SURF Poster SP HS Final_SI

TEMPERATURE EFFECTS ON MARINE INVERTEBRATE PHYSIOLOGYStephanie 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. intestinalisreared 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 Cionaintestinalis 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℃

• Temperaturewasrecordeddaily(℃)• Atubepumpedseasquirtfoodevery

30minutes• Overheadlightskepttheanimalsona

12hourlightcycleuntiltwodaysbeforeourexperiments.Duringthosetwodays,lightsweresettoa24hourcycle.

• OverheadhosesranunfilteredseawaterintoaheadtankwhichfedintoTank1andTank3

• Flowratewasmonitoreddaily

• Animalsrearedintanksatboth18℃ and22℃wereleftinconstantlightforaminimumof24hours

• Theanimalswerethenmovedtothedarkfortwohourswheretheybuiltupinternalgametes,anaturalresponsetolightcycles

• Theanimalswerethenplacedunderlightsagainwheretheythenspawnednaturally,releasingbothspermandeggsfromtheiratrialsiphon

• Spermandeggsfromdifferenttestsubjects(18℃ and22 ℃)werethencrossedinapetridish,washedthroughafiltertoremoveimpurities,andallowedtofertilizeinfilteredseawaterfor15minutes

• Aftereggswerefertilized,theywerethenplacedintobothsalinityandpHstresstestsandincubatedat18℃ for24hours

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. intestinalisembryos to develop normally under these altered temperature, salinity and pH conditions

Figure 1. Ciona intestinalis

• Salinitytestsweresetupbyadjustingfilteredseawatertofourdifferentsalinities(26ppt,28ppt,30ppt,and32ppt,andacontrolsampleoffilteredseawaterwithanunmodifiedsalinityof36ppt)byaddingbothdeionizedwaterandfilteredseawateruntiltherefractometerreadouteachofthefiverespectivesalinities

• pHtestsweresetupbyadjustingfilteredseawatertothreedifferentpHs:7.2,7.55,andthecontrolpHof8.09(pHoffilteredseawater)byaddingHCldropwisetofilteredseawateruntilthepHmeterreadouteachofthethreerespectivepHs

• After24hoursinincubation,theembryoswerethenfixedusingformaldehyde(formalin- 37%)

• Embryoswerethenscoredusingadissectingmicroscopetoassayhowmanyembryos developednormallyandabnormally

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 NarragansettBay 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.,

• Basedonofoursalinitystresstests(Figure3),itappearsthatunderasalinityclosetothatofunfilteredseawater(32-33ppt),animalsrearedat18℃ havea4.7%higherabilitytoproducenormalembryoscomparedtoanimalsrearedat22℃

• Whenthe22℃ eggswerecrossedwith18℃ sperm,themajorityofembryosdevelopednormally(70.7%)

• Thisdatashowsthatspermfromthe22℃ animalscouldbeaffectedbyasalinityof32ppt,as0%producednormalembryonicdevelopmentwhencrossedwith18℃ eggs

• Thisdatatrendpersistsinthecrossfertilizationtrails,with18℃ spermshowingahigherpercentofnormalembryonicdevelopmentthanthe22℃ sperm

• AtourcontrolpHof8.09,the22℃ spermandeggsproducedmorenormalembryosthanthe18℃ spermandeggs

• Thetestingindicatesthatthe18℃ spermproducedlessnormalembryosthanthe22℃ sperm,theoppositepatternindicatedinoursalinitytest

• UnderamoderatelyvariedpHof7.55,the22℃ spermandeggsagainproducedmorenormallydevelopedembryos

• UnderamoreextremedropfromthecontrolpHatapHof7.2,the18℃ spermandeggswerefoundtoproduce20%morenormalembryos

45

25

58.8

27.8

63.5

76.35

41.2

74.1

0

20.85

37

1.9

0

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50

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70

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90

18℃ EGGSX18℃ SPERM

22℃ EGGSX22℃ SPERM

18℃ EGGSX22℃ SPERM

22℃ EGGSX18℃ SPERM

PERC

ENTNORM

ALEMBR

YOS

PERCENTNORMALEMBRYOSUNDER VARIED PH

pH7.2 pH7.55 pH8.09

0.0

2.7

0.0

0.0

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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

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18℃ EGGSX18℃ SPERM

22℃ EGGSX22℃ SPERM

18℃ EGGSX22℃ SPERM

22℃ EGGSX18℃ SPERM

PERC

ENTNORM

ALEMBR

YOS

26ppt 28ppt 30ppt 32ppt 36ppt

PERCENTNORMALEMBRYOSUNDER 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