1. TEMPERATURE EFFECTS ON MARINE INVERTEBRATE PHYSIOLOGY
Stephanie Peramas, Hannah Sheehan, and Dr. Steven Irvine; University of Rhode Island
This material is based upon work supported in part by the National Science Foundation EPSCoR Cooperative Agreement #EPS-1004057.
Background
Due to the expected impact of global climate change, Rhode Island water temperatures are predicted
to rise by as much as 4℃. Studies on the native Rhode Island marine invertebrate Ciona intestinalis,
a species of sea squirts, indicate a potentially negative effect on the species’ reproductive success. In
this project, we looked at the impact of environmental stressors which might hinder the animal’s
embryonic development. This research was conducted by rearing local C. intestinalis animals in both
a projected stressed temperature of 22℃ and the high temperature limit for normal development of
18℃. We then carried out a cross fertilization and further analyzed the impact on embryonic
development.
We focused on the physical development of embryos by fixing them in formaldehyde and scoring
them based on five categories: 1) unhatched and uncleaved eggs, 2) unhatched and cleaved eggs,
3) hatched eggs with normal embryonic tails, 4) hatched eggs with kinked embryonic tails, and
5) hatched masses with no developed structure. Our preliminary studies suggest that C. intestinalis
reared at the elevated temperature produced far fewer viable embryos, often with little or no
development or altered development. However, specimens reared at the 18℃ oceanic temperature
more often produced viable embryos with normal development.
In addition, we conducted further “stress tests” based on modified pH and salinity levels consistent
with the expected effects on the oceanic environment due to global warming. Our results illustrate that
sea squirt embryos reared from animals at the stressed temperature of 22℃ are less likely to survive
exposure to other stressors (changes in pH and salinity, for example). Both of these stressors are
predicted effects of global warming. These results suggest that the reproductive success of the Ciona
intestinalis species will be hindered by increased water temperatures and other effects of global
warming.
Abstract
Results
Discussion & Conclusions
1. Lopez, C., 2016. Ocean Warming Effects on the Reproductive Proteome of Ciona intestinalis.
2. Lopez, C., 2016. Temperature Effects on the Proteome of Ciona intestinalis.
3. Figure 1: http://www.stefansiebert.photography/1930034-creatures-ii
4. Irvine, S.Q., 2016. Changes in the Ciona intestinalis ovarian proteome due to temperature stress.
References
Acknowledgements
• Sea squirt adults were collected
from South Kingstown and Newport,
Rhode Island and brought to tanks
set up at the University of Rhode
Island’s Graduate School of
Oceanography at the Narragansett
Bay Campus
• Tanks were equipped with chillers,
heaters, pumps, and airstones
• Tank 1 (see Figure 3) was set to 22
℃
• Tank 3 (see Figure 3) was set to 18
℃
• Temperature was recorded daily (℃)
• A tube pumped sea squirt food every
30 minutes
• Overhead lights kept the animals on a
12 hour light cycle until two days
before our experiments. During those
two days, lights were set to a 24 hour
cycle.
• Overhead hoses ran unfiltered sea
water into a head tank which fed into
Tank 1 and Tank 3
• Flow rate was monitored daily
• Animals reared in tanks at both 18℃ and 22℃
were left in constant light for a minimum of 24
hours
• The animals were then moved to the dark for
two hours where they built up internal gametes,
a natural response to light cycles
• The animals were then placed under lights again
where they then spawned naturally, releasing
both sperm and eggs from their atrial siphon
• Sperm and eggs from different test subjects
(18℃ and 22 ℃) were then crossed in a petri
dish, washed through a filter to remove
impurities, and allowed to fertilize in filtered sea
water for 15 minutes
• After eggs were fertilized, they were then placed
into both salinity and pH stress tests and
incubated at 18℃ for 24 hours
Aquarium Tank Setup
Methods
• Ciona intestinalis have an upper temperature limit
for normal reproduction of 18℃ in captivity
• Some global warming projections indicate a 4℃
increase in water temperatures over the next
century
• This projected increase in water temperature may
have a negative effect on the growth and
development of C. intestinalis embryos
• Along with this increase in temperature, there are
expected changes in salinity and pH levels
• This research analyzes the ability of C. intestinalis
embryos to develop normally under these altered
temperature, salinity and pH conditions
Figure 1. Ciona intestinalis
• Salinity tests were set up by adjusting filtered sea
water to four different salinities (26 ppt, 28 ppt, 30
ppt, and 32 ppt, and a control sample of filtered
sea water with an unmodified salinity of 36 ppt) by
adding both deionized water and filtered sea water
until the refractometer read out each of the five
respective salinities
• pH tests were set up by adjusting filtered sea water
to three different pHs: 7.2, 7.55, and the control pH
of 8.09 (pH of filtered sea water) by adding HCl
dropwise to filtered sea water until the pH meter
read out each of the three respective pHs
• After 24 hours in incubation, the embryos were
then fixed using formaldehyde (formalin- 37%)
• Embryos were then scored using a dissecting
microscope to assay how many embryos developed
normally and abnormally
Figure 6. Cross-fertilization diagram of
salinity stress test
Figure 7. Cross-fertilization diagram for pH
stress test
Figure 5. Tank arrangement including head tank at Narragansett
Bay Campus Graduate School of Oceanography
Figure 3. Graph of average percent normal embryos
drawn from salinity stress tests
Figure 2. a) Cleaved egg (labeled with arrow pointing to cleave) and an uncleaved egg, b) embryo with a normal tail, c) embryo with a
kinked tail, d) hatched mass with no developed structures.
2a. 2b. 2c. 2d.
Figure 4. Graph of average percent normal embryos
drawn from pH stress tests
For laboratory assistance and help monitoring our animals, we would like to thank Rose Jacobson, M.S., Evelyn Siler, Chelsea Lopez,
B.S., and Jay Grocott, B.S.
For monitoring our tanks when we were not present, we would like to thank Ed Baker, manager of seawater facilities.
Lastly, for helping us with organizing various events and for allowing us to have this opportunity, we would like to thank Jim Lemire in part
with EPSCoR Rhode Island.,
• Based on of our salinity stress tests (Figure 3), it appears that under a salinity close to that of
unfiltered sea water (32-33 ppt), animals reared at 18℃ have a 4.7% higher ability to produce
normal embryos compared to animals reared at 22℃
• When the 22℃ eggs were crossed with 18℃ sperm, the majority of embryos developed
normally (70.7%)
• This data shows that sperm from the 22℃ animals could be affected by a salinity of 32 ppt, as
0% produced normal embryonic development when crossed with 18℃ eggs
• This data trend persists in the cross fertilization trails, with 18℃ sperm showing a higher
percent of normal embryonic development than the 22℃ sperm
• At our control pH of 8.09, the 22℃ sperm and eggs produced more normal embryos than the
18℃ sperm and eggs
• The testing indicates that the 18℃ sperm produced less normal embryos than the 22℃ sperm,
the opposite pattern indicated in our salinity test
• Under a moderately varied pH of 7.55, the 22℃ sperm and eggs again produced more
normally developed embryos
• Under a more extreme drop from the control pH at a pH of 7.2, the 18℃ sperm and eggs were
found to produce 20% more normal embryos
45
25
58.8
27.8
63.5
76.35
41.2
74.1
0
20.85
37
1.9
0
10
20
30
40
50
60
70
80
90
18℃ EGGS X
18℃ SPERM
22℃ EGGS X
22℃ SPERM
18℃ EGGS X
22℃ SPERM
22℃ EGGS X
18℃ SPERM
PERCENT NORMAL EMBRYOS
PERCENT NORMAL EMBRYOS UNDER VARIED PH
pH 7.2 pH 7.55 pH 8.09
0.0
2.7
0.0
0.0
0.0
2.3
35.5
0.0
7.4
5.7
6.3
66.7
50.0
47.8
0.0
70.7
45.3
78.6
57.1
0
10
20
30
40
50
60
70
80
90
18℃ EGGS X
18℃ SPERM
22℃ EGGS X
22℃ SPERM
18℃ EGGS X
22℃ SPERM
22℃ EGGS X
18℃ SPERM
PERCENT NORMAL EMBRYOS
26 ppt 28 ppt 30 ppt 32 ppt 36 ppt
PERCENT NORMAL EMBRYOS UNDER VARIED SALINITY
Climate change predictions indicate oceanic changes in
temperature, pH, and salinity levels. Preliminary data suggests that
alterations in either pH levels or salinity levels can result in
abnormal embryonic development. Our data not only supports part
of this hypothesis, but goes on to suggest that sea squirts reared at
the elevated water temperatures (22℃) predicted by global
warming could be more likely to exhibit abnormal embryonic
development than animals reared in their typical water temperatures
(18℃). Our data suggests that the forecasted, elevated oceanic
temperature of 22℃ poses an environmental stress on sea squirt’s
reproduction abilities by inhibiting their normal embryonic
development when reared in an altered salinity. However, the data
indicates that pH may not negatively affect normal embryonic
development in animals reared in elevated temperature. This study
does suggest that the environmental stresses of global warming,
including altered temperature, pH, and salinity could hinder C.
intestinalis reproduction by impairing their embryonic development,
leading to abnormal or undeveloped embryos. However, repeated
trials must be done in order to further support this hypothesis.
Tank 1 Tank 3Head Tank