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Plasticity in parental effects confers rapid larval thermal tolerance in the estuarine anemone
Nematostella vectensis
Authors: Rivera, Hanny E.1,2,3*, Chen, Cheng-Yi4, Gibson, Matthew C.4, 5, Tarrant, Ann M.2
Affiliations:
1. MIT-WHOI Joint Program in Oceanography/Applied Ocean Science & Engineering, Cambridge and Woods
Hole, MA, USA.
2. Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA.
3. Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA.
4. Stowers Institute for Medical Research, Kansas City, MO.
5. Department of Anatomy and Cell Biology, The University of Kansas School of Medicine, Kansas City, KS.
* Present address: Boston University, Department of Biology. Boston, MA.
Keywords: Acclimation; Cnidaria; LT50; maternal effects; paternal effects; thermal limits
Summary statement: The estuarine anemone, Nematostella vectensis, quickly responds to
changes in temperature to modulate parental effects that influence thermal tolerance of larvae in
a reversible (plastic) manner.
Journal of Experimental Biology • Accepted manuscript
http://jeb.biologists.org/lookup/doi/10.1242/jeb.236745Access the most recent version at
First posted online on 5 February 2021 as 10.1242/jeb.236745
Abstract:
Parental effects can prepare offspring for different environments and facilitate survival across
generations. We exposed parental populations of the estuarine anemone, Nematostella vectensis,
from Massachusetts to elevated temperatures and quantified larval mortality across a temperature
gradient. We find that parental exposure to elevated temperatures results in a consistent increase
in larval thermal tolerance, as measured by the temperature at which 50% of larvae die (LT50),
with a mean increase in LT50 of 0.3C. Larvae from subsequent spawns return to baseline
thermal thresholds when parents are returned to normal temperatures, indicating plasticity in
these parental effects. Histological analyses of gametogenesis in females suggests these dynamic
shifts in larval thermal tolerance may be facilitated by maternal effects in non-overlapping
gametic cohorts. We also compare larvae from North Carolina (a genetically distinct population
with higher baseline thermal tolerance) and Massachusetts parents, and find larvae from heat-
exposed Massachusetts parents have thermal thresholds comparable to larvae from unexposed
North Carolina parents. North Carolina parents also increase larval thermal tolerance under the
same high-temperature regime, suggesting plasticity in parental effects is an inherent trait for N.
vectensis. Overall, we find larval thermal tolerance in N. vectensis shows a strong genetic basis
and can be modulated by parental effects. Further understanding the mechanisms behind these
shifts can elucidate the fate of thermally sensitive ectotherms in a rapidly changing thermal
environment.
Journal of Experimental Biology • Accepted manuscript
Introduction:
Parental effects encompass a range of mechanisms that can better prepare offspring for the
conditions they may experience. These effects are often informed by the parental environment,
especially if the environment the offspring will experience will resemble that of the parents
(Jensen, Allen and Marshall 2014; Lacey 1998; Qvasnstöm and Price 2001; Putnam et al. 2020).
Anticipatory parental effects allow parents to enhance the phenotypic plasticity of offspring to
better match their future environment (Burgess and Marshall, 2014). Parental effects may
influence offspring throughout their lifetimes, as for instance, occurs in the water flea Daphnia,
which will develop a helmeted anti-predation phenotype if parents were exposed to predators
(Harris et al., 2012). Alternatively, effects may be short-lived, mainly influencing early life
stages, often through mechanisms such as maternal loading of RNA transcripts; increased
energetic reserves (e.g. lipids) in seeds, embryos, and larvae; or modification of the gestational
environment, in order to enhance offspring survival or allow for faster acclimatization to
environmental conditions (see reviews in Marshall and Uller, 2007; Uller, 2008). In an era of
rapid climate change, swift phenotypic modifications facilitated by parental effects may become
indispensable for species survival (Galloway and Etterson, 2007).
The ability of populations to respond to increasing temperatures will play a critical role in
determining the distribution and persistence of species as global temperatures rise (Logan et al.,
2013). In the context of global climate change, phenotypic plasticity – the ability to modulate
physiology, morphology, behavior, or other phenotypes under different environments – has
emerged as a rapid avenue for organisms to survive environmental change, in comparison to the
slower route of selection upon the existing genetic variation and eventual adaptation (Aitken and
Whitlock, 2013; Donelson et al., 2017; Reusch, 2014; Torda et al., 2017). Plasticity in the form
of parental effects or transgenerational plasticity – usually epigenetic or other semi-heritable
changes across generations – is thus being heralded as a potential safety net for vulnerable
species as it allows plasticity to have intergenerational influence (e.g. Jensen, Allen and
Marshall, 2014; Putnam and Gates, 2015; Schunter et al., 2018; Putnam et al., 2020).
The impact of parental effects will depend on the how reliably parental environments predict the
conditions offspring will experience (Burgess and Marshall, 2014; Marshall and Uller, 2007;
Journal of Experimental Biology • Accepted manuscript
Uller et al., 2013), as well as the ability of parents to modify those effects based on changing
conditions (i.e. plasticity). The degree to which plasticity can benefit organisms will depend on
species- and environment-specific interactions (see Via and Lande, 1985; Reed, Schindler and
Waples, 2011; Kelly, 2019 for such considerations). Environments that are highly variable,
especially at short time scales can often promote plasticity over adaptation for a specific
environmental optima (Bonamour et al., 2019; Chevin and Lande, 2015). In other words, there
can be selection for plasticity in place of selection for higher thermal optima, for instance. In the
context of temperature, the timing and duration of thermal variation relative to reproductive
cycles, as well as the persistence of parental effects across future larval cohorts may facilitate the
progression from acclimatization to adaptive processes, especially for thermally sensitive
organisms (Putnam and Gates, 2015; Seebacher et al., 2015). Organisms that have both short and
multiple reproductive cycles across their lifetime represent interesting case studies for
investigating the role and effectiveness of parental effects in modulating offspring fitness across
environmental conditions and ecologically relevant timescales.
Nematostella vectensis (Stephenson, 1935) is also a highly tractable experimental organism for
studying development and ecophysiology (Darling et al., 2005). This estuarine anemone lives
burrowed in the sediment of coastal salt marshes along the eastern and western coasts of North
America and parts of the United Kingdom (Reitzel et al., 2008). Populations show strong genetic
divergence, even across short geographic distances, suggesting limited gene flow or strong
adaptation (Reitzel et al., 2008). Nematostella vectensis is able to fully regenerate from a small
body fragment, a process that facilitates asexual reproduction and recovery from injury (Stefanik
et al., 2013), and that can be used to generate clonal lineages by bisecting adults (Reitzel et al.,
2007). Populations of N. vectensis can be easily maintained in laboratory conditions, and
reproductive cycles can be reliably induced in two- to three-week intervals (Fritzenwanker and
Technau, 2002; Stefanik et al., 2013). Females eject egg bundles that are fertilized externally by
sperm released by males, facilitating controlled crosses (Hand and Uhlinger, 1992). Fertilized
embryos develop into swimming planula larvae within 48 hours and metamorphose into a
primary polyp 7-10 days later (Darling et al., 2005). While reproductive timing in the wild has
not been studied, laboratory animals can be spawned year-round. Given their wide geographic
Journal of Experimental Biology • Accepted manuscript
distribution and highly variable habitats, parental effects could serve as a fast and effective way
to modulate the thermal limits of larvae during spawning cycles.
As N. vectensis inhabit shallow salt marsh pools, they experience substantial daily (as high as
20C) and seasonal thermal variation (as much as 40C between winter lows and summer highs)
(Reitzel et al., 2013; Sachkova et al., 2020; Tarrant et al., 2019). Populations across latitudes also
show different thermal tolerance thresholds during larval, juvenile, and adult stages (Reitzel et
al., 2013). For example, the temperature at which 50% of individuals die (LT50) varies by nearly
2C between juveniles (~10 days post fertilization) from Massachusetts and those from South
Carolina (Reitzel et al., 2013). Southern populations also show faster growth and higher
survivorship at warmer temperatures, suggesting some level of adaptation to temperatures across
latitudes (Reitzel et al., 2013). However, the degree of plasticity in these thermal thresholds or
how these may be influenced by parental effects in early larvae is not known.
Here, we leverage the thermal range of N. vectensis and expose parents to an increasing
temperature regime during gamete maturation to quantify temperature’s influence on parental
effects (both maternal and paternal), larval thermal tolerance, as well as adult heat tolerance. We
test whether parental effects persist through subsequent spawning events and explore possible
causes for induction of larval thermal tolerance. We further compare the impact of parental
effects on larval thermal tolerance to differences between genetically distinct N. vectensis
populations from Massachusetts (MA) and North Carolina (NC). Our work examines how
plasticity in parental effects, via mechanisms such as epigenetic mechanisms or transcript
loading may alter thermal physiology to determine thermal thresholds across life history stages,
and the consistency of these patterns across geographically and genetically distinct populations.
Materials and methods:
Animal collection and husbandry
Laboratory populations of N. vectensis were originally collected from the Great Sippewissett
Marsh, MA (41.59N, -70.63W) and Fort Fisher, NC (33.95N, -77.93W) (thermal
experiments). Populations were kept in glass containers under a 12:12, light:dark cycle, in
filtered natural seawater diluted with deionized water to 15 PSU. Water changes were conducted
Journal of Experimental Biology • Accepted manuscript
every two weeks and animals were fed freshly hatched brine shrimp larvae 4-5 times per week.
Animals from Massachusetts (MA) were maintained at the Woods Hole Oceanographic
Institution. North Carolina (NC) animals were reared under comparable conditions at the
University of North Carolina-Charlotte, until being transferred to MA six months before
experiments (N=8 individuals). All animals had been acclimated to laboratory conditions for at
least two years. Animals used for thermal experiments were placed under constant darkness at
least two weeks prior to the start of experiments to reduce any confounding effects associated
with variability in light levels across treatments (all constant dark).
Animals used for gametogenesis analyses were collected from Rhode River, MD (38.87 N,
76.54 W) and provided by Mark Martindale and Craig Magie. Anemones were cultured in 12
PSU artificial seawater (ASW) and acclimated to lab conditions for more than five years (Ikmi
and Gibson, 2010). Female anemones were maintained at 18C with ambient light, fed once per
month with Artemia nauplii until a week before the induction of spawning, and daily until the
day before spawn induction. The slight differences in husbandry practices between MA, NC, and
MD populations simply reflect variations in husbandry protocols between different labs.
Similarly, the spawning cues used for histological quantification of gametogenesis (MD) and
physiological assays (MA and NC) represent minor variations in lab-specific protocols that are
described in subsequent sections.
Histological quantification of gametogenesis in females
One day before the induction of spawning, five females representing the “before spawn” time
point were anesthetized and fixed in 7% MgCl2 in ASW (w/v) and 4% paraformaldehyde/ in
ASW (v/v) at room temperature for one hour. After an hour, the aboral end was opened and fresh
fixative was added for overnight fixation at 4C. Remaining females were induced to spawn, as
described below ("Spawning" section), except that the morning after the overnight light exposure
the females were cold-shocked by replacing room temperature culturing ASW with 18C ASW
and placed under ambient light conditions. Five females that were observed releasing egg
bundles were anesthetized and fixed for the “after spawn” timepoint as described above.
Journal of Experimental Biology • Accepted manuscript
After fixation, samples were washed in PBST (phosphate-buffered saline with 0.2% Triton X-
100, v/v), five times (10 mins per wash) at room temperature. Samples were then incubated in
1:5,000 diluted SYBR™ Green I (S7567, Thermo Fisher Scientific, Waltham, MA, USA) and
1:1,000 diluted SiR-Actin (CY-SC001, Cytoskeleton, Inc., Denver, CO, USA) in PBST at 4C
overnight, then washed three times (10 minutes per wash). Female mesenteries were then
dissected out and immersed in modified Scale A2 (4M urea and 80% glycerol in PBS, v/v), and
imaged using a Leica Sp5 confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA)
with 4 m per z-stack step to quantify the size and number of oocytes before and after spawning.
The area and density of oocytes from 5 anemones per-timepoint (three mesentery views per-
anemone, 30 total views; total 496 oocytes before spawn and 469 oocytes after spawn) were
manually circled and quantified using ImageJ/FIJI imaging software (Rueden et al., 2017;
Schindelin et al., 2012). The density of oocytes within mesenteries was measured semi-
automatically by FIJI macro, in which maximum projected images of F-actin channel
(mesenteries) were filtered by Gaussian Blur (Sigma = 8), thresholded with the same value to
automatically select oocyte tissue and manually curated to measure the area. Oocyte data were
normally distributed (Shapiro-Wilk test, p = 0.1513); therefore, an unpaired, two-tailed, t-test
was used to quantify differences in oocyte area before and after spawn (N=5 anemones).
Establishment of clonal lineages and genotype-controlled parental populations
To control for genetic variability between parents, experiments were conducted with clonal
parental populations whenever possible (see individual experimental descriptions below). To
create clonal lineages, individuals were repeatedly bisected across the body column, and allowed
to regenerate completely until lineages reached 20-40 individuals. A total of 53 lineages were
created. Multiple individuals from each lineage were combined to incorporate genetic diversity
into genotype-controlled parental populations (i.e. the same number of individuals per lineage
were present in each population). Genotype-controlled (but diverse) populations, as opposed
single-sex clonal lineages were used for experiments due to time constraints in generating clonal
lineages and growing them to maturity in the numbers required for the desired larval outputs. In
addition, mixed populations more closely simulate wild populations, which would contain more
than a single genotype. A detailed enumeration of lineages/individuals in each MA experimental
Journal of Experimental Biology • Accepted manuscript
population is provided in Table S1. NC animals were bisected to obtain a total of 35 female
individuals (N=3 lineages) and 60 male individuals (N=5 lineages).
Heat stress regime
We examined in situ temperature data from loggers deployed at both our MA and NC anemone
source sites and previously published in Sachkova et al., (2020). Overall summer temperatures in
NC are warmer than in MA (Fig. S1A), with mean daily summer (June-Sept) temperatures
significantly higher in NC than MA as expected (p<0.01, two sample t-test, N=122 days; Fig.
S1B). However, the daily temperature range was comparable between the two sites (p=0.16, two
sample t-test, N=122 days; Fig. S1C). Using two Precision™ Dual Chamber 188 water baths
(Thermo Fisher Scientific, Waltham, MA, USA), animals were transitioned from 20C to 33C,
at a rate of ~3C/day, held at 33C for four days, simulating mean daytime (9 AM-9 PM)
temperatures (32.76C) during a midsummer week in Woods Hole, MA (Fig. 1A) and then
returned to 20C at the same rate, prior to spawning. The range of temperatures experienced
during the treatment (13C) is similar to the average daily temperature range that N. vectensis
would experience in the field, both in MA and NC (Fig. S1C). HOBO™ Tidbit loggers (Onset
Computer Corporation, Onset, MA, USA) were used to track treatment temperatures every half
hour during experimental incubations. Parental populations exposed to the heat stress regime are
hereafter called the short-term heat stress (STHS) parental treatment. Water changes were
conducted every other day for both STHS and control animals. Each bowl was fed the same
ration of brine shrimp nauplii daily (0.2 grams). Control animals were kept at 20C in a Low
Temperature Incubator (Thermo Fisher Scientific, Waltham, MA, USA), humidified to prevent
evaporation. All animals were kept under constant dark conditions for at least two weeks prior to
and throughout the heat stress regime.
Spawning for physiological assays
To clear gametes and promote gametogenesis during experimental conditions, parents were
induced to spawn two weeks prior to the start of each experimental incubation. Parental
populations were again induced to spawn on the day following the end of the heat stress regime.
Spawning was induced following the protocols detailed in (Fritzenwanker and Technau, 2002).
Briefly, N. vectensis were individually fed mussel gonad tissue. After 5-6 hours, their water was
Journal of Experimental Biology • Accepted manuscript
changed and anemones were placed under a bright full spectrum light for 12 hours overnight at
room temperature. The following morning a half-water change was made, and they were placed
in the dark at 20C. Induction of spawning for histological assays varied slightly from these steps
and is described above.
Bowls were then checked for egg bundles every half hour. For mixed male-female bowls, egg
bundles were kept in the bowls for at least a half hour after release. For crosses between
differently treated males and females, egg bundles released by females were transferred to male
bowls to allow for fertilization for at least a half hour. Fertilized egg bundles were then
distributed across 6-well culture plates for development in 15 PSU water in the 20C incubator,
in the dark. All gametes were incubated at the same temperature to minimize differences in larval
thermal tolerance due to developmental plasticity or effects of temperature on developmental
timing, allowing us to isolate parental effects as the cause of thermal tolerance differences. The
word cohort is used throughout the manuscript to refer to larvae resulting from a single parental
treatment/parental population combination (e.g. larvae from parental population G1, with both
parents exposed to STHS would be one larval cohort).
Massachusetts parental effects experiment
To investigate if parental heat exposure could increase larval thermal tolerance, we compared
larvae from STHS parents to those from control parents (Fig. 1B). Nine paired, genotype-
controlled parental populations derived from the Massachusetts stock were used in this
experiment. For six of the nine trials, parental populations were genetically identical across
treatments, and for three of the nine trials, parental populations were genetically similar across
treatments (i.e. there was at least one unique lineage within a treatment; see Table S1). Parents
were subjected to temperature regimes, cued to spawn, and larval thermal tolerance was assessed
as described below. For each larval cohort 144-192 larvae were assessed (N=3-4 replicate larval
heat stress trials).
Journal of Experimental Biology • Accepted manuscript
To test for the persistence of parental effects following STHS, three (genetically identical)
parental population pairs were placed in the 20C incubator after the initial STHS exposure and
re-spawned after two weeks along with their paired control populations to account for any
impacts in larval quality induced by repeated spawning.
Massachusetts maternal/paternal effects experiment
To investigate whether parental effects were predominantly paternal or maternal, six of the nine
parental populations described above included additional female/male-only parental cohorts to
enable reciprocal crosses of STHS and control males/females (Fig. 2A; Table S1). Fertilization
between STHS x control males/females was achieved by transferring eggs from female-only
bowls into their opposite treatment male-only bowls. Female bowls were checked for the
presence of new egg bundles every half hour, for five hours. For each larval cohort, 144 larvae
were assessed (N=3 replicate larval heat stress trials). In one population (G2), the STHS female x
control male cross failed to produce viable larvae, and the STHS male x control female cross
yielded only 96 viable larvae (N=2 replicate larval heat stress trials). For population G4, the
STHS female x control male cross yielded only 48 viable larvae (N=1 larval heat stress trial).
Genetic vs. parental effects experiment (MA vs. NC)
Given previously established differences in thermal thresholds in Nematostella from
geographically and genetically distinct populations (Reitzel et al., 2013), we wanted to compare
these differences (putatively from local adaptation) to shifts in larval thermal tolerance resulting
from parental effects. To do so, we compared offspring from MA parents to those from NC
parents. Due to the small number of founder NC individuals (8), we generated clones of all
genotypes to create a NC self-breeding population and a population to reciprocally cross with
MA animals. Each parental combination had 14 female individuals (3 genotypes) and 24 males
individuals (5 genotypes).
All four parental groups (MA, NC, MA female x NC male, NC female x MA male) were
maintained under control conditions, spawned, and used to measure control larval thermal
tolerance. After three weeks, the same parents were subjected to the STHS regime and re-
Journal of Experimental Biology • Accepted manuscript
spawned to measure larval thermal tolerance after parental heat exposure (Fig. 3A). For this
experiment a temporal control was used due to the limited number of NC genotypes and the
limits of generating sufficient clones within a reasonable time frame to have separate control and
STHS populations. We should note that during our subsequent spawning of MA individuals to
test for the persistence of parental effects there were no noticeable differences in the thermal
tolerance of larvae from control parents from sequential spawns (Fig. 1F). As such, using a
temporal control for this experiment is unlikely to have introduced any additional effects.
Fertilization of MA x NC hybrids was conducted in the same manner described above for the
maternal/paternal effects experiment. For each larval cohort 192-288 larvae were assessed for
thermal tolerance (N=4-6 replicate larval heat stress trials).
Larval thermal tolerance assays
To determine the larval temperature gradient, a series of preliminary trials were conducted with
larvae from mixed MA parents under control conditions using the temperature gradient from
Reitzel et al. (2013) as a starting point (37-43C). Minimum temperatures were shifted warmer
and the range of the gradient was adjusted to produce smaller temperature steps around the mid
temperature to allow us to better capture small shifts in LT50 values that might be missed by
larger temperature steps.
At 72 hours post fertilization (hpf), eggs that had developed into swimming planula larvae were
individually pipetted into 0.2 mL PCR strip tubes (USA Scientific, Ocala, FL, USA) with 200 l
of 15 PSU water (as per Reitzel et al., 2013), and exposed to a temperature between 39.8 and
42.3C for one hour. Using a Bio-Rad™ C1000 PCR thermocycler (Bio-Rad Laboratories,
Hercules, CA, USA), with two 48-well heating plates. The temperature protocol was as follows:
(1) 1 minute at 25C, (2) 4 minutes at 30C, (3) 4 minutes at 38C, (4) 1 hour at the treatment
temperature: 39.8, 40, 40.4, 40.8, 41.4, 41.8, 42.1, or 42.3C, (5) 4 minutes at 38C, (6) 4
minutes at 30C, and (7) infinite hold at 22C. Each run consisted of six 8-well strip tubes (48
wells total) per trial replicate per larval cohort. Replicate trials for larvae from treatment and
control parents were always exposed simultaneously (one set per heating plate). The assignment
of STHS and control larvae to one of the two heating plates within each run was alternated to
minimize any possible variations between the thermocycler's heating plates.
Journal of Experimental Biology • Accepted manuscript
Following the thermal exposure, larvae were maintained in the same PCR tubes and returned to
the 20C incubator, in the dark. Mortality was scored 48 hours after each trial by examining
larvae under a dissecting scope. By this time, dead larvae had begun to disintegrate and appeared
as fuzzy clumps. To ensure that the small volume (0.2 mL) would not independently impact
larval survival, we maintained larvae (both larvae that were never experimented on as well as
larvae from experiments) in tubes for over two weeks. We saw no mortality in larvae that had not
been exposed to heat stress. Mortality in experimental larvae was exclusively observed only for
those subjected to the higher ends of the temperature range. All surviving larvae also
metamorphosed into polyps, suggesting the effects of maintaining larvae in 0.2 ml tubes until
scoring were negligible, if any. In any case, effects would have been consistent across larval
cohorts which would not have influenced our interpretations.
Statistical analysis of larval survival
For each larval cohort, replicate trials were used to form logistic regression models (LL.2) using
the dose response curve, ‘drc,’ package (Ritz et al., 2015) in R (R Foundation for Statistical
Computing, 2017), to generate survival curves and the calculate the dose (temperature) at which
50% of the larvae died (LT50). In other words, replicate larval trials were used to generate more
precise estimates of LT50 values per larval cohort. Within each parental population, survival
curves between different cohorts were simultaneously inferred (as larvae from genetically
identical parents cannot be considered independent), and standard errors (of the LT50 estimate)
were corrected for simultaneous inference using the glht() function from the ‘multcomp’ package
in R. These LT50 values were then compared between larvae from STHS parents and control
parents as described below:
MA parental effects experiment (including persistence): The LT50 estimates from models for
control and STHS parents were compared using paired t-tests to account for variation in thermal
tolerance between larvae from the same parental populations (N=9 parental populations). We
conducted paired t-tests on just the LT50 values, as this was the main parameter of interest and it
allowed us to account for the effect of parental genotype on effect size, and follows the analyses
conducted in Reitzel et al., (2013), facilitating comparisons with prior work on N. vectensis. In
Journal of Experimental Biology • Accepted manuscript
addition, paired t-tests eliminate the need for multiple comparisons as would have occurred if we
tested each pair of treatment curves against each other. For the MA vs. NC experiment (below)
the latter approach was taken as there was only one set of parental populations examined.
MA maternal/paternal effects experiment: The LT50 estimates were analyzed using a general
linear model with parental treatment as a fixed effect and parental population as a random effect
using the lmer() function from the ‘lme4’ package in R (Bates et al., 2015) (N=6 parental
populations).
Genetic vs. parental effects (MA vs. NC) experiment: For each pair of STHS and control curves
(e.g. NC control, NC STHS) we tested whether response curves were significantly different by
comparing a model in which both treatments were estimated to have the same LT50 and one in
which LT50 estimate could vary by treatment, using likelihood ratio tests through the ‘anova()’
function in R to compare the two model outputs. This method was used because there were a
limited number of unique NC genotypes, therefore it would have been impossible to obtain
independent replication at the level of parental groups between STHS and control treatments. In
this instance, the replication is at the level of the number of larval trials conducted for each
parental cross and treatment combination (N=4-6 replicates).
Quantitative Polymerase Chain Reaction (qPCR) assays of larval gene expression
To test whether larvae from STHS parents exhibited differential expression of genes commonly
involved in stress response pathways, we measured expression of three genes: Heat Shock
Protein 70 (HSP70), Manganese Superoxide Dismutase 2 (MnSOD2), and Citrate Synthase. Four
pairs of genetically identical parental populations were either subjected to the STHS or control
thermal regimes (Fig. 4A, Table S1). Larvae were allowed to develop as described above.
At 72 hpf, 2-5 replicate pools of 200-300 swimming planula larvae from each parental
population and treatment were pipetted into 1.5 mL microcentrifuge tubes (Fig. 4A; Table S2)
for RNA extraction (“baseline” timepoint). Additional larvae were subjected to a 4-hour heat
shock at 35C using a Fisher Scientific™ Isotemp heating plate (Thermo Fisher Scientific,
Waltham, MA, USA). Larvae were sampled for gene expression immediately after the heat
Journal of Experimental Biology • Accepted manuscript
shock (“immediate”) (parental population G2 only), and 18 hours following the end of the heat
shock (“post”) (Table S2). Differences in gene expression at different timepoints were tested
using paired t-tests.
All larvae were immediately processed for RNA extraction using the phenol-chloroform based
Aurum™ Total RNA Fat and Fibrous Tissue Kit (Bio-Rad, Hercules, CA, USA) with on-column
DNase treatment. RNA yields were assessed using a NanoDrop One™ spectrophotometer
(Thermo Fisher Scientific, Waltham, MA, USA) giving a mean yield of 44.9 ng/l and a range
of 12.8-132.2 ng/l in a 15 l total elution volume. For synthesis of complimentary DNA
(cDNA) we used 200 ng of RNA per sample and the iScript™ DNA Synthesis kit (Bio-Rad,
Hercules, CA, USA) and C1000 PCR thermocycler (Bio-Rad, Hercules, CA, USA) with the
following protocol: (1) 5 minutes at 25C (2) 20 minutes at 46C (3) 1 hour at 95C, and (4) 4C
hold.
Primer sequences for Actin, 18S, L10, HSP70, and MnSOD2 were obtained from (Tarrant et al.,
2014) and (Helm et al., 2018) as these were previously used in N. vectensis. The gene sequence
for Citrate Synthase was determined by searching the N. vectensis genome on the JGI database
and selecting the sequence annotated as eukaryotic-type Citrate Synthase. The full sequence was
then submitted to the Primer3 web portal to generate the best primer sequence. Actin, 18S, and
L10 were used as reference genes. Gene accession numbers and primer sequences are listed in
Table S3.
For each gene, expression was measured in two 96-well plates with two technical replicates per
sample, and two across-plate replicated samples, using iTaq™ universal Syber Green Supermix
(Bio-Rad, Hercules, CA, USA), and a CFX96™ thermocycler (Bio-Rad, Hercules, CA, USA)
with the following protocol: (1) 1 min at 95C, (2) 40 cycles of amplification [15 seconds at
95C, 25 seconds at 60C], and (3) a final melt curve from 65C to 95C with a 0.5C increase
every 5 seconds. Raw, uncorrected fluorescence values were used to estimate the starting
template concentration using LinRegPCR (Ramakers et al., 2003). Cross plate variation was
corrected using Factor_qPCR (Ruijter et al., 2015). To obtain final expression values, the
Journal of Experimental Biology • Accepted manuscript
expression of each gene was normalized by dividing the gene’s estimated concentration by the
geometric mean of the references.
Fertilized oocyte size measurements
To test whether the experimental conditions affected egg size and potential energetic
provisioning, we examined egg bundles from all spawning females of four genetically identical
parental population pairs from the MA parental effects experiment. We photographed eggs using
a Zeiss™ Axio Cam 1Cc1 camera and imaging software (Carl Zeiss AG, Oberkochen,
Germany). A stage micrometer was photographed under the equivalent magnification for scale.
The diameter of 10 eggs per bundle was measured using Image J software (N= 230, 260, 160,
and 220 eggs per control population and N=130, 240, 210, and 200 eggs per STHS population,
respectively). Differences in mean diameters between STHS and control females for each
population pair were tested using paired t-tests (N=4 female populations).
Adult heat stress survival assays
To test whether thermal preconditioning resulted in priming of adult anemones to a subsequent
heat shock, we exposed 80 mixed-genotype, MA adult individuals from the general laboratory
populations to the STHS ramp described above and maintained another 80 individuals at 20C.
The day following the end of the STHS ramp, all anemones were subjected to a 6-hour heat
shock at 36C, and then returned to 20C. Mortality was assessed 48 hours following the acute
heat shock. Adults that had ejected mesenteries, were decomposing, or were unresponsive to
touch were scored as dead. Difference in the proportion of surviving anemones by treatment was
tested using a one-tailed, Fisher’s exact test in R for lower survival in control (non-
preconditioned) anemones.
Results:
Oocyte sizes before and after spawning
We examined the timing of gamete maturation to guide the development of protocols during
which gametes would mature under different thermal regimes. Immature oocytes were retained
after spawning, with a significant shift towards smaller oocytes following spawning (~45%
decrease in size, p<0.001, two-tailed, two-sample t-test, N=5 females pre- and post-spawn; Fig.
Journal of Experimental Biology • Accepted manuscript
5B). Oocyte density was higher after spawning suggesting the release of larger, mature oocytes,
and subsequent contraction of the mesenteries following spawning (mean: 21.8 oocytes/mm2
before and 27.3 oocytes/mm2 after spawning). These data suggest selective spawning of mature
oocytes, with retention of smaller immature oocytes that can continuously develop between
spawns. Thus, our protocol for physiological assays included a "clearing spawn" prior to heat
ramp exposure to focus the assays on larvae produced from gametes that matured under
experimental conditions.
MA parental effects
Across the nine genotype-controlled parental population pairs we found a significant (p<0.001,
one-tailed, paired t-test, N=9) increase in LT50 (mean LT50: 0.34C, range: 0.22-0.46C) in
larvae from STHS parents (Fig. 1E). However, once STHS parents were returned to 20C for
two weeks, larvae from the three parental populations that were re-spawned after 2 weeks at
20C no longer showed higher thermal tolerance (p=0.57, one-tailed, paired t-test, N=3; Fig.
1F).
MA maternal vs. paternal effects
Linear model results indicate that maternal effects confer a significant (p<0.05) increase in larval
thermal tolerance (mean LT50: 0.18C, sd: 0.087, N=5), while paternal effects induced no
difference in larval LT50 compared to controls (mean LT50: -0.001C, sd: 0.084, N=6).
Interestingly, when both parents were subjected to STHS, larvae showed the largest increase in
LT50 (mean LT50: 0.369C, sd: 0.082, p<0.01, N=6), indicating there are either synergistic
effects to having both parents exposed to heat stress or negative, epistatic effects when only one
of the parents is exposed.
Genetic effects: North Carolina and Massachusetts crosses
As expected from previous studies, we found that NC purebred larvae had higher larval LT50
than MA purebred larvae under control conditions (LT50: 0.18C, p<0.01, likelihood ratio test,
N=6; Fig. 3D). Hybrid larvae showed intermediate phenotypes, between MA purebred and NC
purebred larvae (N=6; Fig. 3B-C). Exposure of MA parents to STHS, however, resulted in larvae
that had statistically indistinguishable LT50 values to purebred larvae from NC controls (p=0.17,
Journal of Experimental Biology • Accepted manuscript
likelihood ratio test, N=4-6; Fig. 3D), and from NC STHS larvae (p=0.09, likelihood ratio test,
N=4; Fig. 3D).
NC purebred larvae from STHS parents also showed a significant increase in larval LT50
compared to the controls (LT50: 0.2C, p=0.001, likelihood ratio test, N=4-6; Fig. 3D). Hybrid
larvae with a NC mother and a MA father showed a significant, though smaller increase
following STHS (LT50: 0.17C, p<0.01, likelihood ratio test, N=6; Fig. 3D). Hybrids from a
MA mother and NC father showed slightly higher LT50 following STHS, but this difference was
not statistically significant (LT50: 0.13C, likelihood ratio test, p=0.09, N=6; Fig. 3D).
Larval gene expression
HSP70 and MnSOD2 were chosen based on previous work on the response of N. vectensis to a
variety of stressors, such as oxidative stress, UV, and pollutants (Helm et al., 2018; Tarrant et al.,
2014). For instance, HSP70 expression is quickly upregulated after 6 hours of heat stress and
remains elevated 24 hours after recovery (Helm et al., 2018). MnSOD2 levels increase most after
recovery from heat stress, but respond quickly under ultraviolet stress (Helm et al., 2018; Tarrant
et al., 2014). Citrate Synthase was chosen because activity is used as a proxy for mitochondrial
density and aerobic capacity, which can play a role in thermal physiology and would be expected
to increase under prolonged thermal stress (Gibbin et al., 2017; Hawkins and Warner, 2017).
Spawning success across parental cohorts used for this experiment was uneven, therefore,
biological replication depth across different parental groups and larval treatments varied (Table
S2). Statistical tests are conducted with replication at the level of parental population (N=4).
Overall, gene expression levels did not differ between larvae from STHS and control parents at
baseline conditions (p=0.29 for Citrate Synthase, p=0.31 for HSP70, and p=0.25 for MnSOD2,
two-tailed, paired t-tests; Fig. 4B). Eighteen hours following the acute larval heat stress, larvae
from STHS parents showed higher expression of HSP70 and MnSOD2 (p=0.052, p=0.12,
respectively, paired t-test; Fig. 4C), though these were not significantly different. Levels of
Citrate Synthase showed no discernable difference (p=0.68, two-tailed, paired t-test; Fig. 4C).
Journal of Experimental Biology • Accepted manuscript
Gene expression was also measured immediately following the larval heat stress for larvae from
control parental populations (N=4). HSP70 expression was significantly higher immediately
following heat stress relative to baseline in larvae from control parents (p=0.03, two-tailed,
paired t-test, N=4; Fig. 4D), while expression of Citrate Synthase was significantly lower
(p=0.04, two-tailed, paired t-test, N=4; Fig. 4D). Gene expression results across all families and
time points are shown in Fig. S4.
Fertilized oocyte sizes
We did not find any differences in fertilized oocyte diameter between control and STHS mothers
(p=0.28, two-tailed, paired t-test, N=4; Fig. S5).
Pre-conditioning of adult anemones to heat stress
We found a significant difference (p=0.03, Fisher’s exact test) in survival following an acute, 6-
hour heat shock at 36C between control and pre-conditioned (STHS) adult anemones (N=80
anemones per treatment). On average there was 80% survival in control anemones and 92%
survival in STHS anemones, with an odds ratio of 0.38, indicating anemones exposed to the
STHS regime are less likely to die from an acute heat shock, as would be expected if thermal
exposure induced physiological priming in N. vectensis.
Discussion:
We used N. vectensis as a powerful study system to measure the strength and plasticity of
parental effects as well as explore the mechanisms promoting shifts in larval thermal tolerance
across distinct populations. We first explored parental effects in MA populations. For our
experiments we created genetically controlled parental populations, that were replicated across
parental treatments. Though the overall genetic composition of the parental populations across
treatments was the same, one may wonder whether the contributions of individual clonal lineages
to the gamete pool may have varied among treatments, potentially resulting in clonal effects
across parental treatments. However, both the consistency in the relative LT50 values across
larval cohorts (e.g. the ranking of LT50s of larvae from the same parental populations was
consistent across control and STHS parental treatments), and the fidelity in the reaction norm
Journal of Experimental Biology • Accepted manuscript
slope (mean=0.34C; sd: 0.07), strongly suggests the increase in larvae thermal tolerance is a
result of parental effects and is unlikely to stem from differential fecundity among more
thermally tolerant parental clonal lines. In line with our observations, parental effects have also
been shown to promote offspring thermal tolerance in polychaetes (Massamba-N’Siala et al.,
2014), fruit flies (Lockwood et al., 2017), copepods (Vehmaa et al., 2012), corals (Putnam and
Gates, 2015; Putnam et al., 2016), sticklebacks (Shama et al., 2016), and damselfish (Donelson
et al., 2012), among many others.
Despite the increasing popularity of studies in parental effects and transgenerational plasticity,
many studies only test the phenotypes of one cohort of offspring. For organisms with multiple
reproductive cycles it is important to also test the persistence of such effects across subsequent
breeding periods. For instance, in polychaetes the impact of maternal effects depends on when
during oogenesis the mother experiences a particular environment, with exposure early in
oogenesis providing stronger protective parental effects under variable environmental conditions
(Massamba-N’Siala et al., 2014). In our case, we find that once STHS parents are returned to
control temperatures, larvae from the parents’ next spawn return to baseline levels of thermal
tolerance (Fig. 1F), indicating reversible (plastic) parental effects in N. vectensis. The continuous
gametogenic cycle in N. vectensis likely facilitates this process by maintaining a constant pool of
immature oocytes that can be modified by cues from the environment in which they directly
develop (Fig. 5). Given the anemone’s naturally fluctuating environment, this plasticity in
parental effects combined with continual gametogenesis could be more beneficial than adjusting
irreversibly in any one direction (Beaman et al., 2016). Our results underscore the need to test for
reversibility or persistence across breeding periods, as well as monitoring of gametogenesis, in
order to better understand how such mechanisms may help (or fail to help) organisms keep pace
with global climate change.
It is also important to determine what degree of parental exposure or stress yields beneficial
results for offspring. In Massachusetts, N. vectensis experience a wide diel (approximately 15C
during summer months), and seasonal range in temperatures (approximately 40C between
winter and summer temperatures) (Tarrant et al., 2019). Our experiments used laboratory
acclimated animals, which have not been exposed to diel temperature fluctuations for several
Journal of Experimental Biology • Accepted manuscript
years. Parental exposure to diel temperature fluctuations prior to spawning could potentially lead
to different results, as parents would experience high temperatures every day, albeit for a shorter
duration. In such a case, parental anemones may consistently produce larvae with higher thermal
limits, instead of constantly attempting to modulate parental effects. Given the vast seasonal
temperature variation in their natural habitat, it is also possible that under field conditions,
parental effects may more closely track seasonal variation instead of the inter-week differences
tested here. Nevertheless, our results clearly show that N. vectensis is capable of such
modulation. Future studies that examine temperature regimes that more closely match the short-
and long-term variability in field temperatures as well as compare different timescales of
parental exposure to such variability would provide new insights.
We found that parental effects in N. vectensis were consistent across populations, as both NC and
MA parents conferred similar increases in thermal tolerance to their larvae, signifying such
effects are not unique to Massachusetts populations, and may, instead, be an inherent
characteristic of N. vectensis that suits its naturally variable estuarine habitat. The ecological
relevance of these increases is underscored by the fact that the thermal threshold of larvae from
STHS Massachusetts parents was comparable to that of larvae from control North Carolina
parents (Fig. 3D) – a genetically distinct and isolated population that is likely locally adapted to
warmer temperatures (Reitzel et al., 2013). The shallower slope in the reaction norm of hybrid
larvae, however, suggests that epistatic effects may arise when combining distinct populations
and that differences in larval thermal tolerance between sites have a strong genetic basis.
Uncovering the mechanisms behind parental effects can also improve understanding of
organismal physiology under changing conditions. Preliminary experiments (data not shown)
showed a substantially smaller increase in thermal tolerance for juveniles (7 dpf) from STHS
parents, suggesting that gamete provisioning mechanisms (such as increased lipid or antioxidant
content, transcript loading, or early epigenome influences) may be more likely than effects that
last further into development (e.g. induction of developmental plasticity), as the former would
enhance fitness of early larval stages but wane as larvae grow. The timing of gametogenesis in
relation to stress exposure may also play an important role, as has been shown for polychaetes
(Massamba-N’Siala et al., 2014). During the development of germ cells there are critical
Journal of Experimental Biology • Accepted manuscript
windows in which the epigenome is more readily influence by environmental cues (Bale, 2014;
Xavier et al., 2019). During our trials, we spawned animals prior to the start of the experimental
heat ramps, which facilitates clearance of mature oocytes (Fig. 5B). This would imply that the
experimental larvae were primarily derived from oocytes that completed later stages of
maturation in the experimental conditions. As such, STHS parents may have been able to modify
gamete epigenomes in a manner that could facilitate larval thermal tolerance. The timing of
epigenetic programming in N. vectensis, however, is not known. Furthermore, we cannot be
certain that all mature gametes were released during the pre-spawn. It is possible, therefore, that
some of the effects described here arose from a combination of direct parental effects and
environmental effects on developing gametes, as the timing of gametogenesis could not be
entirely constrained to the experimental period, despite our best efforts (see Torda et al. (2017)
and Byrne et al. (2020) for a detailed discussion of such considerations).
We investigated two possible mechanisms for increased larval tolerance: modulation of egg size
and larval gene expression. Larger eggs and larvae could be expected to be more sensitive to
thermal stress associated with oxygen-limitation (Martin et al. 2017). We found eggs from STHS
and control parents did not differ in size (Fig. S5). Anecdotally, egg masses from STHS mothers
sometimes had fewer eggs, but this pattern was inconsistent and not rigorously quantified. A
potential trade-off between egg number and egg size, overall fecundity, or other provisioning
(e.g. lipid content) could be investigated in future studies. Such a trade-off may be responsible
for the range of egg diameters seen in STHS egg clutches (Fig. S5).
As a second possible mechanism, modulation of gene expression might enhance larval
thermal tolerance. In Drosophila, for instance, loading of maternal transcripts for a heat shock
protein into eggs, enhanced embryonic thermal tolerance (Lockwood et al., 2017). Comparison
of egg and sperm transcriptomes in coral also suggest that early development is largely governed
by parentally derived transcripts (Van Etten et al., 2020). Larvae from STHS parents showed
higher levels of HSP70 and MnSOD2 expression eighteen hours after heat stress, suggesting that
sustained expression could be influencing tolerance (Fig. 4C). However, larvae from control
parents showed a prompt return to baseline (pre-stress) levels of gene expression after showing
differences in expression immediately after heat stress (Fig. S4). This pattern, termed
Journal of Experimental Biology • Accepted manuscript
‘transcriptomic resilience,’ has been linked to stress tolerance in other species such as seagrasses
and corals (Franssen et al., 2011; Thomas et al., 2019). As such, it is surprising that
transcriptomic resilience in larval gene expression patterns for N. vectensis is not coupled with
higher thermal tolerance (i.e. it was larvae from control parents that showed this pattern, not
larvae from STHS parents). If gene expression is contributing to larval tolerance, then it is
possible that the dynamics of expression (e.g. timing and magnitude) are more important than
either factor alone. We saw no differences in expression of Citrate Synthase under baseline
conditions or after recovery from heat stress between larvae from control of STHS parents,
suggesting mitochondrial density was not substantially different between larvae from different
parental treatments (Fig. 4C). Interestingly, we do see a decrease in Citrate Synthase expression
when comparing larvae from control parents at baseline and immediately after heat stress, which
could suggest mitochondrial fusion (and decreasing density) under elevated temperatures (Fig.
S4). Previous studies on mitochondrial fission and fusion rates in vertebrate neural cells, estimate
fusion rates of 0.023 fusions/min (Cagalinec et al., 2013). If similar rates can occur in N.
vectensis then around 4-6 fusions might be possible over the span of four hours which could
impact expression levels. However, it is also possible that heat stress may decouple Citrate
Synthase expression from mitochondrial function, especially in developing larvae, so these
results should be interpreted with caution. Due to limitations in sampling size, our study may
have missed other dynamic expression patterns or lacked the power to detect substantial
differences in expression across timepoints. Examining gene expression in larvae under heat
stress is doubly challenging as temperature can accelerate developmental processes that also
prompt widespread changes in gene expression (Jacott and Boden, 2020). Future transcriptomics
studies tracking global gene expression through RNAseq or similar methods, and which compare
individual larvae could reveal more complex patterns and identify candidate genes involved in
increasing larval thermal tolerance. In particular examining gene expression patterns in larvae
from single parent crosses or a split-brood design may provide higher power to detect such
differences.
We found exposure of mothers alone to the STHS regime leads to an increase in larval LT50,
while exposure of fathers alone does not result in any detectable shift in larval thermal tolerance
(Fig. 2C). Maternal effects alone, however, account for only about half of the LT50 increase seen
Journal of Experimental Biology • Accepted manuscript
when both parents are exposed to the STHS regime, suggesting that having both parents under
the same conditions confers additional benefits (Fig. 2C). The mechanisms responsible for this
synergy are unknown but theoretical, antagonistic epistasis between maternal effects and
offspring genotypes have been described (Wolf, 2000). While examples of maternal effects
abound, evidence for direct paternal effects is only beginning to emerge (Crean and
Bonduriansky, 2014; Soubry, 2015). Paternal influences on zygotic phenotypes through transfer
of cytosolic compounds to the fertilized egg or through compounds in the seminal fluids have
been described in humans (Kumar et al., 2013), trout (Danzmann et al., 1999), mice
(Rassoulzadegan et al., 2006), and insects (Avila et al., 2011). Potential influences of maternal
effects on the efficacy of paternal effects have also been conceptually explored (Crean and
Bonduriansky, 2014), and have been shown for paternally inherited QTLs that influence thermal
tolerance in rainbow trout (Danzmann et al., 1999). In addition to transcript loading, the parental
epigenome may influence early offspring performance or gene expression (Bale, 2014; Xavier et
al., 2019), although this influence may be short-lived as the epigenome is often reset in early
development in most animals and plants studied (Feng et al., 2010). It is possible that male N.
vectensis are providing larvae with additional resources or epigenetic differences, but these are
insufficient when decoupled from maternal contributions. Combined with the elevated
expression of HSP70 and MnSOD2 in larvae from STHS parents following heat stress, our
results suggest that RNA loading or epigenetics may contribute to larval thermal performance
and these could be stronger when both parents are exposed to the same STHS regime.
Exposure of N. vectensis to elevated temperatures also substantially increased survival of adult
anemones to acute heat shock, suggesting N. vectensis can also modulate adult physiology to
match thermal conditions. Priming effects such as these have been previously reported for
cnidarians, including corals (Gibbin et al., 2018; Putnam and Gates, 2015) and anemones
(Hawkins and Warner, 2017), and across other taxa such as sculpins (Todgham et al., 2005). A
study of adult thermal acclimation to a range of temperatures (6 - 33C) suggests N. vectensis
rapidly adjusts respiration rates and later increases metabolic capacity (activity of mitochondrial
enzymes) when exposed to different temperatures (Brinkley, Rivera, and Tarrant, unpublished
data), a response that mirrors that found in polychaetes under thermal acclimation (Gibbin et al.,
Journal of Experimental Biology • Accepted manuscript
2018). Studies that focus on mitochondrial physiology in adults and larvae may help identify the
mechanisms responsible for N. vectensis’ rapid acclimation to different thermal regimes.
While plasticity both of adult physiology and parental effects on their offspring can enhance
short-term survival, there may also be trade-offs. For instance, heritable transgenerational
changes that increase offspring’s aerobic scope have been described in spiny damselfish (Ryu et
al., 2018), but F2 generation fish maintained at warmer temperatures were unable to breed,
suggesting a strong trade-off between thermal performance and reproduction (Veilleux et al.,
2018). Such trade-offs should be explored in future studies in order to fully characterize the
potential of parental effects to promote species persistence under changing climate scenarios. We
find that parental effects on thermal tolerance in N. vectensis are substantial, but quickly
reversible (subsequent cohorts lose protection) suggesting N. vectensis responds quickly to its
current environment and may take advantage of parental effects without long-term trade-offs.
Studies that follow multiple generations of N. vectensis through parental heat exposure and track
offspring growth and eventual reproduction could elucidate any potential trade-offs associated
with increased thermal tolerance early in life. A parental strategy that favors short-term gamete
provisioning over longer-term epigenetic changes may be better suited to N. vectensis’ highly
variable, yet broadly predictable (seasonal and tidal) environment.
Studies of N. vectensis reproduction in the field are scarce. Only one study, to our knowledge,
describes gravid gonads in field-collected individuals from Nova Scotia, and only during August
and September (Frank and Bleakney, 1976). A handful of other studies suggest reproduction is
mainly asexual under natural conditions, given the high levels of clonality observed in field
collected anemones (Eckelbarger et al., 2008; Hand and Uhlinger, 1994; Reitzel et al., 2008).
Dedicated studies of N. vectensis’ reproductive cycle in the field, including the effects of natural
thermal variation on reproduction and larval phenotypes, would offer greater understanding of its
parental strategies.
Journal of Experimental Biology • Accepted manuscript
Overall, N. vectensis offers a robust organismal system in which to study thermal responses due
to their wide thermal range, easy husbandry, fast development, ease of spawning, ability to
generate clonal lineages, as well as their well-developed genomic resources. Here, we show N.
vectensis is capable of quickly modulating parental effects to increase larval thermal tolerance.
While maternal exposure can result in significant shifts, exposure of both parents to different
environments results in a larger increase in larval thermal tolerance. In a northern population
(MA), shifts due to parental effects result in larval thermal limits that are comparable to those of
a southern, more thermally tolerant, population (NC). These patterns point to both a genetic and
plastic basis for thermal tolerance in N. vectensis. Given our rapidly changing global thermal
environment, studies that aim to uncover the mechanisms responsible for these rapid shifts in
thermal performance can provide insights into the sensitivity, acclimation, and adaptation
potential of vulnerable species such as marine ectotherms.
Journal of Experimental Biology • Accepted manuscript
Acknowledgements:
We would like to thank Rebecca Helm for initial guidance on experimental questions and design,
Victoria Starczak for guidance on statistical analyses, and Hollie Putnam for input on
experimental designs to investigate the role of parental and transgenerational effects. We further
thank Hadley Clark, Sabine Angier, and Hannah Stillman for their assistance with animal care
and larval trials. We also thank Adam Reitzel and for providing animals from North Carolina and
Whitney Leach for field assistance. We thank David Brinkley, Jehmia Williams, Sarah Davies,
Hannah Aichelman, Nicola Kriefall, Daniel Wuitchik, and James Fifer for feedback on figures.
We also thank members of H.E.R.’s thesis committee (Iliana Baums, Simon Thorrold, Janelle
Thompson, and Anne Cohen) for feedback on data analysis and interpretation. Lastly, we thank
the Cnidofest 2018 meeting for facilitating collaboration between the Tarrant and Gibson labs.
Competing interests:
We have no competing interests.
Funding:
We further thank the Betty and Gordon Moore Foundation [4598 to A.M.T.] for providing
funding for this work. Additional funding for H.E.R. was provided by the National Defense
Science and Engineering Graduate Fellowship Program, Gates Millennium Scholars Program,
the Martin Family Fellowship for Sustainability, and the American Association of University
Women. C.Y.C. and M.C.G. were funded by the Stowers Institute for Medical Research.
Data availability:
Additional methods and results as noted in the paper are in the electronic supplementary
material. Raw data and code for all statistical analyses and figure generation are included in the
authors’ GitHub repositories: https://github.com/hrivera28/Nematostella-ParentalEffects/ and
https://github.com/Penguinayee/Nematostella_ParentalEffects
Journal of Experimental Biology • Accepted manuscript
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Journal of Experimental Biology • Accepted manuscript
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Journal of Experimental Biology • Accepted manuscript
Abbreviations:
LT50 – Lethal Temperature 50, the temperature causing 50% mortality in a population
HSP70 – Heat Shock Protein 70
CS – Citrate synthase
MnSOD2 – Manganese Superoxide Dismutase 2
MA – Massachusetts
NC – North Carolina
MD – Maryland
STHS – Short Term Heat Stress, referring to parental population that underwent the heat stress
regime described in the methods section
ASW – Artificial seawater
FSW – Filtered seawater (natural)
PSU – Practical salinity units (equivalent to g/kg)
Journal of Experimental Biology • Accepted manuscript
Figures
Fig. 1 | Larval thermal tolerance increases when parents experience heat stress during
gametogenesis. (A) In situ temperatures in Sippewissett marsh, Woods Hole, MA during one
week in July 2016; data from Sachkova et al., (2020). Dashed grey line denotes maximum STHS
treatment temperature (33C) (B) Experimental design for MA parental effects experiment. See
Table S1 for population breakdown. STHS – Short Term Heat Stress parental treatment (C)
Temperature regimes experienced by parents during the 10 days prior to spawning, logged every
half hour. (D) Example survivorship curves of larvae from control (blue) and STHS (red) parents
when exposed to acute temperature stress. Ribbon shows 95% confidence interval for logistic
survivorship model. Curves shown for larval cohort G6. Curves for all the cohorts tested are
shown in Fig. S2. (E) Temperature at which 50% of larvae die (LT50). Colors correspond to
genotype-controlled families. Error bars: s.e.m. Mean shift in LT50 is 0.34C (p= 2.25x10-7, two-
tailed, paired t-test, N=9). (F) Larval LT50 for N=3 parental pairs that were re-spawned
approximately 2 weeks after the first experimental spawn (shown in E). Two-tailed, paired t-test
for differences in LT50 between parental treatments were non-significant p=0.57, indicating
parental effects subside during subsequent spawns.
Journal of Experimental Biology • Accepted manuscript
Fig. 2 | Increase in larval thermal tolerance is not attributable to exclusively maternal or
paternal effects. (A) Experimental design. Colored boxes represent genotype-controlled parental
populations. See Table S1 for complete details. F: Female; M: Male (B) Example survivorship
curves of larvae from different parental treatments when exposed to acute temperature stress.
Ribbon shows 95% confidence interval for logistic survivorship model. Curve shown for cohort
G3; curves for all cohorts tested are shown in Fig. S3. (C) LT50 for larvae from each cohort
(N=6, colors), and parental treatment. Linear model showed a significant increase in larval LT50
in larvae with a STHS mother or both STHS parents, but not STHS father only, relative to
controls. * p<0.05, ** p< 0.001
Journal of Experimental Biology • Accepted manuscript
Fig. 3. | MA Parental effects confer thermal tolerance equivalent to native NC larvae.
(A) Experimental design. Animals from NC are shown in red, from MA in blue. Note that unlike
for MA-only experiments (Figs. 1-2), controlling for parental genotypes was achieved
temporally instead of through the use of paired populations. The same groups of parents were
spawned under control conditions and then after the STHS treatment. F: Female; M: Male (B)
Survival curves of larvae from control parents and (C) after parents underwent STHS ramp. (D)
LT50 for each larval cohort and cross. Asterisks denote cohorts for which there was a significant
(p<0.05, likelihood ratio test) increase in larval LT50 following STHS exposure of parents.
Brackets denote cohort pairs that had significantly different LT50 within parental treatment
condition. Cross denotes insignificant difference between MA larvae from STHS parents and NC
larvae from control parents. LT50 of larvae from STHS MA parents is comparable to that of
larvae from NC parents from both control (p=0.17, likelihood ratio test) and STHS conditions
(p=0.09, likelihood ratio test).
Journal of Experimental Biology • Accepted manuscript
Journal of Experimental Biology • Accepted manuscript
Fig. 4. | Larval gene expression under baseline and heat stress conditions.
(A) Experimental design. Colored bowls represented genotype-controlled parental populations.
Filled shapes indicate STHS parents and their larvae. (B) Constitutive gene expression at
baseline in 72 hpf larvae from four control (blue) and STHS (red) clonal parental populations.
Colored points correspond to larval cohort (C) Inducible gene expression patterns 18 hours after
the acute heat shock treat (bottom trajectory in panel A). Larvae from STHS parents have a
slightly stronger induction of HSP70 and MnSOD2 following heat stress (p=0.052, p=0.12,
respectively, two-tailed, paired t-tests, N=4). (D) Larvae from only control parents show
decreased expression of MnSOD2 and increased expression of HSP70 (p=0.04, p=0.03,
respectively, paired t-test, N=4), immediately after heat stress. In panels B-D expression is
shown as the starting concentration after normalizing to expression of housekeeping genes and
correcting for cross-plate variability (see methods).
Journal of Experimental Biology • Accepted manuscript
Fig. 5 | Nematostella vectensis females retain immature oocytes after spawning.
A) Single focal plane of confocal microscopy image of mesenteries containing oocytes. Oocytes
(dashed outline) are distinguished from the surrounding tissue by size, the enlarged nuclei (white
arrow) and nucleolus (asterisks). The retractor muscle fibers (white arrowheads) of the
mesenteries are enriched with F-actin (magenta). Nuclei are labeled by SYBR™ Green I (green).
Scale bar = 100 µm. (B) Mean oocyte area per-animal before and after spawn (N=5 anemones).
After spawn, the oocytes remaining in gonads are significantly smaller (p<0.001, two-tailed,
two-sample t-test).
Journal of Experimental Biology • Accepted manuscript
Figure S1 | In situ temperatures from MA and NC salt marsh sites.
(A) Temperatures logged every 20 minutes from June 1, 2016 until Sept 30, 2016. (B) Boxplot
of mean daily daytime (9 AM to 9 PM) temperatures at both sites over the same summer period.
NC temperatures are significantly higher than MA (p<0.01, two sample t-test, N=122 days) (C)
Boxplot of the mean daily range (daily maximum temperature – daily minimum temperatures) in
MA and NC over the same time period. All data are from Sachkova et al., (2020), data S6.
Journal of Experimental Biology: doi:10.1242/jeb.236745: Supplementary information
Journal of Experimental Biology • Supplementary information
Figure S2 | Survival curves for all larval cohorts from MA parental effects experiment.
Survival of larvae from different parental treatments when exposed to acute temperature stress.
Ribbon shows 95% confidence interval for logistic survivorship model. Parental population
codes correspond to populations described in table S1.
Journal of Experimental Biology: doi:10.1242/jeb.236745: Supplementary information
Journal of Experimental Biology • Supplementary information
Figure S3 | Survival curves for all larval cohorts in MA maternal/paternal effects
experiment.
Survival of larvae from different parental treatments when exposed to acute temperature stress.
Ribbon shows 95% confidence interval for logistic survivorship model. Parental population
codes correspond to populations described in table S1.
Journal of Experimental Biology: doi:10.1242/jeb.236745: Supplementary information
Journal of Experimental Biology • Supplementary information
Figure S4 | Larval gene expression across all timepoints and families.
Gene expression is shown as the starting concentration after normalizing to expression of
housekeeping genes and correcting for cross-plate variability (see methods). Base: Constitutive
expression at 72 hpf without any heat exposure. HS-Im: Immediately following the acute larval
heat shock. HS-Post: 18 hours following the end of the heat shock. Larvae from control parents
are designated with “Ctrl” and those from STHS parents with “STHS.” Expression is faceted by
family (rows) and genes (columns).
Journal of Experimental Biology: doi:10.1242/jeb.236745: Supplementary information
Journal of Experimental Biology • Supplementary information
Figure S5 | Egg diameter does not differ between parental treatments. Diameter of eggs
released by four genetically-controlled, paired parental populations. There is no significant
difference in sizes of eggs by parental treatment (p=0.28, paired t-test).
Journal of Experimental Biology: doi:10.1242/jeb.236745: Supplementary information
Journal of Experimental Biology • Supplementary information
