O. Tills a,⁎, X. Sun b, S.D. Rundle a, T. Heimbach a, T. Gibson a, A. Cartwright a, M. Palmer a, T.

Rudin-Bitterli
a
, J.I. Spicer a
a b
Marine Biology and Ecology Research Centre, Marine Institute, School of Marine Science and Engineering, Plymouth University, Drake Circus, Plymouth PL4 8AA, UK
Jiaozhou Bay Marine Ecosystem Research Station, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 26071, China

article info abstract

Our understanding of how reduced seawater pH affects the behaviour and growth of scyphozoan jellyfish is poor. Here, we
Article history: investigated the effects of simulated Ocean Acidification (OA) (pH = 7.6 for 7 d) on pulsing behaviour (as an index of
Received 18 November 2015
swimming behaviour) and aspects of the morphology of ephyrae of the moon jellyfish Aurelia aurita. Ephyrae exposed to
Received in revised form 16 March
2016 Accepted 22 March 2016 reduced pH had a significantly smaller surface area, central disc area, and lappet length and width than controls. Pulsation rate
Available online 22 April 2016 was significantly lower, and the mean pulse-to-pulse period shorter, in the reduced pH treatment. There was, however, no
significant treatment effect on either the maximum or minimum pulse-to-pulse period, suggesting that the ability for rapid
pulsations was maintained. Ephyrae from the reduced pH treatment displayed a more variable pulsation behaviour, with an
Keywords: Ocean
elevated standard deviation and root mean square of successive difference (RMSSD) in pulse-to-pulse period. In summary,
Acidification
Pulsation
reduced pH simulating future predicted Ocean Acidification conditions, had important effects on aspects of swimming
Bio-imaging behaviour and size of A. aurita ephyra, which may have consequences for survival and the population dynamics of field
populations.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction Doney et al., 2009). For example, direct measures obtained from time series
data collected in the North Atlantic and
The pH of seawater in the world's oceans varies considerably, with current
mean (total scale) values of surface waters ranging between 7.8 and 8.4 (IPCC,
2013) and even lower natural values for some areas such as coastal upwelling
zones (Feely et al., 2008), estuaries (Burnett, 1997) and intertidal rock pools ⁎ Corresponding author.
E-mail address: [email protected] (O. Tills).
(Morris and Taylor, 1983). Oceanic pH values are also affected by http://dx.doi.org/10.1016/j.jembe.2016.03.014 0022-0981/© 2016 Elsevier B.V. All rights
anthropogenic factors. Historic increases in atmospheric CO2, mainly as a reserved.
result of anthropogenic fossil fuel consumption are now well documented
(Friedli et al., 1986; Petit et al., 1999; Stocker et al., 2013). Much of this CO2
dissolves in the oceans and this process has greatly ameliorated atmospheric
warming caused by the accumulation of this greenhouse gas (Sabine et al.,
North Pacific indicate that historic pH reductions at these stations in the
2004). Dissolution of CO2 by the oceans chemically alters seawater by
region of 0.0014 to 0.0024 pH units per annum (Sullivan et al., 2014). Such
changing its carbonate chemistry: the partial pressure of CO2 (pCO2) increases,
rates of reduction in oceanic pH are occurring around 100 times faster than
leading to an increase in bicarbonate, but a decrease in the saturation of CaCO 3
noted during the previous 650,000 years (Raven et al., 2005) and it has been
minerals and pH (Feely et al., 2009). This CO2-driven decrease in seawater pH
predicted that a reduction in the pH of marine surface water by 0.4 units
has been termed Ocean Acidification (OA) (Calderia and Wickett, 2003; could occur by 2100 (Caldeira and Wickett, 2003).
 Reduced seawater pH may result in a reduction in calcium carbonate A. aurita used in this study originated from a population collected from
saturation states and therefore there has been unprecedented attention the coast of San Diego, California and sent to The Deep Aquarium, Hull,
devoted to the impact of OA on shell-forming marine organisms (reviewed UK. Here they were kept and bred for approx. 30 generations. Polyps were
in Orr et al., 2005; Hofmann et al., 2010; Findlay et al., 2011; Gazeau et al., transported from Hull to Plymouth (48 h transit, no mortality) in a number
2013). Considerably less attention has been paid to non-calcifying of sealed plastic vials (vol. = 10 mL) containing previously aerated
organisms, although the work that has been carried out points to important seawater (S = 33, T = 15 °C). Upon arrival in Plymouth, polyps were
OA-related effects on the physiology and life history of a wide range of allowed to settle on Petri dishes (diam. = 100 mm) for 7 days, before the
marine taxa (e.g. algae (Connell and Russell, 2010), non-coralline dishes were suspended in seawater (T = 15 °C) in an aquarium (vol. = 10
cnidarians (Jarrold et al., 2013) and sipunculid worms (Pörtner, 2008)). The L). Water in the aquarium was changed weekly. Polyps were fed Artemia
sensitivity of both calcifying and noncalcifying marine organisms to OA sp. nauplii twice a week ad libitium.
appears to be related to their ability to buffer extracellular pH (Pörtner, Strobilation was induced by increasing the environmental temperature
2008). It is now known that OA can alter various aspects of the function of of the aquarium from 15 °C to 20 °C for 7 days and then reducing the
marine animals (Siebel and
O. Tills et al. / Journal of Experimental Marine Biology and Ecology 480 (2016) 54–61 55

Walsh, 2003; Widdicombe and Spicer, 2008), including extracellular acidbase temperature back to 15
balance (Pörtner, 2008), metabolism (Pörtner et al., 1998), behaviour (Simpson °C (Widmer, 2008). Strobilation began approx. one month after the return
et al., 2011) and growth and development (Dupont and Thorndyke, 2009). to T = 15 °C. The aquarium was checked daily and new ephyrae removed
Scyphozoan jellyfish are important predators of, and competitors with, fish using a 1.5 mL plastic pipette. Newly-produced ephyrae were maintained in
(Purcell et al., 2007; Graham et al., 2014). There is also concern over their a second aquarium (vol. = 30 L) filled with natural seawater (S = 33). A
anthropogenic, environmentally-mediated bloom formation, although the Bio-Orb (Reef One, Norwich, UK) aquarium was used due to its spherical
effect of OA on scyphozoans has received little study. No convincing shape, which optimized water flow and ensured that ephyrae remained
causative mechanism linking jellyfish abundance with OA has been suspended in the water column. Water was changed daily (50%
established. Attrill et al. (2007) published a positive correlation between replacement) and ephyrae were fed ad libitum with Artemia sp. Ephyrae of
decreasing seawater pH and increasing jellyfish (including, but not restricted equivalent body size (total area = 3.02–3.73 mm2, mean = 3.39 mm 2) were
to, Scyphozoa) frequency over the period 1971–1995 in the North Sea. removed and used in the experiment described below seven days after
However, Richardson and Gibbons (2008) could detect no significant strobilation.
relationships between jellyfish abundance and seawater pH using data gathered
from a much wider geographical area and concluded that a role for pH in
structuring zooplankton communities in the North Sea and Atlantic Ocean was 2.2. Exposure to CO2-enriched seawater
tenuous.
Several reviewers have speculated on how reduced pH may affect Effects of CO2-enriched seawater on aspects of the pulsation behaviour and
physiological function in scyphozoans (e.g. Fabry et al., 2008), but also body size of ephyrae were investigated by haphazardly selecting and assigning
emphasise the absence of empirical studies. There is some empirical work ephyrae to control (present day, non CO2enriched seawater pH = 8.10, N = 19),
on the effects of low pH on reproduction, development and morphology of or predicted, OA conditions (CO2-enriched, nominal pH = 7.60, N = 20).
scyphozoans, but it is largely restricted to early life history stages (polyps Ephyrae were maintained individually in modified Falcon tubes (vol. = 50
and ephyrae) (Kikkawa et al., 2010; Klein et al., 2014). Kikkawa et al. mL). Gas was introduced through a pipette tip (diam. = 2 mm) inserted into the
(2010) found that swimming activity of Aurelia spp. ephyrae was impaired bottom of the tube in order to create water circulation that maintained the
by 96 h exposure to pH = 6.896 (pCO 2 = 5000 μatm) and that it ceased suspension of the ephyra in the water column. The pipette tip was connected to
immediately at pH = 6.366 gas-tight tubing that was attached to a manifold of air-line gang valves. Each
manifold was connected to the appropriate treatmentspecific gas supply. Rates
(pCO2 ≥ 30,000 μatm). They concluded that ephyrae were highly tolerant of
of gas flow into each tube were controlled using the gang valve manifold and
very low pH. Winans and Purcell (2010) reported that culturing polyps and
this was standardised at 5 mL min−1. Where required an airline G-clamp was
ephyrae of Aurelia labiata at low pH (7.5 and 7.3) had no signi ficant effect
used to attain finer control. The Falcon culture tubes were fitted with lids
on survival or asexual reproduction. They did find that culture in low pH
containing a pin-hole. The pin-hole was used to facilitate a build-up of the gas
resulted in smaller statoliths, but the number of statoliths in each organ of
mixture in the air space at the top of the tube, which was necessary for the gas
balance (statocyst) remained unchanged.
to equilibrate with the seawater and to subsequently achieve the desired pH,
Here, we investigated the effect of culture in CO2-enriched (reduced pH)
but also ensured a sufficient release of gas from the culture tubes to prevent the
seawater on pulsation behaviour (as an index of swimming behaviour) and
system from pressurising.
some key aspects of the morphology of ephyrae of the moon jellyfish,
Seawater was changed twice weekly, after which ephyrae were fed
Aurelia aurita (Linnaeus) (Cnidaria: Scyphozoa). Seven days post-
strobilation, ephyrae were exposed for a further 7 day period to either decapsulated Artemia sp. eggs (0.2 mL of an 8 × 10 4 individuals mL−1
control (nominal pH = 8.10), or reduced pH (nominally pH = 7.60) concentrate). Seawater in the culture tubes was aerated with either ambient air
conditions; this reduced pH value was chosen to mimic projected OA (control — pCO2 380 μatm, nominal pH = 8.1), or CO2-enriched air (acidified
conditions, i.e. pH levels predicted to occur by 2100 — Scenario A2 of treatment — pCO2 1000 μatm, nominal pH = 7.6). The CO2enriched air was
Caldeira and Wickett (2003). After this period the pulse-to-pulse behaviour produced by introducing air and CO2 gas (BOC, Plymouth, UK) into a Buchner
of individual ephyra was measured and morphological measurements were flask for mixing with a compressed air source, using a multistage CO 2 regulator
made. (EN ISO 7291; GCE Worksop, UK). The level of carbon dioxide in the
CO2enriched air was monitored continuously using a CO 2 analyser (820; LI-
2. Materials and method COR, Lincoln, NE, USA) and, if required, the level of CO 2 flow was manually
adjusted to the desired value on a daily basis. Seawater pH was measured in
2.1. Ephyrae origin and culture each culture chamber daily and seawater tCO 2 was measured in each culture
chamber three times during the course of the experimental period (Table 1).
pH (NBS scale) was measured using a pH electrode (HI-1210B/5, Hanna
56 O. Tills et al. / Journal of Experimental Marine Biology and Ecology 480 (2016) 54–61
Instruments Ltd., Leighton Buzzard, UK) connected to a handheld pH meter Ephyrae were kept in the incubation chamber for a 3 h settling period. This
(HI-98160, Hanna Instruments Ltd.) calibrated daily using pH buffer standards period was sufficient to ensure that the pulsation behaviour observed in the
(Mettler-Toledo Ltd, Leicester, UK). Temperature was measured using a incubation chambers was broadly equivalent to that observed in free-
thermocouple (HH802U, Omega Engineering Inc. Stamford, USA), and swimming ephyra maintained in much larger volumes of seawater (vol. = 50
salinity using a refractometer (S/Mill Hand Refractometer, Atago, Tokyo, L) (Heimbach, Tills and Spicer, in prep). However, because it was not
Japan). Total carbon dioxide (tCO2) was measured on duplicate 50 μL samples uncommon for the ephyra to come into contact with the sides of the incubation
using a CO2-analyser (CibaCorning 965, England). chamber we refer to the activity we observed as pulsation behaviour and not
Owing to the small seawater volumes within each culture chamber total swimming behaviour, even though both activities are broadly equivalent. After
alkalinity (TA) was only measured twice during the experimental period, and the acclimation period a sequence of 8000 digital images (1024 × 1024 pixels,
from a neighbouring culture system of larger volume (Table 1). This system 8 bit) was obtained (5.3 min at 25 frames s −1 [fps]) for each ephyra. All images
contained the same seawater, and was supplied the same gas, as used in the were stored as TIFF files written to mirrored hard drives (Seagate Barracuda
ephyra culture system. Seawater samples were stored in 150 mL borosilicate 2TB, 7200 RPM, 64 MB cache) to guard against data loss.
bottles with Teflon caps, treated with 30 μL of a saturated solution of HgCl2
and stored in darkness until they were analysed. Total alkalinity was 2.4. Image analysis
subsequently measured using a Gran Titrator (AS-ALK2, Apollo SciTech Inc,
Bogart, GA). The program CO 2Sys v2.1 (Pierrot et al., 2006) was used to Video recorded by the bio-imaging system was used for quantification of
calculate the saturation state for aragonite and calcite (Ωarg and Ωcal) and ephyra pulsing, and measurement of key morphometric parameters. Manual
concentrations of carbonate [CO23] and bicarbonate [HCO−3 ] (Table 1). The observation was used to generate a time-series of the body pulses exhibited
input parameters were: pH, total alkalinity, temperature and salinity in addition by individual ephyra. Image sequences were observed using ImageJ
to the dissociation constants from Mehrbach et al. (1973) as refitted by (Abàmoff et al., 2004). A macro (supplementary information) was used in
Dickson and Millero (1987). ImageJ to record the frame index at which each pulsation began, via manual
Table 1 pressing of a keyboard spacebar at the time of a pulsation being first
Measured and calculated (grey shaded) physico-chemical parameters (mean ± 1 S.E.) of observed. This macro recorded the frame number of this instance and the
untreated and CO2-enriched seawater in which ephyrae were cultured. Calculated values were
resultant frame index list of the instances for each image sequence was
determined using CO2Sys v2.1 (http://cdiac.ornl.gov/oceans/co2rprt.html) from the
temperature, salinity, pH and TA measurements made during the course of the experiment. transferred to a datasheet. To produce a timeseries of pulsations for each
T (°C) S pH tCO2 Total CO3 HCO3 ΩCa ΩAr image sequence the frame index of a pulsation was divided by the frame rate
Alkalinity (25 frames s−1). Pulsation rate was calculated from these time series data. To
(mmol.L1) (μmol.l-1) (μmol.l )1

investigate variation in the temporal patterns of pulsation behaviour, the

(μmol.l1) timing of consecutive pulsations was analysed via calculation of each pulse-
Control 14.93 34.5 ± 8.11 ± 2.17 ± 2.353 ± 176.8 1909 4.25 2.67 to-pulse period within each time series. These pulse-to-pulse timings were
± 0.04 0.16 0.018 0.04 0.044 analysed via calculation of: the minimum pulse-to-pulse period; the
maximum pulse-to pulse period; mean pulse-topulse period; standard
Simulated 14.89 34.7 ± 7.69 ± 2.15 ± 2.336 ± 73.2 2150 1.76 1.1
deviation in pulse-to-pulse periods; and the root mean square of successive
OA ± 0.03 0.15 0.032 0.150 0.062 differences (RMSSD) (Acharya et al., 2006) in the period between
2.3. Analysis of ephyra pulsing behaviour and morphology pulsations, in each time series.
Morphological measurements of ephyrae (Fig. 1) were made using
Following a 7 day exposure to either the untreated or CO 2-enriched image analysis of a single frame from the image sequence and included:
seawater ephyrae were removed and transferred to individual wells of a 48- lappet length and width; the area of the central disc; and total area,
well microtitre plate (BD Biosciences, Durham, UK), each containing 0.6 mL measured using Image Thresholding to produce a binary image (Fig. 1),
of untreated seawater (Table 1). The microtitre plate was placed into an from which the ImageJ measure particle function was applied. Lappet length
incubation chamber (Okolab, Naples, Italy, T = 15 ± 0.1 °C) within a bio- was measured from the bell margin to the rhophalia (Fig. 1) and did not
imaging system specially designed for time-lapse imaging of aquatic embryos include the ends of the lappet as these were sometimes at an angle to the
and larvae (Tills et al., 2013). The system comprises an aluminium frame in section between the bell and the rhophalia.
which a machine vision camera (Pike 421B, Allied Vision Technology,
Stradtroda, Germany) connected to a zooming lens system (VHZ20R, 2.5. Statistical analyses
Keyence, Milton Keynes, UK) was inverted and mounted beneath a motorized
stage (Optiscan, Prior Scientific, Cambridge, UK), upon which the incubation The effects of 7 d exposure to CO 2-enriched seawater on morphological
chamber containing the ephyrae was positioned. Dark-field cold lighting was and pulsation behaviour metrics were analysed in R using an ANOVA
achieved by an LED ring light mounted above the incubation chamber. The employing the aov function. Where necessary Boxcox transformation was
motorized stage and camera were controlled using the open-source ImageJ applied to variables to address non-normally distributed data and these
based software MicroManager 1.4 (Edelstein et al., 2010) on an Apple MacPro transformed variables were used in subsequent analysis. Morphological
running OS X 10.6. variables were analysed using the model:
 Fig. 1. Micrographs of Aurelia aurita illustrating measurements made of central disc area, total area, lappet length and lappet width.
O. Tills et al. / Journal of Experimental Marine Biology and Ecology 480 (2016) 54–61 57

Fig. 2. Effect of 7 day exposure to CO2-enriched seawater on ephyrae of A. aurita. A. Total and central disc area and B. lappet length and lappet width. Values are expressed as means ± 1 S.E.
*P b 0.05, **P b 0.01, ***P b 0.001. comparison of treatment effects on ephyra pulsation behaviour these seven
ephyrae are excluded to enable a comparison of movement in ephyrae of a
similar size range in both control and treatment groups.
morphologicalVariable ~ CO2enrichment. To account for possible size effects
After seven-days of culture in CO 2-enriched seawater ephyrae exhibited a
on pulsation behaviour, ephyra total area was included as a covariate in the
significantly reduced pulse rate (control — 24 ± 2.1 min−1, acidification — 18.2
models testing for effects of CO 2 enrichment on swimming behaviour:
± 0.98 min−1, Fig. 4) and a longer mean pulse-to-pulse period (control — 2.7 ±
swimmingVariable ~ CO2enrichment + totalArea + totalArea
0.26 s, acidification — 3.4 ± 0.16 s). There was, however, no significant effect
CO2enrichment.
of ephyra size on either pulse rate or mean pulse-to-pulse period (Table 2),
although pulse rate was higher in the seven outlier ephyrae than in the
3. Results
remaining smaller ephyrae (Fig. 3). This may suggest that over a greater size
range a positive relationship between size and pulse-rate is detectable. As the
3.1. Morphology
CO2enriched treatment did not produce sufficient individuals equivalent in size
Survival was 100% in both control (n = 19) and acidified (n = 20) to the six largest control individuals, we were unable to explore this
treatments. Ephyrae exposed for seven days to CO2-enriched seawater had a relationship any further.
significantly smaller overall area (Figs. 1 and 2; control = 7.12 ± 0.31 mm2 While there was a clear influence of CO2 enrichment on the pulsation rate
[mean ± 1 S.E.], CO2-enriched = 3.96 ± 0.96 mm 2), which was the there were no significant effects of this treatment on either the minimum
consequence of a smaller, central disc area (control = 4.5 ± 1.9 mm 2, (control — 0.357 s ± 0.03 s, CO2-enriched — 0.408 ±
CO2enriched = 1.82 ± 0.16 mm2), and reduced mean lappet length (control = 0.04 s) or maximum (control — 15.39 s ± 1.59 s, CO2-enriched — 20.03 ± 1.99
0.674 ± 0.037 mm2, CO2enriched = 0.514 ± 0.031 mm2) and width (control = s) pulse-to-pulse periods (Table 3). There was, however, a significant positive
0.455 ± 0.023 mm2, CO2enriched = 0.363 ± 0.016 mm2) (Table 2). relationship between the size of ephyrae and the minimum pulse-to-pulse
period, but only within the CO 2-enriched treatment (Fig. 5). Conversely,
3.2. Swimming behaviour within the control treatment there was a negative relationship between the
maximum pulse-to-pulse period and total ephyra area (Fig. 5). These
Following a seven-day exposure to treatments of either CO2enriched treatment-specific differences in the relationships between pulse-to-pulse
seawater or a control treatment of control seawater, seven ephyrae from the parameters and size suggest a more complex influence of culture under CO2
control treatment had a total surface area approximately double that of all enrichment than simply an overall reduction in pulsation rate.
other ephyrae from this study (Fig. 3). Therefore, in making a formal
Table 2 Results of ANOVAs testing for effects of exposure to CO 2-enriched seawater for seven days
on the morphology of Aurelia aurita ephyrae.
58 O. Tills et al. / Journal of Experimental Marine Biology and Ecology 480 (2016) 54–61
Factor df SS MS F P Factor df SS MS F P

Total area Pulsation rate

CO2 enrichment CO2 enrichment 1 0.0004779 0.0004779 6.927 0.0137⁎
1 0.0765 0.0765 7.99 0.0083⁎⁎
Error 29 0.2872 0.0096 Ephyra area 1 0.0000802 0.0000802 1.163 0.2901
CO2 enrichment × 1 0.0001039 0.0001039 1.506 0.23
Central area × Ephyra area
CO2 enrichment Error
1 0.2452 0.2452 6.33 0.0174⁎ 28 0.0019321 0.000069
Error 29 1.1618 0.0387 Mean pulse-to-pulse period
CO2 enrichment 1
Lappet length 2511 2510.8 6.35 0.0177⁎
Ephyra area 1 542 541.8 1.37 0.2516
CO2 enrichment
1 0.2046 0.2046 11 0.0024⁎⁎ CO2 enrichment × 1 305 305.3 0.772 0.387
Error 29 0.5579 0.0186 × Ephyra area Error
28
Lappet width 11071 395.4
CO2 enrichment Minimum pulse-to-pulse period
1 5.369 5.369 11.76 0.0018⁎⁎ CO2 enrichment 1 0.559 0.5586 0.971 0.3328
Error 29 13.696 0.457 Ephyra area 1 2.876 2.8764 5.002 0.0335⁎
CO2 enrichment × 1 1.292 1.2917 2.246 0.1451
⁎ P b 0.05. × Ephyra area
⁎⁎ P b 0.01. Error 28
16.102 0.5751
⁎⁎⁎ P b 0.001.
Maximum pulse-to-pulse period
CO2 enrichment 1 0.000853 0.000853 2.729 0.10972
Ephyra area 1 0.003161 0.0031613 10.113 0.00358⁎⁎
CO2 enrichment × 1 0.000139 0.0001388 0.444 0.5107
× Ephyra area
Error 28
0.008753 0.0003126
Standard deviation of pulse-to-pulse period
CO2 enrichment 1 0.00363 0.00363 5.619 0.0249⁎
Ephyra area 1 0.004188 0.004188 6.483 0.0167⁎
CO2 enrichment × 1 0.00106 0.00106 1.641 0.2107
× Ephyra area
Error 28
0.018087 0.000646

Fig. 3. Total area and pulse rate of Aurelia aurita ephyrae following a seven-day culture in either
control, or CO2-enriched seawater. Seven ephyrae with significantly larger total area are circled.
To enable a comparison between treatment groups of similar sized ephyrae these seven ephyrae
were omitted from our formal comparison.

Fig. 4. Effect of 7 day exposure to CO 2-enriched seawater on aspects of Aurelia aurita ephyrae swimming behaviour. A. Pulse rate and B. Pulse-to-pulse parameters (mean pulse-to-pulse period,
minimum pulse-to-pulse period, maximum pulse-to-pulse period and the root mean square of successive differences (RMSSD) in pulse-to-pulse period). Values are expressed as
mean ± 1 S.E. *P b 0.05, **P b 0.01, ***P b 0.001.

Standard deviation in pulse-to-pulse timing was influenced significantly by

CO2-enrichment and the size of ephyrae. Ephyrae from the CO 2-enriched
treatment had a greater standard deviation in pulse-to-

Table 3
Results of ANCOVAs testing for effects of exposure to CO 2-enriched seawater for seven days on
the swimming behaviour of Aurelia aurita ephyrae.
 RMSSD pulse-to-pulse period O. Tills et al. / Journal of Experimental Marine Biology and Ecology 480 (2016) 54–61 59
CO2 enrichment 1 0.001812 0.0018123 6.044 0.0204⁎
velocity.
Ephyra However,
area the link
1 between pulsation
0.002255 rate,0.0022546
velocity and7.519 size remains
0.0105⁎ and not size, of statolith has
been shown to affect swimming behaviour.
unclear as they did
CO2 enrichment × not record
1 pulsation rates. Mangum
0.000693 0.0006931 2.311 0.1396 Furthermore the smaller size of statoliths in ephyrae in reduced pH cannot be
× Ephyra area
Error 28
0.008396 0.0002999

Fig. 5. Effects of seven-day culture in CO 2-enriched seawater on A) minimum and B) maximum pulse-to-pulse periods in Aurelia aurita ephyrae of different sizes. Results of treatment-

⁎ P b 0.05. ⁎⁎ account for the difference in pulsation rate solely on the basis of size
P b 0.01. ⁎⁎⁎ P differences. Costello and Colin (1994) reported that A. aurita medusae
b 0.001. exhibited a positive relationship between size and flow
pulse period (control — 2.68 ± 0.2 s, CO2-enriched — 3.84 ± 0.32 s). The specific regression analysis are presented, when significant.

root mean square of successive differences (RMSSD) quantifies the

differences between consecutive pulse-to-pulse periods and so provides a
longer term measure of variability. In the CO 2-enriched treatment the et al. (1972) studied the physiological effects of temperature on the early
RMSSD in pulse-topulse period was greater than in untreated seawater developmental stages of several species, including A. aurita. They discovered
(control — 2.84 ± 0.29, CO2-enriched — 3.74 ± 0.31), suggestive of a less that ephyrae of A. aurita had a pulsation rate of 33 beats min −1 at 15 °C,
predictable behaviour over consecutive pulsations. Visual examination of a similar to the rate we report here, although details of ephyrae size were not
time series for pulsations exhibited by ephyrae from the control or CO 2- reported.
enriched seawater treatment supports the presence of both a reduction in In common with a previous study (Kikkawa et al., 2010), the overall
pulsation rate, and greater variation in the pulsation behaviour within the rate of pulsation was depressed in reduced pH. We also uncovered
CO2-enriched treatment (Fig. 6). differences in the fine detail of how pulsations were affected.
CO2enrichment increased the mean length of the pulse-to-pulse period, but
not the minimum or maximum pulse-to-pulse period, suggesting that
4. Discussion
ephyrae exposed to reduced pH may still be able to swim at the same
Knowledge of the sub-lethal effects of reduced pH and elevated CO 2 on velocity as controls. Furthermore, both standard deviation in pulse-to-pulse
jellyfish is key to understanding the potential effects of OA in driving timing and the RMSSD in pulse-to-pulse timing, a statistic used in cardiac
medicine for quantifying variation in beatto-beat variability in heart rate
population fluctuations in the wild. Here we have demonstrated significant
(Malik et al., 1996), were greater in ephyrae exposed to reduced pH. An
effects of reduced pH on both the body size and pulsation behaviour of A.
increase in variation in pulse-to-pulse timing suggests a more variable and
aurita ephyrae, even though there was no undersaturation of calcite or
less predictable swimming behaviour.
aragonite.
Rhopalia are an assemblage of sensory structures that appear and
In common with Kikkawa et al. (2010) we observed a decrease in
develop towards the end of the lappets in early ephyrae and are important
ephyrae pulsation rate in reduced pH conditions, although there were also
for detecting orientation and producing swimming movements (Arai,
important differences between our two studies. Here, we recorded a
2001). Statocysts contain statoliths, mineralised masses, and it is these that
decrease in pulsation rate from 27 ± 1.7 pulses min−1 in untreated seawater,
stimulate the sensory cells of the statocyst. Reduction of statocysts,
to 18.2 ± 0.98 pulses min −1 in response to 7 d exposure to pCO 2 = 1000
naturally or experimentally, has due to dissolution, as they are comprised
μatm, whereas Kikkawa et al. (2010) reported a decrease from approx. 90
of calcium sulphate hemihydrate (Plaster of Paris), which is neither
pulses min−1 to approx. 72 pulses min−1 following 4 d exposure to pCO2 =
dependent on carbonate concentrations or made more soluble by even
5000 μatm. Hence, we detected severe acid conditions (Becker et al., 2005; Shukla et al., 2008). As Winans
CO2-induced decreases in pulsation rate at much less severe pCO 2 values and Purcell (2010) point out, the implications of smaller statoliths (smaller
(pH = 7.60, pCO2 = 1000 μatm) than used by Kikkawa et al. (2010) (pH = as a result of a secondary effect of low pH on feeding and thus energy
6.896, pCO2 = 5000 μatm) at the same temperature. Ephyrae in the current available to build structures) for swimming behaviour warrants further
study exhibited lower pulsation rates in control seawater compared to the study.
ephyrae used by Kikkawa et al. (2010). This difference may be explained There is another line of enquiry that still may implicate the rhopalia as
by differences in the culture conditions and the size of ephyra used. Here, having a role in the swimming behaviour response of ephyrae to reduced pH.
ephyrae were cultured individually for the duration of the study, whereas The nervous system of scyphozoans includes a motor nerve net that controls
Kikkawa et al. (2010) cultured ephyrae in groups of c. 10. This may have swimming, and how that control is exercised is comparatively well known
led to differences in water chemistry, food availability and hydrodynamics (Romanes, 1876; Horridge, 1954; Lerner et al., 1971; Satterlie and Nolen,
that might have affected swimming behaviour. The ephyrae used in the 2001; Nakanishi et al, 2009). The swimming contraction we have investigated
current study were also considerably larger (central disc area = 4.5 ± 1.9 has its genesis in a network of distributed pacemakers located in the rhopalia.
mm2) than those used by Kikkawa et al. (2010) (central disc diameter = 1.76 It is known that the activity of each solitary pacemaker is irregular; the fastest
mm2), However, we found some evidence for a positive relationship pacemaker controls the output of the swim-signal, and by linking irregular
between pulsation rate and size, which makes it unlikely that we can pacemakers the swimming produced is the result of regular contractions. As
60 O. Tills et al. / Journal of Experimental Marine Biology and Ecology 480 (2016) 54–61
we found not just a CO2-related reduction in bell contraction frequency, but Acknowledgements
that the contraction also became more variable and less predictable, we
hypothesise that low pH, directly or indirectly, may affect the function of We thank Michael Schmid for assistance in writing the ImageJ macro used
individual, and the linkage of multiple, irregular pacemakers. This idea to quantify the swimming behaviour of individual ephyra.
warrants further study. This work was partly supported by the National Basic Research Program

represents a single pulsation.

been shown to result in swimming abnormalities, reduced swimming, and and lappet length and width) than controls. Just as
cessation of swimming when they are absent (Spangenberg, 1968). Given Winans and Purcell (2010) hinted that smaller statoliths in ephyrae cultured
the role of statocysts in determining swimming behaviour, it is tempting to under reduced pH, may have resulted from a negative effect of pH on feeding,
link the pH-related decrease in statolith size in Aurelia ephyrae, as reported we suggest that the smaller ephyra size in our reduced-pH treatment was due
by Winans and Purcell (2010), to the gross and subtle pH-related differences either to pH-related decreases in feeding or metabolism, or a redirecting of
in movement we report for a congeneric Aurelia ephyra. However the energy away from growth towards meeting the increased cost of maintenance
absence, activities
Ephyrae kept in CO2-enriched seawater for 7 d were significantly smaller, of China (No. 2014CB441504),the Strategic Priority Research Program of the
Chinese Academy of Sciences (No. XDA11030204) and Plymouth Marine
and had a smaller surface area (resulting from smaller both central disc area
Institute. [SW]
(e.g. Wood et al., 2008). The relationship between the morphometrics of
ephyrae and fluid dynamics during their swimming behaviour is complex.
Appendix A. Supplementary data
Viscous boundary layers of seawater surrounding the ephyra occlude water
passing between the lappets, thereby increasing swimming efficacy by
Supplementary data to this article can be found online at http://dx.
boundary layers of water acting as paddles (Feitl et al., 2009). Morphogenesis doi.org/10.1016/j.jembe.2016.03.014.

Fig. 6. Time series plots of the temporal pattern with whichpulsationsoccur in five Aurelia aurita ephyrae (a–e) after culture for seven daysin either control or CO2-enriched seawater. Each vertical l
in ephyrae has also been shown to be a plastic trait that can consequently
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