′27″W), Valdivia, Chile. The subtidal
capsules were obtained while snorkeling during an extreme
low tide (0.1m) at a depth of 1m, to ensure that the capsules
had been continuously submerged and never exposed to air.
Capsules were also collected from the highest intertidal
where they occurred, ~ 0.5m above mean low lower water
(MLLW), attached to rock surfaces not directly exposed to
the sun, usually in crevices. The tidal regime in Calfuco is
mixed semi-diurnal.
We selected capsules deposited by a number of differ-
ent females, to avoid potential maternal effects. To do this,
collections were made from a number of different widely
separated groups of capsules. The capsules were taken to the
laboratory, cleaned, and placed in aquaria with circulating
seawater (salinity 29–31) that was taken from where the cap-
sules were collected. For our studies, we used only capsules
that were close to hatching, as indicated in part by capsule
color (changing from yellow in recently laid capsules to a
dark brown when the embryos were close to hatching) and
embryo size (when capsule walls were sufficiently transpar-
ent), but mainly by observing the condition of the hatch-
ing plug, which shows distinct exterior deterioration when
hatching is imminent. The egg capsules of this species are
flattened, with concave and convex sides, reaching lengths
up to ~ 15mm, excluding the stalk (Fig.1).
Packaging condition
To identify a potential differential packaging of embryos
within intertidal and subtidal egg capsules, we examined
advanced capsules-containing only pre-hatching juveniles
(i.e., all nurse eggs had been consumed by the developing

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embryos, and the embryos were distinctly brown; n = 47
intertidal, n = 46 subtidal capsules) and determined the
relationship between capsule size (length and surface area)
and the number of juveniles in each capsule. At hatching,
the capsules were photographed using a camera attached
to a stereomicroscope. Using a reference ruler with the
computer, we later estimated capsule length and surface
area using the program Image J. The capsular length cor-
responded to the maximum distance from the apical plug
(hatching area) to the base of the capsule (excluding the
attachment peduncle, Fig.1). Capsule surface area was
estimated using one side of the capsule, by capturing
the images with Micrometrics SE Premium software for
each capsule and analyzing the images using the ImageJ
software. Simultaneously, we quantified the number of
advanced embryos (only pre-hatching juveniles) in each
capsule. With this information, a packaging index was
calculated, associating the area (one capsule face) or the
maximum length of the capsule with the corresponding
number of embryos. This was done for both intertidal and
subtidal egg capsules.
Carry‑over eects
A total of 47 intertidal capsules and 46 subtidal capsules,
obtained from a number of different mothers, were placed
in small glass tanks with recirculating seawater (salinity
29–31) from the site where the egg capsules were collected.
Seawater temperature was 11.0–11.8°C. The photoperiod
used corresponded to the natural light:dark cycle for that
time of year (~ 11h light: 13h dark).
Each capsule was placed individually in a miniaquarium
(4.5 × 6.5cm), numbered for identification, and enclosed in
a small mesh bag (400-µm pore diameter) to retain the juve-
niles at hatching. All mesh bags were cleaned frequently
to eliminate fouling and to avoid pore occlusion. The cap-
sules were checked daily for hatching. The hatching date
was considered to be the day that the first juvenile emerged
from each capsule. At that time, we quantified the following
for each hatched capsule: the number of juveniles, juvenile
shell lengths, and rates of juvenile oxygen consumption (see
below).
Using photographs taken with a magnifying stereomi-
croscope at 10× magnification and processing software, we
determined maximum shell lengths for all juveniles associ-
ated with each hatched egg capsule. The number of juveniles
and their shell lengths was recorded following each measure-
ment of oxygen consumption.
Oxygen consumption rates (OCR)
Oxygen consumption rate was quantified for all juveniles
obtained from each of the 47 intertidal egg capsules and the
46 subtidal egg capsules. OCR was determined on the day
of hatching (day 1) and 1, 2, and 3weeks later.
For each OCR measurement, all juveniles that hatched
from each capsule were placed inside a hermetically sealed
8-mL glass respirometer chamber filled with filtered seawa-
ter (12°C and salinity 30 ± 1) sterilized with UV light. The
water had previously been saturated by bubbling with air;
we waited at least 15minafter the bubbling was stopped
before filling the chambers, to allow for the elimination of
mini-bubbles. The capsules were placed inside the cham-
bers, which were then closed, while they were underwater, to
prevent the entry of air. The respiration chambers were then
carefully removed from the water and sealed with parafilm
to prevent any inward diffusion of oxygen. During meas-
urements, the chambers with juveniles were kept inside a
thermobath system to avoid changes in temperature. Oxygen
concentration readings were performed using a non-invasive
Fibox3 oxygen-sensing system (Precision Sensing gmbH).
Measurements in each chamber were made at the beginning
of the experiment and at 1-h intervals for 2–3h. During each
set of measurements, two chambers without egg capsules
served as controls.
Capsule area
Len
g
th
Attachment peduncle
2.5 mm
Fig. 1 Acanthina monodon: sketch of an egg capsule showing meas-
urement used to estimate capsule size and surface area

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After each measurement, the juveniles in each aquarium
were counted and measured. Juvenile OCR’s were expressed
as oxygen consumed h−1mg−1 dry juvenile biomass. Total
dry biomass was obtained from juveniles of the same age
and condition, maintained in parallel with the experimental
individuals. The ratio between shell length and total dry bio-
mass was used to estimate the biomass of the experimental
snails. The measured OCRs were converted to an individual
of “standard” dry total weight (tissue + shell), following
Bayne etal. (1987).
Juvenile biomass
Total juvenile dry weights were determined using individu-
als hatched from parallel groups of intertidal and subtidal
egg capsules. The hatchlings were maintained in the same
way as the experimental juveniles (see mortality section).
Each week after hatching took place, we sampled 3–10
groups of snails for weighing. For each sample group, five
juveniles of similar age were collected haphazardly from
a pool of juveniles of the same age, and maximum shell
lengths were determined (see the previous section on image
processing). Measured shells were quickly rinsed with dis-
tilled water to remove adhering salt, and placed in small,
numbered, pre-weighed aluminum foil cups. The juveniles
were then dried at 60°C for 24h and weighed. In this way,
the average total dry weight (shell + soft tissue) of the
snails in each treatment was obtained weekly. The total dry
weight of juveniles used during the measurements of oxy-
gen consumption could then be estimated from shell-length
measurements.
Hatching size
Newly hatched juveniles from the intertidal and subtidal
capsules (n = 46 intertidal, 47 subtidal) were photographed
using a stereomicroscope at 10× magnification to assess
sizes at hatching.
Juvenile culture
During the post-hatching period, juveniles werefed adlibi-
tum on juvenile mussels (Perumytilus purpuratus; Soto etal.
2004); the snails began consuming the food immediately
after hatching, using their accessory boring organ (Carriker
and Grubers 1999). The size of the prey was increased as the
juveniles grew. The mussels were collected from the same
place that we collected the egg capsules of A. monodon.
Juvenile mortality
At the time of hatching, the number of juveniles was quan-
tified for each of the 93 capsules collected (46 subtidal, 47
intertidal). The number of survivors was then determined
every week for the next 4weeks, thus providing weekly mor-
tality and survival data.
Statistical analyses
Homogeneity of variance was assessed using a Levene test.
Values that did not meet this assumption were transformed
(area and capsular length; juvenile survival) before further
analysis.
Data concerning the area and length of the capsules col-
lected from the intertidal and subtidal zones, as well as the
number of juveniles hatched per capsule, were compared
using one-way ANOVA. Index of capsule packaging: the
relationships between capsule area and number of juveniles
hatching and between capsule length and number of juve-
niles per egg capsule for capsules of intertidal and subtidal
origin were also examined by using a one-way ANOVA.
The relationship between the source of egg capsules and
age after hatching on juvenile survival was analyzed using
a two-way ANOVA.
OCR data were analyzed using the Kolmogorov–Smirnov
and Bartlett tests to determine whether the data met the
assumptions of normality and homoscedasticity, respec-
tively. When necessary, data were transformed using the
reciprocal of the value. Comparisons of OCR between juve-
niles hatched from intertidal and subtidal capsules through
the first 3weeks after hatching were made using ANCOVA
with permutations. These analyses were performed using
RStudio, library LmPerm.
Results
Capsule packaging
Subtidal and intertidal egg capsules of Acanthina mono-
don showed no significant differences in length (one-way
ANOVA: F1,86 = 1.52; P = 0.22; n = 88) nor in the capsu-
lar area (one-way ANOVA: F1,87 = 2.80; P = 0.09; n = 89)
(ESM 1). Similarly, the mean number of embryos hatched
capsule−1 did not differ significantly for capsules that had
been collected subtidally and intertidally (one-way ANOVA:
F1,80 = 0.10; P = 0.74; n = 82) (ESM 2). Moreover, we found
no significant differences in the relationships between cap-
sule length and the number of encapsulated juveniles at
hatching (one-way ANOVA: F1,79 = 0.77; P = 0.38; n = 81),
or between capsule surface area and the number of embryos
in the capsule (one-way ANOVA, F1,79 = 1.74; P = 0.19;
n = 81) (ESM 3). Thus, there was no significant difference
in how juveniles were distributed among egg capsules, by
any measure, for capsules collected from the two environ-
ments, intertidal and subtidal.

Marine Biology (2018) 165:155
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Carry‑over eects
Hatching size
Juvenile hatching size was not significantly affected by the
environment in which the embryos had developed (one-
way ANOVA: F1,2165 = 0.3; P = 0.58; n = 2167). Mean SL
at hatching was 963.5 ± 65.5 for subtidal juveniles and
956.8 ± 61.9µm (mean ± SD) for intertidal juveniles (ESM
4).
Mortality
Although substantial mortality was recorded for juveniles
in all the treatments, juveniles hatching from intertidal egg
capsules had significantly greater mortalities than those
from subtidal capsules (two-way ANOVA: zone × week:
F1,4 = 2.63; P = 0.03; zone: P < 0.0001; week: P < 0.0001;
n = 81, Fig.2). In both cases, the greatest absolute number
of juvenile deaths occurred in the first week of post-hatch-
ing juvenile life, with the number of surviving offspring per
egg capsule declining by ~ 50% in the first week and reach-
ing < 10% by week 4 (Fig.2).
Oxygen consumption rate (OCR)
Oxygen consumption rate of A. monodon juveniles differed
significantly according to the origin of the capsules that they
hatched from (subtidal or intertidal, ANCOVA permutation
test, F1,320 = 5.63, P < 0.0001) and level of juvenile develop-
ment (ANCOVA permutation test, F3,320 = 3.39, P = 0.0018,
Fig. 3). For both groups, juvenile OCRs significantly
increased with age. For 1-day-old juveniles, the mean OCR
was > 60% higher for those hatching from subtidal egg cap-
sules than those from intertidal egg capsules (Fig.3). Differ-
ences in the mean OCR for intertidal and subtidal juveniles
were still significantly different 21 d after hatching, and they
were 53% higher in subtidal juveniles.
Discussion
The availability of oxygen limits the size and shape of
aquatic egg masses and the packaging of embryos within
those masses (Strathmann and Strathmann 1995). In cap-
sules deposited intertidally, the capsule wall may limit the
inward diffusion ofoxygen into the capsule, possibly result-
ing in oxygen stress for the encapsulated embryos. In the
particular case of A. monodon, there were no obvious differ-
ences in the packaging of the embryos in capsules collected
from the intertidal and subtidal sites: there were no differ-
ences in the size of the capsules or in mean capsule surface
area, nor in the number of juveniles hatching per capsule.
However, these results are only valid for comparisons of
embryo packaging during the later portion of the reproduc-
tive period that was studied, which was based solely on
capsules-containing juveniles near hatching. We still know
nothing about the packaging of “nurse eggs” into subtidal
and intertidal egg capsules; those nurse eggs provide food
for the embryos during encapsulation (Gallardo 1979). Cap-
sules deposited intertidally or subtidally may contain differ-
ent numbers of nurse eggs per embryo, something that could
be examined in a future study.
Intertidal Subtidal
N of surviv
ors hatched egg capsule
-1
0
10
20
30
40
50
60
70
day 1
day 7
day 14
day 21
day 28
aa
b
b
c
bc
de
cd
e
d
Source of capsule
Fig. 2 Acanthina monodon. Survivors during first month as inde-
pendent juveniles, after release from capsules collected from
intertidal and shallow subtidal (two-way ANOVA: zone × week:
F1,4 = 2.63; P = 0.03; zone: P < 0.0001; week P < 0.0001; n = 81). Dif-
ferent letters on bars indicate significant differences. Source of cap-
sule = zone
Juvenile age (d)
Standardiz
ed mean oxygen consumption rate (mgO
2
h
-1
)
0
1
2
3
4
5
6
Subtidal
Intertidal
714 21 0
Fig. 3 Acanthina monodon. Age-related changes in oxygen consump-
tion (mean ± SD) for juveniles hatching from capsules collected inter-
tidally or subtidally. OCR values standardized to I mg dry weight
(n = 320)

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The results of this study show that, in A. monodon,
hatching size was the same whether the capsules had been
deposited intertidally or subtidally. However, this does not
necessarily mean that time to hatching was the same for
capsules deposited in the two environments; embryos with
reduced growth rates should take longer to reach the same
sizes at hatching (Segura etal. 2014). In this research, there
was no available information on the initial packing (nurse
eggs: embryonic eggs ratio) at oviposition, so future studies
should examine this issue.
Many benthic marine invertebrates show high mortali-
ties during the early juvenile life (Gosselin and Qian 1997;
Hunt and Scheibling 1997). In our experiments, we saw
substantial post-hatching mortalities regardless of whether
the juveniles were from intertidal or subtidal egg capsules,
even though all juveniles in our study were well-fed, and
the flowing seawater that we used was taken directly from
the area where they naturally develop. Moreover, prey was
effectively consumed by the early juveniles from the first day
after hatching. Even so, post-hatching survival was higher
for juveniles with a subtidal origin than for those from cap-
sules collected intertidally. Intertidal individuals had ~ 52%
lower survival over the first month after hatching than indi-
viduals hatching from the subtidal egg capsules, suggesting
a carry-over effect of the environmental stresses experienced
by the intertidal embryos during their development. To our
knowledge, this is the first study to document such findings
for a direct-developing species.
However, such carry-over effects have been well
described for species with free-living larvae. For example,
an equivalent impact on mortality was identified in the bryo-
zoan Watersipora subtorquata after the larvae were exposed
for a short time to sublethal levels of Cu++ in seawater: col-
ony survival was reduced dramatically after such exposure
in the larval stage (Ng and Keough 2003). Changes in larval-
feeding pulses can also affect subsequent juvenile survival.
Thus, exposing early stage larvae of the mussel Mytilus
galloprovincialis to permanent or periodic pulses of low-
food concentrations resulted in significantly higher juvenile
mortality in field transplants, compared with the survival of
juveniles that had received abundant food during larval life
or even just at an early stage of larval development (Phillips
2004). Feeding the larvae of Crepidula onyx at a low-food
concentration (1 × 104 cells mL−1) also resulted in higher
mortality for metamorphosed juveniles kept under labora-
tory conditions (Chiu etal. 2007). In addition, exposing lar-
vae of the polychaete Capitella teleta (formerly Capitella
sp. I; Blake etal. 2009) for as little as 24h to low salinities
(10–12) significantly reduced subsequent juvenile survival
and growth (Pechenik etal. 2001), and field-transplanted
juveniles of the polychaete Hydroides diramphus showed
reduced survival if their larvae had been reared at reduced
food concentrations (Allen and Marshall 2010). Pechenik
etal. (2002) suggested that although larvae of Crepidula
fornicata may fully recover from periods of the early nutri-
tional stress, the resulting juveniles may exhibit poor ini-
tial growth due to impaired gill function, reduced digestive
capability, or reduced assimilation efficiency. Implications
of these results are potentially worrisome, considering the
increasing evidence of global climate change and increasing
pollution (see review Pechenik 2018).
The extent to which stresses experienced during periods
of encapsulation create similar latent effects in other species
is largely unknown. Results of the present study indicate
that the environment in which encapsulated development
takes place can impact post-hatching juvenile survival and
physiology, and thus potentially impact subsequent popula-
tion dynamics (see review Pechenik 2018); the issue seems
well worth further exploration.
Such stresses can also have subtle effects on juvenile
physiology. In A. monodon, juveniles of intertidal origin
showed a much lower OCR than subtidal juveniles of equiv-
alent age. These differences in OCR between the two groups
were present throughout the experimental period; we saw
no recovery in OCR over the 3weeks included in this study.
These results thus extend results from the previous research
on free-living larvae, showing that sublethal stressors expe-
rienced during encapsulated development can substantially
impact life after hatching. Temporary maternal isolation
from the outside environment due to hypoxic conditions dur-
ing brooding of C. dilatata impacted post-hatching juvenile
shell growth rates, as well as rates of ingestion and respira-
tion (Chaparro etal. 2014). In the intertidal environment,
egg capsules of A. monodon can be exposed to air for as long
as 3–4h at the more extreme tides, which could impact rates
of oxygen diffusion, and cause capsule desiccation problems,
as well.
Capsules of A. monodon that were exposed to air for 3h
in lab experiments lost up to 60% of their initial weight,
something that is likely to impose a high level of stress on
the encapsulated offspring (L. Salas, Pers. Obs.). Intracap-
sular oxygen availability may also be an important source
of stress in intertidal areas, particularly during low tides; in
this species, capsule walls can be as thick as 42µm, although
they become thinner as the embryos continue to develop
(Buchner-Miranda J, Pers. Obs). Desiccation during low
tides (up to 3–4h in extreme tidal events) will also poten-
tially expose embryos to osmotic stress.
In summary,the environmental stresses experienced by
encapsulated intertidal embryos ofA. monodoncan clearly
have a substantial impact on post-hatching, juvenile survival,
and OCR, effects that could well impact recruitment for the
next generation. The previous studies have shown strong var-
iation in growth rates, survival, and competitive ability, and
tolerance to heat, desiccation, and pollution among juvenile
marine invertebrates of many species (reviewed by Pechenik

Marine Biology (2018) 165:155
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Page 7 of 8 155
2006). At least some of that variability might be due to vari-
ation in the quality of offspring arriving at particular sites,
or at particular times, and that variation in quality might
well be caused in large part by experiences that the animals
have had as the developing embryos or larvae (reviewed by
Pechenik 2006, 2018). Further studies must be conducted
in the lab to determine exactly what sorts of environmental
stresses cause the effects documented here for field-collected
encapsulated offspring of A. monodon.
Funding This work was supported by the Fondo Nacional de Investi-
gación Científica y Tecnológica-Chile (Fondecyt) through the Grant
1180643 to OC.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Human/animal rights statement All applicable national, state, and
University guidelines for the care and use of animals were followed.
Only invertebrates were used in this study.
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