1. Uptake of anthropogenic carbon dioxide (CO2) by the ocean has led to a
30% drop in pH of surface waters since the start of the Industrial
Revolution. This process, known as ocean acidification (OA), results
when CO2 reacts with water to form carbonic acid. OA is of major
ecological concern because it interferes with pH-sensitive biological
processes, including fertilization. Recent research in our lab suggested
that predicted near-future(50-100 y) levels of atmospheric CO2 will
negatively affect gamete function (e.g., sperm speed, motility, and
fertilization success) in the free-spawning sea urchin, Arbacia
punctulata. However, a recent review noted that OA more weakly
affects fertilization in studies that mixed sperm and eggs from more
than one mating pair. To test the hypothesis that group spawning
reduces the negative effects of OA, we measured fertilization success
under 1x and 2.5x-current CO2 conditions using single-and multiple-
male crosses. Surprisingly, we did not find a significant effect of CO2 on
fertilization in single- or multiple male crosses and the latter did not
show greater resistance to the effects of increased CO2. However,
multiple-male crosses had significantly lower fertilization than the
average fertilization of the individual crosses involving the same males.
These results reflect some mechanism that reduces fertilization success
for females that spawn in larger aggregations, which could have
important implications for differences in optimal aggregation strategies
for males and females.
Fertilization in single- and multiple-male spawnings under elevated CO2:
implications for reproductive success in response to ocean acidification
Tess C. Dooley and Robert D. Podolsky
Department of Biology, Grice Marine Laboratory, College of Charleston, Charleston SC 29412; podolskyr@cofc.edu
Abstract
Introduction
• Gametes of free spawning organisms are subject to
ambient water conditions (e.g., salinity,
temperature, pH) during fertilization
• Ocean acidification (OA) is a decline in pH resulting
from the production of carbonic acid as CO2 reacts
with seawater:
H2O + CO2 ↔ H2CO3 ↔ 2H+ + CO3
-2
• Fertilization success in some sea urchins is strongly
affected by pH changes predicted for the near-
future (50 to 100 y)
• A recent review (Byrne 2011) noted that studies
that use single male-female crosses tend to show a
larger effect of OA on fertilization than those that
use gamete mixtures from multiple animals
• A suggested explanation is that particular gamete
combinations, made possible by multi-pair
spawnings, are better at resisting effects of OA
• We used a factorial breeding design to examine the
effect of single and multi-male crosses on
fertilization success and on their resistance to OA
Implications
Ecological effects of spawning density
• Our results suggest that females spawning among
many males could experience lower fertilization
success than among fewer males
• On the other hand, spawning among fewer males
could result in sperm limitation, suggesting that
intermediate aggregation densities may be optimal
Evolutionary implications for males and females
• Eggs and sperm are under different selection
pressures, with sperm competing to fertilize the
first egg they encounter and females acting to
counter multiple fertilization
• The mechanism for the multi-male effect is
unknown, but could involve interactions among
sperm or damage to eggs, consistent with these
different selection pressures
Predictions
1. Negative effect of increased pCO2 on fertilization
success
2. Effects of single- vs. multi-male crosses on
fertilization success:
a. Greater effect of pCO2 on single-male than on
multi-male crosses
b. No difference between multi-male vs. mean of
single-male crosses
3. Significant variation among males, among females,
and among male-female combinations in:
a. Fertilization success
b. Sensitivity of fertilization success to pCO2
Acknowledgements
We thank Emily Hall for invaluable assistance with animal collection and
experiments. We also thank the Office of Undergraduate Research and
Creative Activities at the College of Charleston for support through a
SURF Grant and the staff of Grice Marine Lab for assistance.
Results Summary
1. There was a significant effect of sperm concentration
on fertilization (Fig. 2; F2,441 = 1101, p < 0.001), but
no effect of pCO2 (Fig. 2; F1,441 = 3.11, p = 0.078)
2a. There was no significant interaction between pCO2
and male number (single- vs. multi-male cross) (F1,154
= 0.56, p = 0.45)
2b. Percent fertilization was lower in multi-male crosses
than the average of single-male crosses with the
same 3 males, especially at intermediate and low
sperm concentrations (Fig. 3; F1,154 = 25.4, p < 0.001)
3a. Effects of both male identity and female identity on
fertilization percentage were marginally non-
significant (Table 1). The interaction between male
and female identity was not significant
3b. There was no significant effect of male or female
identity or their interaction on the sensitivity of
fertilization success to pCO2 (Table 1)
Methods
15 min
KCl
2 h
fix
3 females x 3 males+sperm mix x 5 blocks
@ 3 [sperm] = 104.5, 105.0, 105.5 ml-1
x 2 [CO2] = 1x (392 ppm, current), 2.5x (980 ppm, future)
Count!
Literature Cited
Byrne, M. 2011. Impact of ocean warming and ocean acidification on
marine invertebrate life history stages: Vulnerabilities and
potential for persistence in a changing ocean. Oceanography and
Marine Biology: An Annual Review, 2011, 49, 1–42.
Fig 1. Each fertilization block used 3 male and 3 female Arbacia punctulata from Sullivan’s Is., SC
(total 5 blocks). Crosses involved single males or a mixture of equal portions of sperm from the 3
males at the same total concentration. Animals were spawned through injection of 0.53M KCl.
M1
M2
M3
M
1 2 3
F1 F2 F3
Arbacia punctulata
Male Female Male x Female
Fertilization Wald Z = 1.91, p = 0.056 Z = 1.93, p = 0.054 NS
Effect of CO2 NS NS NS
Results
Fig. 2. (A) Fertilization under 1x versus 2.5x pCO2 at 3 sperm concentrations. Dashed line
represents equal fertilization at the two pCO2. (B) Average fertilization percentages under 1x and
2.5x pCO2 at 3 sperm concentrations.
0
20
40
60
80
100
4.5 5.0 5.5
%fertilization
Log sperm ml-1
1x
2.5x
B
0
20
40
60
80
100
0 20 40 60 80 100
%fertilization@2.5xpCO2
% fertilization @ 1x pCO2
4.5
5.0
5.5
A log [sperm]
0
20
40
60
80
100
4.5 5.0 5.5
%fertilization
Log sperm ml-1
Average
Mixed
0
20
40
60
80
100
0 20 40 60 80 100
%fertilizationinmulti-malecrosses
% fertilization in single-male crosses
4.5
5.0
5.5
Fig. 3. (A) Average percent fertilization of 3 single-male crosses vs. the percent fertilization of the
multi-male cross using the same individuals at the 3 sperm concentrations. Dashed line represent
equal fertilization (assuming no difference) between the multi- male crosses and the average of
the 3 single-male crosses. All dots below the line indicate a decrease in percent fertilization for
multi-males versus the average of the 3 single-males. (B) Average fertilization percentages of the
average of single-male crosses versus the multi-male crosses at the 3 sperm concentrations.
A B
log [sperm]
Table 1. Effects of gamete quality (contributions of male and female phenotypes) and
compatibility (interaction between male and female phenotypes) on fertilization and on the
effects of CO2 on fertilization. NS = covariance parameter was redundant in mixed-effects model .
Conclusions
• Surprisingly, increased pCO2 did not appear to have
a negative effect on fertilization success
• A mixture of gametes from different males did not
alter the effects of OA on fertilization
• Gamete quality rather than gamete compatibility
appeared to play a role in fertilization success
• Involvement of multiple males had a detrimental
effect on fertilization success of females