1. The effect of pH on settlement and metamorphosis in response to adult cues in an
invasive gastropod
Tess C. Dooley1,2 and Anthony Pires1,3
1. Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250
2. Department of Biology, Grice Marine Laboratory, College of Charleston, Charleston SC 29412;
dooleytc@cofc.edu
3. Department of Biology, Dickinson College, Carlisle PA17013
Abstract
The average pH of ocean surface waters has dropped by about
30%, due to absorption of anthropogenic CO2 emissions. In the
past decade, much research has been conducted examining the
effect of this “ocean acidification” on marine organisms. Larvae
seem especially sensitive. Acidification has been shown to affect
chemosensory mechanisms and behavior of clownfish larvae, but
little is known about how acidification may affect the sensory
biology of marine invertebrate larvae. Like many marine
invertebrates, the gastropod Crepidula fornicata settles and
metamorphoses in response to chemical cues associated with
favorable habitat for juveniles. Other studies on marine
invertebrates have found decreased settlement and
metamorphosis at lower pH, but none have measured how pH
affects settlement and metamorphosis in response to cues from
adult conspecifics. We tested the effect of pH on settlement and
metamorphosis of four broods of larvae of C. fornicata that were
derived from different parents. pH had a significant overall effect
on both settlement and metamorphosis in the presence of adults,
but not in the direction expected. Larvae settled and
metamorphosed at higher frequency at pH 7.5 and 7.7 than at pH
8.0. While this pattern was seen in three of the four broods tested,
response to pH treatment varied between broods. Further
research is needed to determine if differences between broods
remain consistent throughout the larval period, and might
therefore reflect adaptations to variable pH regimes in estuarine
environments.
1 hr
Juvenile
Larva
OR
pH treatments: 7.5, 7.7, 8.0
@ 20oC
400µm
Juvenile Velum
Introduction
• Ocean acidification (OA) is a decline in pH and
available carbonate resulting from the production
of hydrogen ions and bicarbonate as CO2 reacts
with seawater:
• OA impairs the ability of larval clownfish to detect
suitable habitat for settlement and to avoid
predators by affecting their response to cues1,2
• Chemical cues from conspecific adults induce
settlement and metamorphosis in Crepidula
fornicata3
• Native to the Atlantic coast of the U.S. and invasive
in the Pacific Northwest, Europe and Japan4
• First introduced into the Puget Sound in
Washington more than 80 years ago4
• Puget Sound experiences highly variable pCO2
5
References
1. Munday, P. L., Dixson, D. L., Donelson, J. M., Jones, G. P., Pratchett, M. S., Devitsina, G. V and Døving, K. B. 2009. Ocean
acidification impairs olfactory discrimination and homing ability of a marine fish. Proc. Natl. Acad. Sci. U. S. A. 106: 1848–
1852.
2. Dixson, D. L., Munday, P. L. and Jones, G. P. 2010. Ocean acidification disrupts the innate ability of fish to detect predator
olfactory cues. Ecol. Lett. 13: 68–75.
3. McGee, B. L. and Targett, N. M. 1989. Larval habitat selection in Crepidula (L.) and its effect on adult distribution patterns.
J. Exp. Mar. Bio. Ecol. 131: 195–214.
4. Blanchard, M. 1997. Spread of the slipper limpet Crepidula fornicata (L. 1758) in Europe. Current state and consequences.
Sci. Mar. 61: 109–118.
5. PMEL Carbon Program. 2015. Twanoh. [Online]. NOAA. Available: http://www.pmel.noaa.gov/co2/story/Twanoh [2015,
Aug. 4].
Predictions
1. Settlement will decrease as pH decreases
2. Metamorphosis will decrease as pH
decreases
1. Culturing:
a. Brooded larvae released
by adults
b. 500 larvae collected
(~400mm)
c. Larvae fed for two
weeks (Isochrysis)
2. Metamorphosis Assay
a. Once competent (~900-
1200mm), 20 larvae
placed in treatment tank
with adult stack for one
hour
b. Larvae and juveniles
collected and counted
Figure 1. Adult C. fornicata were collected from Totten Inlet, Puget Sound, WA. Assays were
performed in the treatment tanks at the Ocean Acidification Environmental Laboratory at
Friday Harbor Laboratories. At the end of the assays, larvae and juveniles were counted and
settlement and metamorphosis were quantified: Settlement: all individuals on the adult
when removed from container; Metamorphosis: complete loss of velum.
Methods
http://i688.photobucket.com/albums/vv244/stargate985/IMG_9094.jpg
Results
Figure 2. Rates of (A) settlement and (B) metamorphosis of C. fornicata for four different
broods. Comparisons made using ANOVA and Tukey multiple comparison intervals (a = 0.05).
Different letters between treatments in Brood 9 indicate statistical differences. Asterisk (*)
indicates statistical difference between treatments. Error bars represent +1 SEM.
7.5 7.7 8.0
0
20
40
60
80
100
pH Treatment
Metamorphosis(%)
Brood 8
Brood 9
Brood 12
Brood 16
*
a a
b
7.5 7.7 8.0
0
20
40
60
80
100
pH Treatment
Settlement(%)
*
a
b
a
A B
7.5 7.7 8.0
pHTotal 7.48-7.54 7.66-7.74 7.91-8.03
Total Alkalinity (mmol kgsw
-1) 2103-2124 2101-2114 2100-2115
pCO2 (matm) 1405-1613 853-1009 422-532
Wcalcite 1.17-1.32 1.77-2.08 2.96-3.58
Waragonite 0.75-0.85 1.14-1.33 1.91-2.30
Table 1. Range of seawater properties during metamorphosis assays. pCO2, Wcalcite and
Waragonite calculated from the measured values of total alkalinity (TA), pH, temperature, and
salinity.
Results summary
1. pH significantly affected settlement (ANOVA,
p = 0.032; Figure 1A)
a. Settlement was significantly lower at pH 8.0
than at 7.5 (Tukey multiple comparison, p =
0.025)
b. Brood 9 was the only brood where pH had a
significant effect. Settlement was significantly
lower at pH 8.0 than at 7.5 or 7.7 (Tukey
multiple comparison, p < 0.01)
2. pH significantly affected metamorphosis (ANOVA,
p = 0.035; Figure 1B)
a. Metamorphosis was significantly lower at pH 8.0
than at 7.7 (Tukey multiple comparison, p =
0.038)
b. Brood 9 was the only brood where pH had a
significant effect. Metamorphosis was
significantly lower at pH 8.0 than at 7.5 or 7.7
(Tukey multiple comparison, p < 0.005)
Discussion
• The effect of pH on settlement and metamorphosis
was the opposite of what was predicted
• Settlement and metamorphosis were robust across
the range of pH tested, which are pH we would
expect to occur during the reproductive period in
Totten Inlet
• High variability between broods: due to differences
between broods or unknown variation?
• Future Research:
o Test the limits of their tolerance to pH
o Understand the physiology that enables this
tolerance
o Test broods multiple times to determine if
response is repeatable within a brood
Acknowledgements
I would like to thank Taylor Shellfish Company for providing us with access
to the adult C. fornicata used in this experiment. I thank Tony Pires for
mentoring me in this project. I would also like to thank Sam Bogan,
Christine Choy, Michele Mei, Rob Burns, and Jan Pechenik for their
assistance in the lab. This study was funded by the National Science
Foundation (NSF CRI-OA-1416690) and the Blinks-NSF REU-BEACON
program (DBI 1262239).