Oceans Acidic and Low in Oxygen: Lessons from Estuarine

Organisms

Lou Burnett & Karen Burnett BurnettL@cofc.edu, BurnettK@cofc.edu

Grice Marine Lab Hollings Marine Lab

Points covered

High CO2 and Low O2 often occur together

Lesson 1: oysters Lesson 2: crustaceans

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http://www.riverlaw.us/fishkills.html

http://thewatchers.adorraeli.com/

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http://www.wlox.com/story/22730148/jubilee-fish-kill-lures-families-to-beach-for-easy-to-catch-seafood

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http://www.oceanservice.noaa.gov/education/kits/estuaries/media/supp_estuar10d_disolvedox.html

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World-wide Oxygen Levels

Diaz & Rosenberg 2008 Science

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Low oxygen, the insidious killer…………….. Nearly always accompanied by elevated CO2.

Oxygen Levels

Lerberg et al. 2000 Estuaries

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Oxygen and CO2

Data provided by Phil Dustan, CofC; published in Burnett 1997 Am. Zool.

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11 redrawn from Melzner et al. 2013 Mar Biol

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Melzner et al. 2013 Mar Biol

13 redrawn from Melzner et al. 2013 Mar Biol

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Dissolved oxygen and pH co-vary

Taken from “Acidification in coastal waters – an example from Waquoit Bay, Massachusetts” Daniel C. McCorkle Woods Hole Oceanographic Institution NAML “Briefing on the Hill” October 2014 http://www.naml.org/policy/briefings.php

Ocean Surface Water

pH CO2 (µatm) preindustrial 8.2 250 present 8.1 390 2100 7.7 – 7.8 700-1000

hypercapnia = ↑CO2

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Coastal South Carolina Water

pH CO2 (µatm) preindustrial 8.2 250 present 8.1 390 2100 7.7 – 7.8 700-1000 present 6.0-8.1 300->20,000

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hypercapnia = ↑CO2

Cochran & Burnett 1996 JEMBE http://cdmo.baruch.sc.edu/

How to understand effects

Habitat Natural history

life cycles habits of organisms

oysters close and are exposed to air crabs and shrimp are active; move and migrate

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Crassostrea virginica

Lesson 1: Oysters

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Dwyer & Burnett 1996 Biol. Bull. Allen & Burnett 2008 JEMBE

That’s CO2 at 20,000 µatm!

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[Ca+2] = 4 mM 11 mM

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Dwyer & Burnett 1996 Biol. Bull. Allen & Burnett 2008 JEMBE

Boyd & Burnett 1999 J. Exp. Biol.

Reactive O2 Intermediate Production in Oyster Hemocytes

+hypercapnia

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Milardo Thesis, CofC

“Dermo” Oyster Disease In vitro growth

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24 Milardo Thesis, CofC

“Dermo” Oyster Disease In vitro growth

Oyster Take-Home Lesson

Being hot and air exposed has some disadvantages.

Intertidal air exposure imparts some protection against “Dermo.”

Some field observations support this.

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A note on larval biology

bivalve larvae are sensitive to both hypercapnia and hypoxia

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Treatment “Ambient” CO2 0.25 torr 0.035 kPa 354 µatm

Treatment “Mid” CO2 0.49 torr 0.066 kPa 651 µatm

Treatment “High” CO2 1.14 torr 0.152 kPa 1,500 µatm

%

Surv

ival

veliger pediveliger metamorphosed

%

Surv

ival

%

Su

rviv

al

hard clam Mercenaria mercenaria

bay scallop Argopecten irradians

Eastern oyster Crassostrea virginica

Days Talmage & Gobler 2009 L&O

Oyster Settlement in Hypoxia

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Baker & Mann 1992 Biol Bull

What about crustaceans?

Atlantic Blue Crab Callinectes sapidus

Pacific Whiteleg Shrimp Litopenaeus vannamei

Lesson 2

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Shrimp Survival

Mikulski et al. 2000 JEMBE 30

Shrimp Survival

Mikulski et al. 2000 JEMBE 31

Hemocytes are abundant

gills of shrimp – Litopenaeus vannamei

10 to 50 x 106 mL-1

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lamellae

Photos Courtesy of Don Lovett

Lamellar Gills

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Gill Lamella

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seawater

seawater

hemolymph

Photo Courtesy of Don Lovett

Gill Lamella

hemocyte aggregation

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seawater

seawater

hemolymph

Elimination of Hemocytes

hemocyte aggregation

Jennifer Ikerd

36 Photo Courtesy of Don Lovett

Immune response has some down sides!

Immune response itself decreases O2 transport hemocytes clog gills ↑ vascular resistance ↓ oxygenation at gills ↓ O2 uptake ↓ exercise performance

Burnett et al. 2006 Biol. Bull. Marissa Roman, REU student 2012

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Immune responses & other functions are affected by hypoxia and hypercapnia!

Boleza et al. 2001 Fish Shellfish Immunol. Burgents et al. 2005 Biol. Bull. Tanner et al. 2006 Comp. Biochem. Physiol. Macey et al. 2008 Fish Shellfish Immunol. Hardy et al. 2013 Am. J. Physiol.

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Shrimp on a Treadmill

David Scholnick Pacific University

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Blue Crab Exercise

Thibodeaux et al. 2009 J. Exp. Biol. 40

Walking Exercise

H2O

Stover et al. 2013 Biol. Bull. 41

Walking Exercise

42 Stover et al. 2013 Biol. Bull.

Fatigue Behaviors in Blue Crabs

20% air sat.

50% air sat.

100% air sat.

43 Stover et al. 2013 Biol. Bull.

Fatigue Behaviors in Blue Crabs

20% air sat.

50% air sat.

100% air sat.

50% air sat. + 2% CO2

44 Stover et al. 2013 Biol. Bull.

Adaptations to Low Oxygen are Muted by CO2

Organisms respond to chronic low oxygen in similar ways. ↑ concentration of respiratory pigment

(e.g., hemoglobin, hemocyanin) ↑ oxygen affinity of pigment ↑ vascularization of tissues

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Effects of Hypoxia on the Atlantic Blue Crab, Callinectes sapidus

25 days at Po2 = 30-35% air saturation

Normoxia Hypoxia

Hemocyanin (g mL-1) 3.11 4.40

Oxygen affinity Increase in affinity due to lactate urate calcium

Increase in affinity due to structural change in

hemocyanin

deFur et al. 1990 Biol. Bull.

Hemocyanin O2 Affinity

Po2 (torr)

%O

2 Sa

tura

tion

100

50

0

• P50 the Po2 required to half saturate Hc with O2

• High affinity in hypoxia = good!

P50 is a measure of O2 affinity

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Hemocyanin O2 Affinity

pH

Log

P50

(to

rr)

high affinity

low affinity

better for hypoxia

not so good for hypoxia

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CO2-specific Effect

P 50

(tor

r)

pH

Mangum & Burnett 1986 Biol. Bull.

Pco2 1.5 torr

Pco2 15 torr

large ↑ in O2 affinity due to CO2

Atlantic blue crab Callinectes sapidus

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Transcriptomic Responses

News Flash… Hypoxia-induced production of hemocyanin is muted by CO2!

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CO2 Turns Off Hemocyanin Synthesis

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Construct Stranded Library

~400 bp

Sequenced Transcriptome (Illumina)

RNAseq

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6

6

6

6

6

4 h 24 h

Normoxia

Hypoxia

Hypercapnic Hypoxia

Gene networks

Hemocyanin Regulation

High Throughput RNA Sequencing (RNA-Seq)

Johnson et al., in revision, Physiological Genomics

Lessons from Estuarine Organisms

life in O2 and CO2 environments more extreme and more variable than the open ocean

immune functions are compromised by hypoxia and made worse by hypercapnia

immune response itself reduces performance performance is strained by hypoxia hemocyanin function may be limited by hypercapnia molecular approaches will provide answers to many

questions!!! 52

Support

NSF Karen & Lou Burnett & Summer REU Program

Oyster Disease Research Program (NOAA)

US Dept. of Agriculture

SC Sea Grant

Oceans & Human Health Initiative (NOAA)

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Oceans Acidic and Low in Oxygen: Lessons from Estuarine

Organisms Lou Burnett & Karen Burnett

BurnettL@cofc.edu, BurnettK@cofc.edu http://burnettl.people.cofc.edu

Grice Marine Lab Hollings Marine Lab

Citations Allen, S. M., and L. E. Burnett. 2008. The effects of intertidal air exposure on the respiratory physiology and

the killing activity of hemocytes in the Pacific oyster, Crassostrea gigas (Thunberg). J. Exp. Mar. Biol. Ecol. 357:165-171.

Baker, S. M., and R. Mann. 1992. Effects of hypoxia and anoxia on larval settlement, juvenile growth, and juvenile survival of the oyster, Crassostrea virginica. Biol. Bull. 182:265-269.

Boleza, K. A., L. E. Burnett, and K. G. Burnett. 2001. Hypercapnic hypoxia compromises bactericidal activity of fish anterior kidney cells against opportunistic environmental pathogens. Fish Shellfish Immunol. 11:593-610.

Boyd, J. N., and L. E. Burnett. 1999. Reactive oxygen intermediate production by oyster hemocytes exposed to hypoxia. J. Exp. Biol. 202:3135-3143.

Burgents, J. E., K. G. Burnett, and L. E. Burnett. 2005. Effects of hypoxia and hypercapnic hypoxia on the localization and the elimination of Vibrio campbellii in Litopenaeus vannamei, the Pacific white shrimp. Biol. Bull. 208:159-168.

Burnett, L. E. 1997. The challenges of living in hypoxic and hypercapnic aquatic environments. Am. Zool. 37:633-640.

Burnett, L. E., J. D. Holman, D. D. Jorgensen, J. L. Ikerd, and K. G. Burnett. 2006. Immune defense reduces respiratory fitness in Callinectes sapidus, the Atlantic blue crab. Biol. Bull. 211:50-57.

Cameron, J. N. 1978. Effects of hypercapnia on blood acid-base status, NaCl fluxes, and trans-gill potential in freshwater blue crabs Callinectes sapidus. J. Comp. Physiol. 123:137-141.

Cochran, R. E., and L. E. Burnett. 1996. Respiratory responses of the salt marsh animals, Fundulus heteroclitus, Leiostomus xanthurus, and Palaemonetes pugio to environmental hypoxia and hypercapnia and to the organophosphate pesticide, azinphosmethyl. J. Exp. Mar. Biol. Ecol. 195:125-144.

deFur, P. L., C. P. Mangum, and J. E. Reese. 1990. Respiratory responses of the blue crab Callinectes sapidus to long-term hypoxia. Biol. Bull. 178:46-54.

Diaz, R. J., and R. Rosenberg. 2008. Spreading dead zones and consequences for marine ecosystems. Science 321:926-929.

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Citations Dwyer, J. J., III, and L. E. Burnett. 1996. Acid-base status of the oyster Crassostrea virginica in response

to air exposure and to infections by Perkinsus marinus. Biol. Bull. 190:139-147. Hardy, K. M., K. G. Burnett, and L. E. Burnett. 2013. The effect of hypercapnic hypoxia and bacterial

infection (Vibrio campbellii) on protein synthesis rates in the Pacific whiteleg shrimp, Litopenaeus vannamei. Am. J. Physiol.-Reg. I. 305:R1356-R1366.

Lerberg, S. B., A. F. Holland, and D. M. Sanger. 2000. Responses of tidal creek macrobenthic communities to the effects of watershed development. Estuaries 23:838-853.

Macey, B. M., I. O. Achilihu, K. Burnett, and L. Burnett. 2008. Effects of hypercapnic hypoxia on inactivation and elimination of Vibrio campbellii in the Eastern oyster, Crassostrea virginica. Appl. Environ. Microbiol. 74:6077-6084.

Mangum, C. P., and L. E. Burnett. 1986. The CO2 sensitivity of the hemocyanins and its relationship to Cl- sensitivity. Biol. Bull. 171:248-263.

Melzner, F., J. Thomsen, W. Koeve, A. Oschlies, M. Gutowska, H. Bange, H. Hansen, and A. Körtzinger. 2013. Future ocean acidification will be amplified by hypoxia in coastal habitats. Mar. Biol. 160:1875-1888.

Mikulski, C. M., L. E. Burnett, and K. G. Burnett. 2000. The effects of hypercapnic hypoxia on the survival of shrimp challenged with Vibrio parahaemolyticus. J. Shellfish Res. 19:301-311.

Rathburn, C. K., N. J. Sharp, J. C. Ryan, M. Nealy, M. Cook, R. W. Chapman, L. E. Burnett, and K. G. Burnett. 2013. Transcriptomic responses of juvenile Pacific whiteleg shrimp, Litopenaeus vannamei, to hypoxia and hypercapnic hypoxia. Physiol. Genomics 45:794-807.

Scholnick, D. A., K. G. Burnett, and L. E. Burnett. 2006. Impact of exposure to bacteria on metabolism in the penaeid shrimp Litopenaeus vannamei. Biol. Bull. 211:44-49.

Stover, K. K., K. G. Burnett, E. J. McElroy, and L. E. Burnett. 2013a. Locomotory fatigue and size in the Atlantic blue crab, Callinectes sapidus. Biol. Bull. 224:63-67.

Stover, K. K., K. G. Burnett, E. J. McElroy, and L. E. Burnett. 2013b. Locomotory fatigue during moderate and severe hypoxia and hypercapnia in the Atlantic blue crab, Callinectes sapidus. Biol. Bull. 224:68-78.

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Citations Talmage, S. C., and C. J. Gobler. 2009. The effects of elevated carbon dioxide concentrations on the

metamorphosis, size, and survival of larval hard clams (Mercenaria mercenaria), bay scallops (Argopecten irradians), and Eastern oysters (Crassostrea virginica). Limnol. Oceanogr. 54:2072-2080.

Tanner, C. T., K. G. Burnett, and L. E. Burnett. 2003. Effects of hypoxia and pH on phenoloxidase activity in the blue crab, Callinectes sapidus. Integr. Comp. Biol. 43:1043-1043.

Thibodeaux, L. K., K. G. Burnett, and L. E. Burnett. 2009. Energy metabolism and metabolic depression during exercise in Callinectes sapidus, the Atlantic blue crab: effects of the bacterial pathogen Vibrio campbellii. J. Exp. Biol. 212:3428-3439.

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