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© INVERTEBRATE ZOOLOGY, 2022Invertebrate Zoology, 2022, 19(1): XXXXXX
Severe seawater acidification causes a significant
reduction in pulse rate, bell diameter, and acute
deterioration in feeding apparatus in the scyphozoan
medusa Cassiopeia sp.
R. Thayer1*, I.A. Brunetz2, S.J. Daniel2, C.M. Wigal3, K.E. Nazor2*
1 Tennessee Aquarium,1 Broad Street, Chattanooga, TN 37402, U.S.A.
2 McCallie School, Scientific Research Program Biology, 500 Dodds Ave, Chattanooga TN 37405,
U.S.A.
3 University of Tennessee at Chattanooga, College of Engineering and Computer Science, 615
McCallie Ave, Chattanooga, TN 37403, U.S.A.
* Corresponding author: rst@tnaqua.org
Rachel Thayer: ORCID 0000-0002-5023-4118
Ian Brunetz: ORCID 0000-0001-7121-2033
Shrayen Daniel: ORCID 0000-0001-5136-4929
Cecelia Wigal: ORCID0000-0003-0328-3213
Karah Nazor: ORCID 0000-0003-4005-5279
ABSTRACT: The detrimental effect of ocean acidification (OA) on marine animals with
carbonate exoskeletons or shells is an issue drawing increased attention in marine biology
and ecology, yet few studies have focused on the impact on gelatinous organisms like
scyphozoan medusae. Here, we examined the physiological tolerance of Cassiopea sp., an
abundant genus of scyphozoans valuable for their role as bioindicators and for having
similarities to other cnidarians, to OA by conducting three, 12-week trials using CO2
diffusers and electronic pH controllers to incrementally lower the water to test pHs of 7.5
and 7.0. The impact of reduced pH on the survival, pulse rate, bell diameter, and
reorientation and settlement abilities of Cassiopea sp. medusae were measured weekly.
Cassiopea sp. was tolerant to pH 7.5 while further reduction of the pH to 7.0 resulted in
22.22% mortality rate, which was significantly different from the control and treatment pH
7.5. Significant differences between the treatment pH 7.0 and control first occurred on day
23.5 with a 50% reduction in the pulse rate, and on day 36 with a 16.6% reduction in bell
diameter, while pH 7.5 had no effect. By the final time point of 66 days in treatment pH 7.0,
there was an 87% reduction in pulse rate and a 36% reduction in bell diameter versus
control. Reduced pH 7.0 caused bell malformations, inhibited swimming abilities, and
deterioration of the oral arm feeding apparatus, but had no effect on the orientation and
settlement assay. Observations indicate that asexual reproduction via planuloid production
and strobilation was unaffected by pH reduction, though polyps in treatment pH 7.0 gave
rise to ephyrae with inverted bells. Combined, findings from this study demonstrate
Cassiopea sp. to be resilient to the end of century ocean acidity prediction of pH 7.6, and
vulnerable to more severe OA to pH 7.0.
How to site this article: Thayer R., Brunetz I.A., Daniel S.J., Wigal C.M., Nazor K.E. 2022.
Severe seawater acidification causes a significant reduction in pulse rate, bell diameter, and
acute deterioration in feeding apparatus in the scyphozoan medusa Cassiopeia sp. // Invert.
Zool. Vol.19. No.1. P.XXXXXX. doi: 10.15298/invertzool.19.1.XX
KEY WORDS: Ocean acidification, Cassiopea sp., medusa, climate change, Scyphozoa,
pulse rate.
2R. Thayer et al.
Ñèëüíîå çàêèñëåíèå ìîðñêîé âîäû âûçûâàåò
çíà÷èòåëüíîå óìåíüøåíèå ïóëüñàöèè, äèàìåòðà
êîëîêîëà è óõóäøåíèå ñîñòîÿíèÿ ðîòîâîãî àïïàðàòà ó
ñöèôîèäíîé ìåäóçû Cassiopea sp.
Ð. Òàéåð1*, I. È. Áðóíåö2, Ñ. Äàíèýëü2, Ê. Âèãàë3, Ê. Íàçîð2
1 Tennessee Aquarium,1 Broad Street, Chattanooga, TN 37402, U.S.A.
2 McCallie School, Scientific Research Program Biology, 500 Dodds Ave, Chattanooga TN
37405, U.S.A.
3 University of Tennessee at Chattanooga, College of Engineering and Computer Science, 615
McCallie Ave, Chattanooga, TN 37403, U.S.A.
* Àâòîð, îòâåòñòâåííûé çà ïåðåïèñêó: rst@tnaqua.org
ÐÅÇÞÌÅ: Ïàãóáíîå âîçäåéñòâèå çàêèñëåíèÿ îêåàíà íà ìîðñêèå æèâîòíûå ñ èçâåñ-
òêîâûì ýêçîñêåëåòîì èëè ïàíöèðåì ïðîáëåìà, ïðèâëåêàþùàÿ çíà÷èòåëüíîå
âíèìàíèå â ìîðñêîé áèîëîãèè è ýêîëîãèè. Îäíàêî òîëüêî íåìíîãî÷èñëåííûå èññëå-
äîâàíèÿ ïîñâÿùåíû èçó÷åíèþ âëèÿíèÿ èçìåíåíèÿ êèñëîòíîñòè ñðåäû îáèòàíèÿ íà
òàêèå æåëåîáðàçíûå îðãàíèçìû êàê ñöèôîèäíîé ìåäóçû. Íàìè ïðîâåäåíî èññëåäî-
âàíèå ôèçèîëîãè÷åñêîé óñòîé÷èâîñòè ê çàêèñëåíèþ ñöèôîèäíûõ ìåäóç-áèîèíäèêà-
òîðîâ, îòíîñÿùèõñÿ ê Cassiopea sp. Èññëåäîâàíèå âêëþ÷àëî ïðîäîëæàâøèåñÿ 12
íåäåëü òðè ýêñïåðèìåíòà ñ èñïîëüçîâàíèåì ðàññåèâàòåëåé CO2 è ýëåêòðîííûõ
èçìåðèòåëåé pH, äëÿ ïîíèæåíèÿ êèñëîòíîñòè âîäû (pH) äî çíà÷åíèé 7,5 è 7,0.
Âëèÿíèå ïîíèæåííîãî pH íà âûæèâàåìîñòü, ïóëüñàöèþ, äèàìåòð êîëîêîëà, à òàêæå
ñïîñîáíîñòü ê ïåðåâîðà÷èâàíèþ è îñåäàíèþ ìåäóç Cassiopea sp. èçìåðÿëè åæåíå-
äåëüíî. Ìåäóçû Cassiopea sp. ïîêàçàëè óñòîé÷èâîñòü ê ðÍ 7,5. Ñíèæåíèå ðÍ äî 7,0
ïðèâîäèëî ê óâåëè÷åíèþ ñìåðòíîñòè äî 22,22%. Çíà÷èòåëüíûå ðàçëè÷èÿ ìåæäó
óñëîâèÿìè ðÍ 7,0 è êîíòðîëüíûìè îòìå÷åíû íà 23-é äåíü è ïðîÿâëÿëèñü â ñíèæåíèè
÷àñòîòû ïóëüñàöèè íà 50%. Íà 36-é äåíü îòìå÷åíî óìåíüøåíèå äèàìåòðà êîëîêîëà
ìåäóç íà 16,6%. Íà 66 äåíü ýêñïåðèìåíòà â óñëîâèÿõ ðÍ 7,0 îòìå÷åíî ñíèæåíèå
÷àñòîòû ïóëüñàöèè íà 87% è óìåíüøåíèå äèàìåòðà êîëîêîëà íà 36%. Ïîíèæåíèå
êèñëîòíîñòè ðÍ äî 7,0 âûçûâàëî èçìåíåíèÿ â ðàçâèòèè êîëîêîëà, ñíèæàëî ïëàâà-
òåëüíóþ àêòèâíîñòü è óõóäøàëî ðàáîòó ïèùåâîãî àïïàðàòà, íî íå âëèÿëî ñóùåñòâåí-
íî íà ñïîñîáíîñòü ê ïåðåâîðà÷èâàíèþ è îñåäàíèþ. Ñïîñîáíîñòü ê áåñïîëîìó
ðàçìíîæåíèþ ïëàíóëàìè è ñòðîáèëÿöèè íå ìåíÿëàñü çàìåòíî ïðè ñíèæåíèè pH,
îäíàêî â óñëîâèÿõ íèçêîãî pH (7,0) ôîðìèðîâàëèñü ýôèðû ñ ïåðåâåðíóòûìè êîëîêî-
ëàìè. Òàêèì îáðàçîì, ìåäóçû Cassiopea sp. óñòîé÷èâû ê ïðîãíîçèðóåìûì íà êîíåö
âåêà óñëîâèÿì êèñëîòíîñòè îêåàíà (pH 7,6), íî óÿçâèìû ïðè áîëåå ñóùåñòâåííîì
çàêèñëåíèè îêåàíà (pH 7,0).
Êàê öèòèðîâàòü ýòó ñòàòüþ: Thayer R., Brunetz I.A., Daniel S.J., Wigal C.M., Nazor K.E.
2022. Severe seawater acidification causes a significant reduction in pulse rate, bell
diameter, and acute deterioration in feeding apparatus in the scyphozoan medusa Cassiopeia
sp. // Invert. Zool. Vol.19. No.1. P.XXXXXX. doi: 10.15298/invertzool.19.1.XX
ÊËÞ×ÅÂÛÅ ÑËÎÂÀ: çàêèñëåíèå îêåàíà, Cassiopea sp., ìåäóçà, èçìåíåíèå êëèìà-
òà, ñöèôîèäíûå, ïóëüñàöèÿ.
3Severe seawater acidification and the scyphozoan medusa Cassiopeia sp.
Order Rhizostomeae, reside in shallow, benthic
environments in the Atlantic Ocean, Caribbean
Sea, Gulf of Mexico, and the Pacific Ocean
(introduced) (Holland et al., 2004). Cassiopea
sp. are involved in nutrient cycling of reefs and
mangrove habitats, as their bell pulsing pulls
nutrients up from the sediment on which they lay
(Jantzen et al., 2010). Cassiopea sp. are eukary-
otic mixotrophs, with both polyp and medusae
life stages containing photosynthetic endosym-
biont zooxanthellae, the dinoflagellate Symbio-
diniaceae (LaJeunesse et al., 2018), which pro-
vides its host with a source of energy. Since they
can harbor more than one clade, Cassiopea sp.
are models for studying the uptake of Symbiod-
iniaceae by corals, as the planulae do not yet
contain endosymbionts, and later life stages
acquire them from surrounding waters (Lampert,
2016). Cassiopea sp. is a prolific species that is
easily cultured in captivity. For this reason, and
the aforementioned likenesses to other cnidari-
ans, Cassiopea sp. was chosen for this study.
While the current ocean pH in native ranges
of Cassiopea sp. is between pH 7.68.3, with
average global surface pH of 8.1 (Barbero et al.,
2019, Jiang et al., 2019), and the projected
average surface pH of the ocean in the years
2050 and 2100 is 7.8 and 7.6, respectively
(Feely et al., 2009; Kennedy, 2010), these val-
ues indicate averages across the ocean as a
whole, and samples in the native ranges are
already measuring pH 7.6 (Barbero et al., 2019),
with global pH likely to continue to decrease
with projected CO2 emissions (Jiang et al., 2019).
The fluctuations in pH can be more drastic in
areas with known upwelling, bringing water
saturated with dissolved inorganic carbon (DIC)
up to the surface (Wu et al., 2019), the nearest
to the native range of Cassiopea sp. being the
Southern Caribbean upwelling system (Rueda-
Roa, Muller-Karger, 2013).
Published research on the effect of OA on
the medusae stage of any scyphozoan, cubozo-
an, or hydrozoan species is lacking. A previous
study by Chelsky et al. (2015) examined the
effects of low pH on frozen specimens of Cato-
stylus mosaicus, to determine the effects on
decomposition. Chuard et al. (2019) presented
Introduction
Ocean acidification (OA), the gradual de-
crease of the average pH of the ocean over time,
is an issue of increased importance in marine
biology and ecology. Since the Industrial Rev-
olution, the oceans have absorbed approximate-
ly 28% of anthropogenic CO2 and the average
pH of the ocean has decreased by about 0.1
units, becoming 30% more acidic (Kleypas et
al., 1999, Sabine et al., 2004; Feely et al., 2009;
Kennedy 2010; Jiang et al., 2019). Determining
the ecological effects of reduced sea pH levels
is crucial to predict future changes in marine
ecosystems at every level of the food web, and
their societal impacts.
The dissociation of carbonic acid in the sea
leads to increasing bicarbonate concentrations
at the expense of carbonate concentrations, cre-
ating problems for many shell and skeleton
builders (Kleypas et al., 1999; Kroeker et al.,
2013), therefore, much of the focus of OA
research has been on marine animals with calci-
um carbonate exoskeletons or shells including
corals, crustaceans, and zooplankton such as
pteropods (Orr et al., 2005, Gazeau et al., 2007;
Comeau et al., 2009; Kleypas, Yates 2009;
Hofmann et al., 2010; Whitely, 2011; Anders-
son, Gledhill, 2013; Jokiel et al., 2016; Campoy
et al., 2020). Fewer groups have examined the
biological effects of OA on gelatinous organ-
isms, and most lines of evidence suggest that
jellyfish are more resistant to OA than many
taxa, though they are not entirely unaffected.
Previous research has focused on the genus
Aurelia (common name: moon jellyfish) planu-
lae larvae, polyps, and ephyrae and, viewed
collectively, demonstrate the resistance of the
genus to this environmental stressor (Kikkawa et
al., 2010; Winans, Purcell, 2010; Alguero-Mu-
niz et al., 2016; Tills et al., 2016; Treible et al.,
2018; Goldstein et al., 2017; Dong, Sun, 2018).
Jellyfish are vital parts of their ecosystems
and are prey for several species, including sea
turtles, ocean sunfish, teleosts and seabirds
(McInnes et al., 2017; Hays et al., 2018). Cas-
siopea sp. (common name: upside-down jelly-
fish), a member of the Class Scyphozoa and
4R. Thayer et al.
approximately the same age since they came
from the same group of ephyrae captively raised
at the Tennessee Aquarium. The pH of the water
in the holding system is maintained at 8.08.2.
Three trials of 1213 weeks in duration were
conducted. Nine 10-gallon (37.85 liters) aquar-
iums were used per trial and were placed in
groups of three tanks that fit under one aquarium
light, referred to as the A, B, and C groups. Each
set of three A tanks, three B tanks, and three C
tanks included one tank maintained at control
pH and two test tanks, where the pH was incre-
mentally lowered to either pH 7.5 or pH 7.0 by
diffusing CO2 into the test tanks using large
micro CO2 bubble diffusers and Milwaukee
MC122 pH controllers. CO2 delivery was acti-
vated when water pH levels climbed above the
set value and shut off when the pH reached the
set value. The test pHs of 7.5 and 7.0 were
chosen based on current global pH projections
and to ensure there would be no overlap be-
tween test groups, given the accuracy of the pH
controllers was +/ 0.2 pH units. The pH probes
were calibrated using pH standards 4 and 7. Any
variation in control pH occurred naturally and
was recorded, giving an overall control pH
range of 8.08.3.
Five jellyfish of similar bell diameter (ap-
proximately 60 mm) were placed in each tank
containing cycled artificial sea water prepared
in deionized water at 35 ppt (parts per thousand)
and baseline data was recorded. The test tanks
were kept at control pH of 8.08.3 for ~2 weeks
before lowering the pH to allow the animals to
acclimate to the experimental systems and data
was not collected during this time. The pH
reduction was performed incrementally, to al-
low the subjects to acclimate to changes in pH,
by decreasing the pH controller dial setting by
0.2 units pH every approximately 34 days.
Once the test pHs were reached for their respec-
tive tanks, the water was maintained at that pH
for the duration of the experiment. Data were
pooled for the three trials for statistical analy-
ses. The average number of days since the initial
lowering of the pH was calculated and these are
the values seen on the x-axis in Figures 13.
After first introducing CO2, pH 7.5 was reached
the first evidence of the potential lethal effects
of OA on the medusae of the wild-caught cubo-
zoan Carybdea xaymaca. One group has previ-
ously reported the effect of OA on Cassiopea
sp., where treatment (pH 7.9, 7.6) of medusae
for four weeks did not impact endosymbiont
density or the bell diameter, while the effect on
the pulse rate, orientation ability, and the polyp
and ephyrae stages were not examined (Weeks
et al., 2019). To test the hypothesis that expo-
sure of Cassiopea sp. to seawater acidification
for 12 weeks would cause increased mortality
rates, a decrease in bell diameter and pulse rate
and a disruption of the reorientation and settling
abilities, we conducted three trials in which CO2
gas diffusers and electronic pH controllers were
used to incrementally lower the pH of the water
to reach test pHs of 7.5 and 7.0. These pH values
were chosen based on current pHs in areas of
upwelling where Cassiopea sp. is found, and to
ensure that there was no overlap in pH between
the test groups based on the accuracy of the pH
controller used. The effect of pH treatment (7.5,
7.0) on the pulse rate, bell diameter, orientation
and settling abilities in medusae were measured
weekly and compared to animals in the control
tanks maintained at pH 8.08.3, that of normal
seawater. Since the polyp and medusae stages of
the life cycle are equally necessary for the
survival of the species (Lucas et al., 2012), the
condition of polyps and the ephyrae was also
monitored.
Materials and Methods
EXPERIMENTAL DESIGN. Cassiopea sp.
have been cultured at the Tennessee Aquarium
since 2008, sourced from multiple captive raised
populations over the years that they have been
maintained at the Aquarium. They have pro-
duced an unknown, but presumably large, num-
ber of generations as they readily produce new
polyp colonies in captivity, which strobilate
frequently throughout the year. Their use for
this research was approved by Tennessee Aquar-
ium Conservation Institute Animal Health and
Welfare Committee (AHW Approval Number:
18-07). Jellyfish used in these studies were
5Severe seawater acidification and the scyphozoan medusa Cassiopeia sp.
Fig. 1. Pulse rate of Cassiopea sp. as a function of lowering of the pH over time. Average pulse rate of subjects
in three trials (y-axis) vs. average number of days exposed to lowered pH (x-axis); baseline represents the
first day of induction of CO2. Each data point represents the mean values ± S.E.M. of n = 45, except for day
5 (n = 15) and days 15.5 and 23.5 (n = 30). The regression line for each treatment group and control group
is shown. Control group black circles, pH 7.5 group medium grey squares, pH 7.0 group light grey
triangles. pH 7.5 was reached by day 7.67. pH 7.0 was reached by day 22.34.
Ðèñ. 1. Èçìåíåíèå ïóëüñàöèè Cassiopea sp. â çàâèñèìîñòè îò ñíèæåíèÿ ðÍ ñ òå÷åíèåì âðåìåíè.
Ñðåäíÿÿ ÷àñòîòà ïóëüñàöèè â òðåõ ýêñïåðèìåíòàõ (îñü Y) â ñðàâíåíèè ñî ñðåäíèì êîëè÷åñòâîì äíåé
íàõîæäåíèÿ â óñëîâèÿõ ïîíèæåííîãî pH (îñü X); èñõîäíûé óðîâåíü ïåðâûé äåíü íàñûùåíèÿ CO2.
Êàæäàÿ òî÷êà äàííûõ ñðåäíèå çíà÷åíèÿ ± ñòàíäàðòíàÿ îøèáêà ñðåäíåãî èç n = 45, çà èñêëþ÷åíèåì
5-ãî äíÿ (n = 15) è äíåé 15,5 è 23,5 (n = 30). Ëèíèÿ ðåãðåññèè ïîêàçàíà äëÿ ýêñïåðèìåíòàëüíûõ è
êîíòðîëüíîé ãðóïï. Îáîçíà÷åíèÿ: êîíòðîëüíûå óñëîâèÿ ÷åðíûå êðóæêè, ðÍ 7,5 ñåðûå êâàäðà-
òû, ðÍ 7,0 ñâåòëî-ñåðûå òðåóãîëüíèêè. ðÍ 7,5 áûë äîñòèãíóò ê äíþ 7,67; ðÍ 7,0 ê äíþ 22,34.
rotifers. A mesh divider was secured with aquar-
ium grade silicone to separate the subjects from
the tank filter. Weekly water quality testing was
performed to measure salinity, ammonia, ni-
trates, and nitrites, to eliminate any extraneous
effects on the subjects or the pH of the test tanks.
It should be noted that jellyfish show more
growth when housed in larger volume aquatic
systems, with larger filters and protein fraction-
ators, all of which allow for more food to be
added to the system without deleterious effects
to water quality (AZA Aquatic Invertebrate
TAG, 2013). Underfeeding in sub-optimal hous-
at an average of 8 ± 1.5 (S.E.M.) days and pH
7.0 was reached at an average of 22.7 ± 3.3
(S.E.M) days for all trials. Trial 1 included two
additional end testing dates (average days 73
and 80 in lowered pH) not used in any statistical
analysis, though photomicrographs from trial 1
day 80 are included in Fig. 4 and Supplementary
file 1.
Water temperature was maintained at 25
26°C and lighting was set on a standardized
photoperiod cycle of 12:12. Subjects were fed
48-hour old Artemia nauplii (enriched with Reed
Mariculture Shellfish Diet 1800) and frozen
6R. Thayer et al.
Fig. 2. Bell diameter of Cassiopea sp. as a function of lowering of the pH over time. Average bell diameter
(mm) of subjects from three trials (y-axis) vs. average number of days exposed to lowered pH (x-axis);
baseline represents the first day of induction of CO2. Each data point represents the mean values ± S.E.M.
of n = 45, except for days 7, 15.5, and 23.5 (n = 30). The regression line for each treatment group and control
group is shown. Control group black circles, pH 7.5 group grey squares, pH 7.0 group light grey
triangles. pH 7.5 was reached by day 7.67. pH 7.0 was reached by day 22.34.
Ðèñ. 2. Äèàìåòð êîëîêîëà Cassiopea sp. â çàâèñèìîñòè îò ñíèæåíèÿ ðÍ ñ òå÷åíèåì âðåìåíè. Ñðåäíèé
äèàìåòð êîëîêîëà (ìì) ìåäóç â òðåõ èñïûòàíèé (îñü Y) â îòíîøåíèè ê ñðåäíåìó êîëè÷åñòâó äíåé
íàõîæäåíèÿ â óñëîâèÿõ ïîíèæåííîãî pH (îñü X); èñõîäíûé óðîâåíü ïðåäñòàâëÿåò ñîáîé ïåðâûé äåíü
íàñûùåíèÿ CO2. Êàæäàÿ òî÷êà äàííûõ ïðåäñòàâëÿåò ñðåäíèå çíà÷åíèÿ ± ñòàíäàðòíàÿ îøèáêà
ñðåäíåãî èç n = 45, çà èñêëþ÷åíèåì òðåõ äíåé: 7, 15,5 è 23,5 (n = 30). Ëèíèÿ ðåãðåññèè ïîêàçàíà äëÿ
ýêñïåðèìåíòàëüíûõ è êîíòðîëüíîé ãðóïï. Îáîçíà÷åíèÿ: êîíòðîëüíûå óñëîâèÿ ÷åðíûå êðóæêè, ðÍ
7,5 ñåðûå êâàäðàòû, ðÍ 7,0 ñâåòëî-ñåðûå òðåóãîëüíèêè. ðÍ 7,5 áûë äîñòèãíóò ê äíþ 7,67; ðÍ
7,0 ê äíþ 22,34.
bon (DIC) is defined as the total amount of
carbon dioxide, carbonic acid, and bicarbonate
in seawater and is expressed as:
DIC = [H2CO3] + [HCO3
] + [CO3
2]
The average amount of CO2 was used to
determine the dissolved inorganic carbon (DIC),
pCO2, and ÙAr for each tank using the USGS
CO2calc software (Robbins et al., 2010) and
using constants by Mehrbach et al. (1973) and
pH on a total scale.
PULSE RATE. A one-minute video was
taken with an iPhone camera weekly of undis-
ing conditions of the experimental tanks could
result in inhibited or reverse growth (Hatai, 1917).
CARBONATE CHEMISTRY. Carbonate
chemistry was assessed for each tank. In the
case of anthropogenic sources of OA, a clear
trend is observed: as CO2 dissolves into the
ocean, the pH decreases, the [CO2 (aq)] and
[HCO3
] increases, and [CO3
2] decreases (Bark-
er, Ridgwell, 2012). Once test pH levels were
reached, the total CO2 in each tank was mea-
sured using the HACH Carbon Dioxide Test Kit
(Model CA-23). Total dissolved inorganic car-
7Severe seawater acidification and the scyphozoan medusa Cassiopeia sp.
Fig. 3. Reorientation and settling ability of Cassiopea sp. as a function of lowering of the pH over time.
Average time (sec) for the group to reorient and settle from three trials (y-axis) vs. average number of days
exposed to lowered pH (x-axis); baseline represents the first day of induction of CO2. Each data point
represents the mean values ± S.E.M. for nine tanks (three tanks per treatment pH (7.5, 7.0) for a total of three
trials) except for the following day 7: n = 6; day 11.6: n = 7; day 15.5: n = 6; day 23: n = 6 (except for pH
7.5 n = 5); day 30: n = 8; control day- 58: n = 8. The regression line for each treatment group and control
group is shown. Control group filled black circles, pH 7.5 group medium grey squares, pH 7.0 group
light grey triangles.
Ðèñ. 3. Èçìåíåíèå ñïîñîáíîñòè ê ïåðåâîðà÷èâàíèþ è îñåäàíèþ Cassiopea sp. â çàâèñèìîñòè îò
ñíèæåíèÿ ðÍ. Ñðåäíåå âðåìÿ (ñ) äëÿ ãðóïïû, ïåðåâîðà÷èâàíèå è îñåäàíèå ïîñëå òðåõ îïûòîâ (îñü
îðäèíàò) â çàâèñèìîñòè îò êîëè÷åñòâà äíåé íàõîæäåíèÿ â óñëîâèÿõ ïîíèæåííîãî ðÍ (îñü àáñöèññ);
èñõîäíûé óðîâåíü ïðåäñòàâëÿåò ñîáîé ïåðâûé äåíü íàñûùåíèÿ CO2. Êàæäàÿ òî÷êà ñðåäíèå
çíà÷åíèÿ ± ñòàíäàðòíàÿ îøèáêà ñðåäíåãî äëÿ äåâÿòè ðåçåðâóàðîâ (òðè ðåçåðâóàðà äëÿ ðàçíûõ óñëîâèé
pH (7,5, 7,0), âñåãî òðè èñïûòàíèÿ), çà èñêëþ÷åíèåì äíÿ 7ãî: n = 6; à òàêæå äíÿ 11,6: n = 7; äíÿ 15.5:
n = 6; äëÿ 23-é: n = 6 (êðîìå pH 7,5 n = 5); äíÿ 30-ãî: n = 8; êîíòðîëüíîãî 58-ãî äíÿ: n = 8. Ëèíèÿ
ðåãðåññèè ïîêàçàíà äëÿ ýêñïåðèìåíòàëüíûõ è êîíòðîëüíîé ãðóïï. Îáîçíà÷åíèÿ: êîíòðîëüíûå
óñëîâèÿ ÷åðíûå êðóæêè, ðÍ 7,5 ñåðûå êâàäðàòû, ðÍ 7,0 ñâåòëî-ñåðûå òðåóãîëüíèêè.
food was given to the subjects.
BELL DIAMETER. Each week, jellyfish
were gently removed from their tanks and placed
subumbrellar side up in glass bowls containing
their respective tank water to measure diameter
(mm) of the fully open bell in real time. After
animals had settled within 23 minutes, each
jellyfish was positioned in the bowl over a thin
plastic ruler and was observed for several pulse
cycles to visually assess the difference between
turbed subjects and the video was reviewed to
count the number of bell pulses per minute
(ppm) for each animal. In healthy jellyfish,
pulse rate can be affected by the size of the
jellyfish, surrounding currents, presence of food
in the water, proximity to conspecifics, periods
of rest, or if the jellyfish is actively swimming or
relocating (Hamlet et al., 2011; Hamlet, Miller,
2014; Ohdera et al., 2018). To control for these
variables, videos were taken at 4:00 p.m. before
8R. Thayer et al.
Fig. 4. Deterioration in the feeding apparatus of Cassiopea sp. in response to treatment pH 7.0. AC
images of baseline subjects in good condition; A exumbrellar photograph of baseline subject; B food
internalization following capture by digitate cirri at a mid-oral arm furcation; C digitate cirri and
cassiosome nests at distal end of oral arm; D photograph of lateral view of baseline subject; E
photograph of lateral view of gum dropped subject from trial 2C tanks on final day 81. FH scaled group
photographs of exumbrellar view of trial 2 final day 81 subjects; F control; G pH 7.5; H pH 7.0 with
inset of subject. IK photomicrographs of subjects on trial 2 final day 81; I feeding appendages on distal
end of an oral arm of a control subject; J cassisome nests on oral arm of a pH 7.5 subject; K t-rexed
oral arms from a pH 7.0 subject.
Yellow arrows ornamental vesicular appendage; cyan circles distal end of oral arm, cyan arrows digitte cirri
lining oral collar of secondary mouths; fuchsia arrows cassiosome nests; orange arrow food internalization at
secondary mouth; neon arrow exposed oral discs.
Ðèñ. 4. Äåãðàäàöèÿ ïèùåâîãî àïïàðàòà Cassiopea sp. â îòâåò íà ñîäåðæàíèÿ ïðè ðÍ 7,0. ÀÑ
èñõîäíîå ñîñòîÿíèå ìåäóç; À âèä ìåäóçû ñî ñòîðîíû ýêñóìáðåëëû; B ïîãëîùåíèå ïèùà ïîñëå
çàõâàòà ïàëüöåâèäíûìè óñèêàìè; C ïàëüöåâèäíûå óñèêè è ãíåçäà êàññèîñîì íà äèñòàëüíîì êîíöå
ðîòîâîé ëîïàñòè; D ôîòîãðàôèÿ èñõîäíîãî ñîñòîÿíèÿ ìåäóçû ñáîêó; Å âèä ìåäóçû íà 81-é äåíü
ýêñïåðèìåíòà 2Ñ. FH ýêñóìáðåëëà ìåäóçû â ýêñïåðèìåíòå 2, 81 äåíü ýêñïåðèìåíòà; F
êîíòðîëü; G ðÍ 7,5; H ðÍ 7,0. IK ìåäóçû, ýêñïåðèìåíò 2, äåíü 81; I ïèòàþùèå ïðèäàòêè
íà äèñòàëüíîì êîíöå ðîòîâîé ëîïàñòè ìåäóçû â êîíòðîëüíûõ óñëîâèÿõ; J ãíåçäà êàññèñîìû íà
ðîòîâîé ðóêå ìåäóçû, ðÍ 7,5; K ðîòîâûå ëîïàñòè ìåäóçû, pH 7,0.
Æåëòûå ñòðåëêè âåçèêóëÿðíûé ïðèäàòîê; êðóæêè äèñòàëüíûé êîíåö ðîòîâîé ëîïàñòè, ãîëóáûå ñòðåëêè
ïàëüöåâèäíûå öèððû, âûñòèëàþùèå ðîòîâûå âîðîòíè÷êè âòîðè÷íûõ ðòîâ; ïóðïóðíûå ñòðåëêè ñêîïëåíèÿ
êàññèîñîì; îðàíæåâàÿ ñòðåëêà ïîëîæåíèå ïèùè âî âòîðè÷íîì ðòå; çåëåíàÿ ñòðåëêà ðîòîâîé äèñê.
9Severe seawater acidification and the scyphozoan medusa Cassiopeia sp.
the diameter of the fully open bell during the
recovery vs. the actively contracted phase of the
pulse. In some cases, where the individual did
not settle on the bottom or did not pulse, the
subject was gently moved to the bottom of the
bowl and placed over the ruler for measurement.
REORIENTATION AND SETTLEMENT
ASSAY. Each week, a plastic pitcher was used
to collect one liter of water from the surface of
each tank and then poured back into the tank
from a consistent distance of approximately 5
cm above the surface of the water in order to
create a current strong enough to lift the jellyfish
from their resting locations on the bottom of the
tank. The elapsed time (sec) until all animals in
the tank settled normally (subumbrellar side up
on the bottom of the tank, exumbrellar side
suctioned to the walls of the tank, or exumbrel-
lar side up at the surface of the water) was
recorded in real time. Settling on the surface of
the water was defined as when a subject ap-
peared to have come to rest on the surface
tension of the water and was no longer actively
swimming away from that position. Any abnor-
malities including landing exumbrellar side up,
not pulsing to reach the bottom, or inability to
settle before or after the test were noted.
ANATOMICAL OBSERVATIONS. To
track morphological features over time, a week-
ly group photo was taken (iPhone or Google
Pixel Camera) of subjects from each tank after
they had been placed in glass bowls. Photomi-
crographs were taken using an iPhone camera
through the ocular of a Zeiss Stemi 2000 dissec-
tion microscope or using a Google Pixel camera
through the ocular of a Leica dissecting micro-
scope. Anatomical features observed include
the feeding apparati (secondary mouths and the
digitate cirri lining the oral collar), whether the
distal ends of the oral arms extended past the
margin of the bell or were drawn in closer to the
center (referred to as t-rexing), vesicular ap-
pendages (ornate appendages, sometimes hous-
ing cassiosome nests, that extend from the oral
arms and are believed to play a role in hydrody-
namics in Cassiopea sp.), flattening/uncurling
of the margin of the bell during both the active
phase of the pulse cycle, bell malformations
including inversion or the doming shape of the
bell (the gumdrop effect), presence of rhopa-
lia and statocysts, presence of zooxanthellae,
and polyp stalk length and oral disc width.
Feeding was documented by imaging jellyfish
5 minutes after being fed Artemia while in a
glass bowl.
POLYP OBSERVATIONS. Polyps were
grown on filtration BioBalls or plastic grating
placed in each tank. To assess growth of polyp
colonies, the number of polyps present on the
BioBalls placed in the A tanks and 7.0 B tank of
trial 1 were counted from photomicrographs
(taken with an iPhone camera through the ocular
of a Zeiss Stemi 2000 dissection microscope) at
baseline and the final day. Since polyps were
counted only for trial 1, data were not statistical-
ly analyzed. Planuloid production and strobila-
tion were noted, but not quantitated. Photo-
graphs (taken with Nikon D3S camera and Mi-
cro-Nikkor 105 mm f/2.8 lens) of polyps grow-
ing on plastic grating on the final day in trial 3
C tanks were used to measure polyp stalk length
(from the base of calyx to the base of polyp
stalk) and across the oral disc using ImageJ
(National Institutes of Health, https://imagej.
nih.gov/ij/). Since this analysis was performed
on polyps in trial 3 only, data were not statisti-
cally analyzed.
STATISTICAL ANALYSIS. Subject mor-
tality rate and pH treatment effect on bell diam-
eter, pulse rate, and reorientation and settlement
assay were analyzed using GraphPadPrism, ver-
sion 9.2.0. Statistical significance was evaluat-
ed at 0.05 alpha level.
MORTALITY RATE. Overall mortality per
trial (9 tanks per trial, 5 subjects per tank) was
calculated by adding the number of deceased
subjects on the final day of each trial and divid-
ing by the total starting number (n = 45). Ani-
mals were considered to be living if they pulsed
at the rate of one pulse per minute (ppm). A one-
way ANOVA test was used to test for the effect
of pH treatment (control, pH 7.5, and pH 7.0) on
subject mortality rate. When significant differ-
ence was found, a Tukeys multiple comparison
post hoc analysis was performed to identify
significance difference between pairs of fac-
10 R. Thayer et al.
patterns of DIC redistribution between its vari-
ous species as in anthropogenic driven OA
(Table 1). As CO2 was added to the systems, the
pH decreased (to experimental levels of 7.5 and
7.0), the [CO2 ( aq)] and [HCO3
] increased, and
[CO3
2] decreased, in all pH 7.5 and 7.0 tanks
compared to all trial 2 control tanks. We ob-
served the same trend in trial 3 for all pH 7.5 and
7.0 tanks compared to all trial 3 controls.
MORTALITY RATES. The mortality rate
for treatment pH 7.5 was 2.20% (± 0.11%
S.E.M.), pH 7.0 was 22.22% (±0.31% S.E.M.),
and control was 2.50% (± 0.13% S.E.M.). To
test the hypothesis that pH reduction causes a
difference in the mortality rates, we performed
a one-way ANOVA test between the three fac-
tors of control, pH 7.5, and pH 7.0 for the nine
tanks and found a significant difference be-
tween the mortality rate based on the pH treat-
ment factor (p= 0.0027, DF = 2, F = 7.720). To
determine which pH treatment had an effect on
the mortality rate, we performed a Tukeys
multiple comparison post hoc test (control v. pH
7.5, control v. pH 7.0, and pH 7.5 v. pH 7.0) and
found that mortality rates were significantly
different between treatment pH 7.0 and control
group (adjusted p = 0.0083, [95% confidence
interval: 0.7325 to 0.7603]) and between treat-
ment pH 7.0 and pH 7.5 (adjusted p = 0.0058,
[95% confidence interval: 1.724 to 0.2759]).
One outlier (trial 3C control tank) was removed
from the data set (z-score = 2.53), decreasing the
control group n from 45 to 40 for calculating the
mortality rates and number of tanks from nine to
eight for the ANOVA and post hoc tests. Three
deaths were observed in the trial 3C control tank
due to factors independent of the treatment.
Water quality for this tank was within normal
parameters and the cause of the mortality is
unknown.
PULSE RATE. To test the hypothesis that
pH reduction would cause a decreased pulse
rate, we performed an ordinary 2-way ANOVA
based on the factors of 1) pH reduction and 2)
trial. The factor of pH had a significant effect on
the pulse rate (p = 0.001; DF= 2; F (2,18) =
0.001). The trial number did not have a signifi-
cant effect on the pulse rate (p= 0.0646; DF = 2;
tors. The adjusted p-values and 95% confidence
intervals were reported.
BELL DIAMETER, PULSE RATE, RE-
ORIENTATION AND SETTLEMENT AS-
SAY. To test for the effect of pH treatment on
bell diameter, pulse rate, and the reorientation
and settlement assay, we first ran an ordinary 2-
way ANOVA based on the factors of 1) pH
reduction and 2) trial and reported the p-value,
degrees of freedom (DF), and the F-value (if
relevant). When significance was found, a
Tukeys multiple comparison post hoc analysis
was performed with adjusted p-values and DF
reported. To determine the timing of when the
pH reduction caused significant changes, we
performed a multiple comparisons unpaired t-
test on the test points and listed the p-values for
each in a table. In the pulse rate analysis, each
data point represents the mean values ± the
standard error of the mean (S.E.M.) for n = 45,
except for day 5 (n = 15) and days 15.5 and 23.5
(n = 30). In the bell diameter analysis, each data
point represents n = 45, except for days 7, 15.5,
and 23.5 (n = 30).
REORIENTATION AND SETTLEMENT
ASSAY. Each data point represents the mean
values ± S.E.M. for nine tanks (three tanks per
treatment pH (7.5, 7.0) for a total of three trials)
except for the following day 7: n = 6; day 11.6:
n = 7; day 15.5: n = 6; day 23: n = 6 (except for
pH 7.5 n = 5); day 30: n = 8; control day 58: n =
8. To determine if there was a difference in the
rate of change in the bell diameter, pulse rate, or
orientation and settling ability between the pH
treatments over time pH (7.5, 7.0), we per-
formed a simple linear regression and reported
the equations, the p-value, the R2 value, and the
95% confidence intervals (C.I.). A basic t-test
was performed to determine whether the slopes
of the linear regression lines were significantly
different between the treatment and control
groups. The p-value and F-value were reported.
Results
CARBONATE CHEMISTRY. Water car-
bonate chemistry measurements for each test
pH were taken and are in accordance with the
11Severe seawater acidification and the scyphozoan medusa Cassiopeia sp.
TCO2 (ìmol/
kg SW)
pCO2
(ìatm)
CO3 (ìmol/ kg
SW)
HCO3 (ìmol/
kg SW)
CO2 (ìmol/
kg SW) ÙAr
Trial 2 Control A 526.698 116.852 49.558 473.748 3.392 0.782
Trial 2 7.5 A 561.706 414.998 17.6 532.058 12.048 0.278
Trial 2 7.0 A 720.62 1642.63 6.967 665.967 47.687 0.11
Trial 2 Control B 563.249 96.333 40.856 390.56 2.797 0.644
Trial 2 7.5 B 498.891 368.589 15.632 472.558 10.7 0.247
Trial 2 7.0 B 600.509 1368.841 5.805 554.965 39.738 0.092
Trial 2 Control C 533.536 118.369 50.201 479.898 3.436 0.792
Trial 2 7.5 C 830.098 613.291 26.01 786.283 17.804 0.41
Trial 2 7.0 C 1003.324 2287.044 9.7 927.23 66.395 0.153
Trial 3 Control A 519.678 115.294 48.897 467.434 3.347 0.771
Trial 3 7.5 A 533.536 394.186 16.718 505.375 11.443 0.264
Trial 3 7.0 A 720.62 1642.63 6.967 668.967 47.687 0.11
Trial 3 Control B 266.075 59.031 25.035 239.326 1.714 0.395
Trial 3 7.5 B 699.833 517.049 21.929 662.894 15.01 0.346
Trial 3 7.0 B 1143.291 2606.95 11.053 1056.582 75.657 0.174
Trial 3 Control C 461.928 102.482 43.464 415.489 2.975 0.686
Trial 3 7.5 C 554.323 409.543 17.369 525.065 11.889 0.274
Trial 3 7.0 C 637.471 1453.094 6.163 589.124 42.184 0.097
Table 1. Summary of carbonate species in trial 2 and 3 tanks. Data computed from measurement of aver-
age total CO2 in each tank over multiple weeks at experimental pH.
Òàáëèöà 1. Ñîäåðæàíèå óãëåêèñëîãî ãàçà è èîíîâ êàðáîíàòíîé ñèñòåìû â îïûòíûõ ðåçåðâóàðàõ 2
è 3. Äàííûå ðàññ÷èòàíû íà îñíîâå èçìåðåíèÿ ñðåäíåãî îáùåãî CO2 â êàæäîì ðåçåðâóàðå â
ýêñïåðèìåíòàëüíûì óñëîâèÿõ pH.
addition, by the final time point of 66 days in
reduced pH (7.0), there was a 87.43% reduction
in pulse rate (Fig. 1, 20.82927 vs. 2.61765 ppm,
p = <0.000001).
To determine if there was a difference in the
rate of change in the pulse rate over time for the
different treatments pH (7.5, 7.0), we performed
a simple linear regression (Table 3). The high R2
values indicate that the equations for treatment
pH 7.5 and 7.0 highly predict test results shown
in the data, while the equation for control has
less of a fit to the data. Then we used a basic t-
test to determine whether the slopes of the linear
regression lines were significantly different (Ta-
ble 4). There is a significant difference in the
slopes between control and pH 7.0 (p = <0.0001)
and between pH 7.5 and pH 7.0 (p = <0.0001).
F (2, 8) = 0.064). To determine which pH
treatment (7.5, 7.0, or both) had an effect on the
pulse rate, we performed a Tukeys multiple
comparison post hoc test. There was no signif-
icant difference between the control and pH 7.5
(adjusted p-value = 0.0895, DF= 0.23) or be-
tween pH 7.5 and pH 7.0 (adjusted p-value =
0.0801, DF= 0.23). A significant difference was
found between the control and pH 7.0 (adjusted
p-value = 0.0007, DF= 0.23). To determine
when the pH reduction caused a significant
difference in pulse rate between the control and
pH 7.0 group, we performed a multiple com-
parisons unpaired t-test (Table 2) and found that
significant differences first occurred at day 23.5
with a 50.19% reduction in the pulse rate (Fig.
1, 22.43 vs. 12.93 ppm, p = 0.000068). In
12 R. Thayer et al.
Table 2. Multiple comparisons unpaired t-test for control vs pH 7.0 for pulse rate, bell diameter, and re-
orientation assay.
Òàáëèöà 2. Ñðàâíåíèå ïàðàìåòðîâ ïóëüñàöèè, äèàìåòðà êîëîêîëà è ñïîñîáíîñòè ê
ïåðåâîðà÷èâàíèþ äëÿ êîíòðîëüíîé ãðóïïû è óñëîâèé ñ pH 7,0 ìåòîäîì ìíîæåñòâåííîãî
ñðàâíåíèÿ íåçàâèñèìûõ ïåðåìåííûõ ïðè ïîìîùè t-êðèòåðèÿ.
p-value
Test Point
(days) Bell Diameter Pulse Rate Reorientation and
Settlement Assay
Baseline 0.53096 0.896472 0.637612
7 0.31566 0.896472 0.544815
11.67 0.45332 0.896472 0.827486
15.5 0.72744 0.133302 0.089557
23.5 0.16663 0.000068 0.483468
29.67 0.12661 <0.000001 0.276113
36.67 0.00194 <0.000001 0.424257
44 0.00174 <0.000001 0.146041
50.67 0.00064 <0.000001 0.107585
58.67 6.9E-05 <0.000001 0.537995
66 1.3E-05 <0.000001 0.749215
between the bell diameter of control and pH 7.5
(adjusted p-value = 0.9929, DF= 0.23), while
significant differences were found between the
bell diameter of control and pH 7.0 (adjusted p-
value = 0.0150, DF= 0.23) and between pH 7.5
and pH 7.0 (adjusted p-value = 0.0118, DF=
0.23). To determine which trial number (1, 2, or
3) had an effect on the bell diameter, we per-
formed a Tukeys multiple comparison post hoc
test. There was no significant difference found
between trials 1 vs. 2 (adjusted p-value = 0.422,
DF= 0.23), while significant differences were
found between trial 1 vs. 2 (adjusted p-value =
0.0158, DF= 0.23) and between trial 2 vs. 3
(adjusted p-value = 0.0010, DF= 0.23). Trial 3
is likely different because of the mortality (n =
However, there is no significant difference be-
tween the slopes of the control and pH 7.5
(p=0.1753).
BELL DIAMETER. To test the hypothesis
that pH reduction would cause a decrease in bell
diameter, we performed an ordinary 2-way
ANOVA based on the factors of 1) pH reduction
and 2) trial. The factor of pH had a significant
effect on the bell diameter (p = 0.0063; DF= 2;
F (2, 18) = 6.800). The trial number had a
significant effect on the size of the bell diameter
(p-value = 0.0011; DF = 2; F (2, 18) = 10.21). To
determine which pH treatment (7.5, 7.0, or
both) had an effect on the bell diameter, we
performed a Tukeys multiple comparison post
hoc test. There was no significant difference
13Severe seawater acidification and the scyphozoan medusa Cassiopeia sp.
pH Level
Test Analysis
Result Control 7.5 7.0
Best Fit Line
Equation
Y = 0.1296*X +
27.18
Y =
0.1985*X +
29.89
Y = 0.4486*X +
27.69
R2 0.5806 0.8081 0.9193
Pulse Rate
Slope
Confidence
Interval
0.2126 to
0.04653
0.2714 to
0.1256
0.5489 to
0.3484
Best Fit Line
Equation
Y = 0.2417*X +
57.24
Y =
0.2404*X +
60.38
Y = 0.4662*X +
58.31
R2 0.9492 0.8689 0.9849
Bell Diameter
Slope
Confidence
Interval
0.2839 to
0.1996
0.3108 to
0.1700
0.5097 to
0.4227
Best Fit Line
Equation
Y = 0.1363*X +
22.95
Y =
0.04791*X +
30.38
Y = 0.05695*X +
27.35
R2 0.5598 0.05428 0.05556
Reorientation
and Settlement
Assay
Slope
Confidence
Interval
0.04517 to 0.2275 0.1029 to
0.1987
0.1201 to
0.2340
Table 3. Linear regression analysis of all pH levels for pulse rate, bell diameter, and reorientation assay.
Òàáëèöà 3. Ëèíåéíûé ðåãðåññèîííûé àíàëèç âñåõ óðîâíåé pH äëÿ ïóëüñàöèè, äèàìåòðà êîëîêîëà
è ñïîñîáíîñòè ê ïåðåâîðà÷èâàíèþ.
and that by the final time point of 66 days in
reduced pH (7.0), there was a 36.04% reduction
in bell diameter (Fig. 2, 42.77 vs. 41.77, p =
0.000013).
To determine if there was a difference in the
rate of change in the bell diameter over time for
the different treatments pH (7.5, 7.0), we per-
formed a simple linear regression test (Table 3).
The high R2 values indicate that the equations
for the control, pH 7.5, and pH 7.0 fall relatively
in line with the data. Next, we used a basic t-test
3) that occurred in the trial 3 control A tank.
Though this tank was excluded from the data for
mortality calculations (outlier z-score = 2.53), it
was not removed from any other data set. To
determine when the pH reduction caused a sig-
nificant difference in the bell diameter between
the control and pH 7.0 group, we performed a
multiple comparisons unpaired t-test (Table 2)
and found that significant differences first oc-
curred at day 36 with 16.63% reduction in the
bell diameter (Fig. 2, 42 vs. 50 mm, p = 0.001935)
14 R. Thayer et al.
pH Level Comparison
Test Analysis Result
Control vs 7.5 Control vs 7.0 7.5 vs 7.0
P value 0.1753 <0.0001 <0.0001
Pulse Rate
F value 1.991 30.74 49.04
P value 0.9709 <0.0001 <0.0001
Bell Diameter
F value 0.001366 70.26 38.11
Table 4. T-test analysis between the regression lines for each test pH vs. control and for pH 7.5 vs. 7.0
for pulse rate and bell diameter.
Òàáëèöà 4. T-òåñò ëèíèé ðåãðåññèè äëÿ êàæäîãî òåñòèðóåìîãî pH â ñðàâíåíèè ñ êîíòðîëåì è äëÿ
pH 7,5 è 7,0 äëÿ ñêîðîñòè ïóëüñàöèè è ðàçìåðà êîëîêîëà.
ANATOMICAL OBSERVATIONS. Group
photographs of all subjects taken at baseline and
on the final day of the experiment are arrayed in
Supplementary files 13 to draw comparisons
of any anatomical changes that occurred within
groups over time and between groups and trials.
Baseline Cassiopea sp. specimens of good con-
dition have abundant vesicular appendages (Fig.
4, yellow arrows), oral arms that extend past the
margin of the bell (Fig. 4A) with numerous
cassiosome nests (Fig. 4, fuchsia arrows) and
secondary mouths (Fig. 4B), the latter encircled
by digitate cirri (Fig. 4C, cyan arrows). Fig. 4B
depicts internalization of Artemia following
capture by a secondary mouth (orange arrow)
located at a middle oral arm furcation. Baseline
subjects maintain a mostly flat bell, typically
with the exumbrellar side against the substrate
or walls of the tank, with the bell margin slightly
curled inward during the resting phase of the
pulse cycle (Fig. 4D), while subjects in pH 7.0
treatment exhibited inversion of the bell to an
unnatural gumdrop shape (Fig. 4E).
Compared to control and treatment pH 7.5,
final day pH 7.0 subjects were smaller and had
t-rexed oral arms that were bare in appearance
(Fig. 4 FH, images to scale, except for zoomed
in pH treatment 7.0 animal in Fig. 4H on right).
Photomicrographs (Fig. 4 IK) show that final
day pH 7.0 subjects had a loss of feeding ap-
pendages (cyan circles) and cassisosome nests
to determine whether the slopes of the linear
regression lines were significantly different (Ta-
ble 4). There was a significant difference in the
slopes between the control and pH 7.0 (p =
<0.0001) and between pH 7.5 and pH 7.0 (p =
<0.0001). The high p-value between the con-
trol and pH 7.5 (p = 0.9709) suggests that the
control and pH 7.5 bell diameter declined at a
similar rate.
REORIENTATION AND SETTLEMENT
ASSAY. To test the hypothesis that pH reduc-
tion would cause longer times to reorient and
settle, we performed an ordinary 2-way ANO-
VA based on the factors of a) pH reduction and
b) trial. The factor of reduced pH had no signif-
icant effect (p = 0.9430; DF= 2; F (2, 18) =
0.05888) and trial number had no significant
effect on assay performance (p = 0.0011; DF =
2; F (2, 18) = 10.21). Settling times ranged
between 2040 seconds for each group (Fig. 3).
To determine if there was a linear relationship
with respect to time for each treatment for
reorientation and settling ability, we performed
a simple linear regression (Table 3). The rela-
tively low R2 values indicate that for the
control, pH 7.5, and pH 7.0 the pH level does
not explain much of the variation observed in
this assay. One outlier value (trial 3B control tank
value of 147.8 seconds on day 58 (z-score = 2.42)
was removed from the reorientation and settle-
ment assay data set.
15Severe seawater acidification and the scyphozoan medusa Cassiopeia sp.
Number of Polyps
Day Control A Tank pH 7.5 A Tank pH 7.0 A Tank pH 7.0 B Tank
Day 1 (Baseline) 108 131 109 119
Day 92 107 142 136 179
Table 5. Number of polyps counted growing on the spherical cap of a BioBalls in Trial 1.
Òàáëèöà 5. Êîëè÷åñòâî ïîëèïîâ, ðàñòóùèõ íà ñôåðè÷åñêîé ïîâåðõíîñòè â îïûòå 1.
zooxanthellae (small golden-brown cells) were
abundant throughout this tissue. No animals
experienced bleaching as an effect of lowered
pH, and shrunken pH 7.0 subjects often appear
darker in color as their zooxanthellae condensed
in the remaining tissue (Fig. 4H and Supplemen-
tary files 13).
In feeding, the oral arms of the control and
pH 7.5 groups were splayed out providing max-
(fuchsia arrows) seen in the control and pH 7.5
groups, and secondary mouths with little to no
digitate cirri leaving the underlying oral discs
exposed (green arrow).
Photomicrographs of the bell margin show
that, even in t-rexed pH 7.0 subjects, the rhopa-
lia (Supplemental file 4AB, red circles) and
the statocysts (composed of statoliths) (Supple-
mental file 4CD, red arrows) were present, and
Fig. 5. Polyp and ephyrae health in response to treatment (pH 7.5, 7.0). AC photographs of polyps
attached to plastic grating from trial 3 C tanks on day 78: A control; B pH 7.5; pH 7.0. DF
photomicrographs of ephyrae retrieved from trial 3 C tanks on day 78: D control; E pH 7.5; F pH
7.0. Grey arrows indicate whether the oral side of the animal is facing up, down, or sideways.
Ðèñ. 5. Èçìåíåíèÿ ïîëèïîâ è ýôèðîâ â ýêñïåðèìåíòå (ðÍ 7,5, 7,0). ÀÑ ôîòîãðàôèè ïîëèïîâ,
ïðèêðåïëåííûõ ê ïëàñòèêîâîìó îñíîâàíèþ â ðåçåðâóàðàõ íà 78-é äåíü ýêñïåðèìåíòà 3Ñ: À
êîíòðîëüíûå óñëîâèÿ; B ðÍ 7,5; ðÍ 7,0. DF ýôèðû, èçâëå÷åííûå èç îïûòíûõ ðåçåðâóàðîâ (3Ñ)
íà 78-å ñóòêè: D êîíòðîëüíûå óñëîâèÿ; Å ðÍ 7,5; F ðÍ 7,0. Ñòðåëêè óêàçûâàþò íàïðàâëåíèå
ðîòîâîé ñòîðîíû.
16 R. Thayer et al.
days of exposure to low pH. These findings
corroborate the previous body of research that
describes the tolerance of Aurelia sp. planulae
larvae, polyps and ephyrae to OA, and the only
previous research on the effect of OA (pH 7.6
for 4 weeks) on Cassiopea sp. (Weeks et al.,
2019). In contrast, treatment of wild caught box
jellyfish with pH 7.5 (12 hours) resulted in 35%
mortality rates with surviving animals present-
ing with retracted tentacles and totally inhibited
swimming abilities (Chuard et al, 2019). This
study is the first to examine the effect of pH<7.6
on Cassiopea sp. Lethal effects occurred with
treatment pH 7.0 for 66 days with 22% mortality
rates and with surviving jellyfish in poor condi-
tion.
After introducing CO2, pH 7.5 was reached
by an average of 8 days and pH 7.0 was reached
by an average 22.67 days. Significant differenc-
es between the treatment pH 7.0 and control first
occurred on day 23.5 with a 50% reduction in
the pulse rate and was followed by a 16.6%
reduction in bell diameter ~2 weeks later, while
pH 7.5 had no effect. The decline in pulse rate
preceded the day that the test pH 7.0 level was
reached. By the final time point of 66 days in
treatment pH 7.0, there was an 87% reduction in
pulse rate and a 36% reduction in bell diameter
versus control. The pulsing behavior is the pri-
mary mechanism by which jellyfish draw in and
catch prey, so if a jellyfish loses its ability to
pulse properly, its feeding ability will be com-
promised and will reduce in size.
The feeding strategy of Cassiopea sp. em-
ploys the anatomy of the oral arms and various
appendages to create vortices that swirl around
the secondary mouths during each pulse (Ham-
let et al., 2011). Here, t-rexing, loss of vesicular
appendages, inversion and gumdropping of the
bell in pH 7.0 medusae weakened the pulse, and
therefore the currents which deliver prey to the
secondary mouths. While the zooxanthellae were
retained, the nutrition was inadequate as signif-
icant size reduction occurred in treatment pH
7.0. In the only previous work to examine the
effect OA on the pulse rate, treatment of A.
aurita ephyrae at pH 7.6 for 7 days resulted in
slower pulse rates and smaller surface area,
imum exposure of the secondary mouths to the
water column for prey capture (prey seen as
clumps of orange Artemia nauplii), while the pH
7.0 subjects curled their oral arms inwardly
towards the central manubrium with prey con-
centrating at the proximal portion of oral arms
while ingesting (Supplementary file 5).
POLYP OBSERVATIONS. An increase in
the number of polyps growing on Bio Balls in
test tanks (pH 7.5, 7.0) occurred from the base-
line to the final day 92 in trial 1, whereas the
number of polyps in the control tank remained
the same (Table 5). Polyps grown on plastic
grating in the trial 3C control tank had elongat-
ed, healthy tentacles, elliptical to round calyxes,
and elongated stalks (Fig. 5A), treatment pH 7.5
had slightly contracted tentacles, elliptical to
round calyxes, and shortened stalks (Fig. 5B),
and treatment pH 7.0 had shortened or constrict-
ed tentacles, irregularly shaped calyxes, and
shortened stalks (Fig. 5C). No difference in oral
disc diameter between test groups and control
was observed. Planuloid production was ob-
served in all groups throughout the experiment
and polyp colonies were present on the final day
in all groups in all trials. Strobilation occurred
in all groups throughout the experiment in all
trials. Ephryae collected from the control and
pH 7.5 tanks had normally shaped bells with a
flat exumbrellar side (Fig. 5DE), while some
ephyrae in the pH 7.0 tanks had inverted bells
(Fig. 5F).
Discussion
It has been suggested that gelatinous organ-
isms are more tolerant to acidifying and warm-
ing waters than other marine taxa leading to
perceived increases jellyfish blooms occurring
over wider geographic ranges (Attrill et al.,
2007), though other causal factors implicated
include the loss of prey or competitors and that
jellyfish populations are subject to worldwide
oscillations with an ~20-y periodicity (Condon
et al., 2013). This study adds to the line of
evidence that jellyfish are more resistant to OA
than many taxa, with 98% of Cassiopea sp.
medusae surviving and in good health after 66
17Severe seawater acidification and the scyphozoan medusa Cassiopeia sp.
for 36 days had no effect (Treible et al., 2018)
and treatment with pH <4.5 for 240 days caused
tissue degradation (Goldstein et al., 2017). Here,
Cassiopea sp. polyps cultured in reduced pH
(7.5 and 7.0) for 66 days had shortened tenta-
cles, irregularly shaped calyxes, and shortened
stalks, but the specimens appeared in good
condition. The effect of these anatomical changes
on prey capture efficiency was not examined.
Exposure of Aurelia aurita planulae larvae to
pH 7.4 for 240 days in combination with low
temperature of 4°C (Goldstein et al., 2017) and
of Aurelia coerulea to pH 7.3 for 7 days (Dong,
Sun, 2018) increased planulae settlement rates.
Though we did not examine planulae larvae
here, exposure of polyps to 66 days of pH 7.0
did not impact their ability to reproduce asexu-
ally through release of planuloids, which oc-
curred throughout the experiment, even on the
final days. Planuloid tolerance to reduced pH is
demonstrated by the increase in polyp colony
size over time in reduced pH environments (as
observed in trial 1). Though able to strobilate
throughout the experiment, polyps exposed to
treatment pH 7.0 gave rise to some ephyrae with
inverted bells and inhibited swimming abilities,
in support of the work of Kikkawa et al. (2010)
in which Aurelia sp. ephyrae exposed to pH
6.15 for 96 hours had inverted oral arms and
inhibited swimming abilities.
While treatment had no significant effect on
the reorientation and settlement assay, the man-
ner in which the pH 7.0 subjects settled was
different from the other groups. Subjects with
gumdropped bells were observed to have inef-
fective bell contractions during the pulse cycle
which affected the hydrodynamics of their swim-
ming, causing them to sink rather than swim to
reach the bottom, but in about the same amount
of time as controls. A more accurate assessment
would be to record the proportion of animals per
tank that settle normally by pulsing to the bot-
tom (as was observed in the control and pH 7.5
groups) versus sinking (as was observed in the
pH 7.0 groups) and how many are able to recov-
er to a normal position in a given amount of time
after disturbance.
The reorientation test was intended to dis-
cern whether OA would cause deterioration to
central disc area, and lappet length (Tills et al.,
2016) and treatment with pH 7.28 for 7 days
caused slower ephyrae growth (Alguero-Muniz
et al., 2016), however, no previous research has
examined the effect of pH reduction on the pulse
rate of any scyphozoan medusae. Additional
research to explore the effects of reduced pH on
the metabolism of Cassiopea sp. could examine
the difference between how much prey is ingest-
ed at pulse rates affected by reduced pH, and
which subjects are expending versus conserving
energy. Since Cassiopea sp. cycles nutrients in
the substrates of their native environments
through the pulsing behavior, a decline in pulse
rate in native populations would have negative
impacts on the nutrient cycles of these benthic
environments.
Having adapted to a benthic lifestyle, Cassi-
opea sp. lacks lengthy stinging tentacles typi-
cally found in other scyphozoans, and instead
uses oral arms lined with numerous secondary
mouths equipped with digitate cirri and cassio-
somes, both of which release stinging nemato-
cysts. Treatment pH 7.0 caused damage to the
feeding apparati of medusae with a loss of
digitate cirri, thereby limiting the effectiveness
of the secondary mouths. These subjects were
observed to curl the oral arms more inwardly
than the normal position during feeding. Loss of
vesicular appendages and cassiosome nests in
pH 7.0 animals may have also impacted their
ability to capture prey, as they are an integral
part of the Cassiopea sp. feeding strategy (Weeks
et al., 2019; Ames et al., 2020). As the brachial
cavities and secondary mouths are involved in
sexual reproduction of this species, damage to
these structures in severe OA to pH 7.0 could
also inhibit the species ability to brood planu-
lae produced by fertilized eggs, making the
survival of the species dependent on the asexual
reproduction of polyps, thereby limiting genetic
diversity over time. A lack of genotypic varia-
tion in future generations caused by the lethal
effects of OA on the sexually reproductive me-
dusae stage of C. xaymacana has also been
proposed by Chuard et al. (2019).
Previous work on the effects of OA on A.
aurita polyps found that treatment with pH 7.62
18 R. Thayer et al.
in the coral-algae relationship (Hoegh-Guld-
berg, 1999), future studies should include the
effect of both OA and rising temperatures relat-
ed to climate change on survival.
Compliance with ethical standards
CONFLICTS OF INTEREST: The authors de-
clare that they have no conflicts of interest.
Supplementary data. The following materials
are available online.
Supplementary file 1. Photographs of trial 1
subjects taken at the baseline and final day. Col-
umns: Left A tanks, middle B tanks, right C
tanks. Rows: top control, middle pH 7.5,
bottom pH 7.0. Scale bars 2.54 cm.
Supplementary file 2. Photographs of trial 2
subjects taken at the baseline and final day. *Day
number adjusted for time spent in acidic water.
Columns: Left A tanks, middle B tanks, right
C tanks. Rows: top control, middle pH 7.5,
bottom pH 7.0. Scale bars 2.54 cm.
Supplementary file 3. Photographs of trial 3
subjects taken at the baseline and final day. *Day
number adjusted for time spent in acidic water. Col-
umns: Left A tanks, middle B tanks, right C
tanks. Rows: top control, middle pH 7.5,
bottom pH 7.0. Scale bars 2.54 cm.
Supplementary file 4. Intact rhopalia and stato-
cysts observed in pH 7.0 treatment day 71. AB
photomicrographs of the bell margin of two subjects.
CD photomicrographs of statocysts in two sub-
jects. Red circles rhopalia. Red arrow stato-
cysts containing statoliths. C, D shown in 40x mag-
nification. Scale bars 1 mm.
Supplementary file 5. Modified feeding behav-
ior observed in pH 7.0 treatment day 71. A control
subjects; B pH 7.5; C pH 7.0. Images taken of
jellyfish in trial 1B tanks on day 71. Scale bar 10 mm.
Acknowledgements. We would like to thank
Tina Stewart, Senior Lab Technician at the Tennes-
see Aquarium, for her assistance in monitoring water
quality and measuring the total DIC of each treat-
ment, Dr. Dustin Kemp, Professor at the University
of Alabama-Birmingham, for his advice on best
practices for determining DIC, members of the Mc-
Callie Scientific Research Program and the Tennes-
see Aquarium Husbandry staff who assisted in daily
care of the animals, Casey Phillips, Communication
Specialist at the Tennessee Aquarium for photogra-
phy in trial 3, Dr. Kristopher Nazor and Dr. Bertrand
Yeung of Spatial Genomics, Inc., Sharyl Crossley,
the only mineralized part of a jellyfishs anato-
my, the statoliths, small stones composed of
calcium sulfate subhydrate within the statocyst,
which functions in sensory balance to detect
positioning in the water (Becker et al., 2005;
Sötje et al., 2017). Winans and Purcell (2010)
reported that exposure of Aurelia labiata pol-
yps to pH 7.2 for 122 days gave rise to ephyrae
with smaller statoliths. Though the statoliths of
ephyrae released from polyps in experimental
tanks were not examined in this experiment, the
statoliths of Cassiopea sp. medusae were intact
following 66 days of treatment pH 7.0, suggest-
ing that the inability of the pH 7.0 subjects to
actively pulse or swim to settle was unlikely due
to dysfunctional balance machinery. Future work
should determine if pH reduction diminishes the
size of the statocyst or the number of statoliths
present.
Conclusions
Cassiopea sp., and possibly other scyphozo-
ans, are likely to be more resistant to OA to a pH
of 7.5 than many taxa. Our results indicate that
more severe OA to pH 7.0, which may be
possible in the future in parts of this species
range exposed to increasing CO2 pollution or
upwelling, would be lethal. While the polyp
stage of Cassiopea sp. can reproduce asexually
and even strobilate at pH 7.0, they do not always
produce a healthy new generation of Cassiopea
sp. Future OA research on gelatinous zooplank-
ton should implement experimental designs with
stepwise incremental pH reduction and longer
exposure times to each pH unit change with data
collected at each step for each stage of the life
cycle, including effects on gamete production
and planulae larvae and Symbiodiniaceae den-
sity. Incremental pH reduction not only mimics
the gradual change happening in nature, but also
negates the effects of shocking test subjects
by not providing enough time for them to accli-
mate to test conditions (sometimes seen in cap-
tive populations), and unintentionally skewing
results. Furthermore, since Cassiopea sp. are
found in shallow lagoons subject to high tem-
peratures, a known stressor of Symbiodiniaceae
19Severe seawater acidification and the scyphozoan medusa Cassiopeia sp.
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Responsible editor V.N. Ivanenko
