2004 Progress Report: Individual Level Indicators: Molecular Indicators of Dissolved Oxygen Stress in Crustaceans

EPA Grant Number: R829458C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R829458
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: EAGLES - Consortium for Estuarine Ecoindicator Research for the Gulf of Mexico
Center Director: Brouwer, Marius
Title: Individual Level Indicators: Molecular Indicators of Dissolved Oxygen Stress in Crustaceans
Investigators: Brouwer, Marius , Denslow, Nancy
Institution: University of Southern Mississippi , University of Florida
Current Institution: University of Southern Mississippi
EPA Project Officer: Hiscock, Michael
Project Period: December 1, 2001 through November 30, 2005 (Extended to May 20, 2007)
Project Period Covered by this Report: December 1, 2003 through November 30, 2004
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000) RFA Text |  Recipients Lists
Research Category: Water , Ecosystems , Ecological Indicators/Assessment/Restoration


Occurrence of hypoxia in estuarine waters is increasing, and recovery of estuaries, once impacted, is slow. Detection of early effects of hypoxia is needed for timely remedial action to be taken. We have examined the use of hypoxia-responsive gene expression profiles in grass shrimp, Palaemonetes pugio, as early warning signals of impacts of hypoxia.

The objectives of this component of the Consortium for Estuarine Ecoindicator Research for the Gulf of Mexico (CEER-GOM) for Year 3 of the project are to: (1) develop DNA macroarrays and antibodies for use in detection of expression of dissolved oxygen (DO) stress genes and proteins in grass shrimp; (2) test the response of the molecular indicators to chronic hypoxia and diurnal DO cycles in grass shrimp under controlled laboratory conditions; (3) validate gene expression using real-time PCR; (4) determine if the molecular signals can be used as predictive indicators of reduced fitness (condition factor and reproduction) in response to DO stress in grass shrimp under controlled laboratory experiments; (5) characterize DO and salinity parameters in Weeks Bay and Mobile Bay during the 2004 field season; (6) validate response of the DO stress indicators in grass shrimp from hypoxic and reference sites in two CEER-GOM targeted estuaries; (7) compare annual and seasonal variation of stress indicators; and (8) determine reproductive status of field-collected grass shrimp.

Progress Summary:

Macroarrays were constructed using 75 potentially hypoxia-responsive genes, as determined through suppressive subtractive hybridization and direct cloning. There were no robust indicators of hypoxia found in grass shrimp exposed to chronic, moderate (2.5 ppm DO) hypoxia under controlled laboratory conditions, with the exception of strong downregulation of cytosolic manganese superoxide dismutase after 14 days. In contrast, grass shrimp exposed to chronic, severe (1.5 ppm DO) hypoxia showed significant (p < 0.05) upregulation of genes coding for hemocyanin, ATP synthase d and f chains, cytochrome b, and troponin C and I after only 3-day exposure, suggesting an attempt to increase oxygen uptake/transport (hemocyanin), ATP synthesis (ATP synthase and cytochrome b), and locomotion (troponin C and I). After 7-day exposure to chronic, severe hypoxia, the adaptation induced by day 3 becomes insufficient and ATP synthase, hemocyanin, and troponin are no longer upregulated. The major response after 7 days is a significant increase of transcription of genes in the mitochondrial genome (16S mitochondrial rRNA, cytochrome c oxidase 1, and cytochrome b), as well as upregulation of ferritin and heme binding protein. By day 14, a dramatic reversal of this expression pattern is seen, with a significant downregulation of transcription of genes in the mitochondrial genome (16S rRNA, cytochrome c oxidase 1 and 3, and cytochrome b) as well as ferritin. Whether this apparent decrease in transcription levels reflects a “true” downregulation of mitochondrial genes transcription, or an overall decrease in mitochondrial biogenesis, is unknown. In summary, the primary response to severe hypoxia (after 3 days) is upregulation of hemocyanin and ATP synthase synthesis, followed by upregulation of mitochondrial gene transcription after 7 days and downregulation of mitochondrial protein synthesis (or mitochondrial biogenesis) after 14 days. These results suggest grass shrimp respond to hypoxia in a time-dependent manner, and the up- or down-regulation of specific genes can lend insight into the hypoxia history of grass shrimp. Grass shrimp exposed to cyclic hypoxic conditions in the field showed two discernable patterns of changes in gene transcription: (1) significant downregulation of mitochondrial gene transcription indicative of long-term hypoxia exposure at some locations; and (2) downregulation of hemocyanin gene expression by late summer at nearly all locations. These expression profiles returned to “normal” in late fall, suggesting a transition from an oxygen-independent, hypometabolic state in late summer to oxidative metabolism in late fall. Furthermore, hypoxia appears to negatively impact grass shrimp reproduction, as females exposed to chronic moderate hypoxia in the laboratory had a longer interbrood-interval than normoxia females. Female grass shrimp exposed to cyclic hypoxia in the laboratory had reduced fecundity and a longer interbrood interval than normoxic females, and grass shrimp collected from the field showed a lower percentage of gravid females at cyclic hypoxic versus normoxic sites. These results suggest that both whole animal and molecular responses to hypoxia can be combined to provide diagnostic and predictive tools for the identification of effects of hypoxia on estuarine crustacea at the individual and population level.

We have met most of our objectives for Year 3. Novel hypoxia-responsive genes of grass shrimp have been identified. Antibodies for HIF-1α have been developed and tested (Objective 1). Laboratory data for gene expression of chronic hypoxia exposures of grass shrimp have been completely analyzed, and several genes (hemocyanin, ATP synthase, 16S rRNA, cytochrome b, and cytochrome c oxidase) have been shown to be hypoxia responsive under laboratory conditions of severe chronic hypoxia. Data analysis is continuing for gene expression of grass shrimp exposed to cyclic hypoxia under laboratory conditions. Protein expression of HIF-1α has been unsuccessful; protein expression analysis of hemocyanin is ongoing (Objective 2). A new objective was added and completed this year, the validation of gene expression results using real-time PCR. Results showed alpha-tubulin is a good “normalizer” gene, whereas hemocyanin is a robust indicator of short-term severe, chronic hypoxia exposure (Objective 3). Results of whole-animal analysis show condition factor of grass shrimp does not differ with hypoxia exposure (Objective 4). Multiple brood studies in both chronic and cyclic hypoxia showed effects of hypoxia on reproduction. Shrimp exposed to chronic hypoxia took longer to produce their first brood than normoxic shrimp, yet offspring of hypoxia-exposed mothers survived longer than normoxic larvae. Shrimp exposed to cyclic hypoxia produced less broods than shrimp exposed to normoxia. Fecundity of the first brood was significantly lower than normoxic females and hypoxic females took a longer time to produce a second brood. The addition of a second estuarine system (Mobile/Weeks Bay) to the field component of the project required extensive water quality monitoring, and continuous DO data was produced showing cyclic hypoxia in marsh sites in Weeks Bay, as well as chronic hypoxia at some Mobile Bay stations. Additionally, water quality data during the occurrence of a jubilee in July was recorded (Objective 5). Finally, gene expression of field-caught grass shrimp shows similarities across years as well as across estuarine systems, with electron transport, ATP metabolism, and hemocyanin genes showing downregulation during late summer. Hypoxia also appears to impact grass shrimp reproduction in the field, as a lower percentage of females carrying eggs were captured at cyclic hypoxic sites compared to normoxic sites in both estuarine systems (Objective 6). During this year, we added a laboratory experiment (multiple brood study of grass shrimp in chronic DO), as well as expanded a planned laboratory experiment on cyclic DO to evaluate multiple brood production under cyclic hypoxia.

Future Activities:

The focus of the upcoming year will be to resolve problems with the hemocyanin protein analysis and analyze laboratory and field protein data, as well as to focus on a better understanding of gene expression data in cyclic hypoxia (laboratory and field). The addition of quantitative PCR (qPCR) analysis (Objective 3) was deemed necessary for validation of our gene arrays, and we plan to include additional qPCR work on other genes during the coming year to validate the cyclic DO experiments and the field data. Finally, inclusion of grass shrimp protein, gene expression, and reproduction data in a marsh model developed by other components of CEER-GOM will enhance our understanding of indicators of hypoxia in the marsh ecosystem.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 27 publications 5 publications in selected types All 4 journal articles
Other center views: All 171 publications 54 publications in selected types All 48 journal articles
Type Citation Sub Project Document Sources
Journal Article Brouwer M, Larkin P, Brown-Peterson N, King C, et al. Effects of hypoxia on gene and protein expression in the blue crab, Callinectes sapidus. Marine Environmental Research 2004;58(2-5):787-792. R829458C003 (2003)
R829458C003 (2004)
not available
Journal Article Brown-Peterson NJ, Larkin P, Denslow N, King C, Manning S, Brouwer M. Molecular indicators of hypoxia in the blue crab Callinectes sapidus. Marine Ecology Progress Series 2005;286:203-215. R829458C003 (2004)
R829458C003 (2005)
  • Abstract: Marine Ecology Progress Series
  • Supplemental Keywords:

    population, community, ecosystem, watersheds, estuary, estuaries, Gulf of Mexico, nutrients, hypoxia, innovative technology, biomarkers, water quality, remote sensing, geographic information system, GIS, integrated assessment, risk assessment, fisheries, conservation, restoration, monitoring/modeling, benthic indicators, ecological exposure, ecosystem monitoring, environmental indicators, environmental stress, estuarine ecoindicator, estuarine integrity,, RFA, Scientific Discipline, ECOSYSTEMS, Geographic Area, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, Ecology, Ecosystem/Assessment/Indicators, Ecosystem Protection, Aquatic Ecosystem, Aquatic Ecosystems, Ecological Effects - Environmental Exposure & Risk, Environmental Monitoring, Ecological Monitoring, Ecology and Ecosystems, Biology, Ecological Indicators, Gulf of Mexico, monitoring, ecoindicator, ecological exposure, molecular ecology, nutrient dynamics, estuaries, estuarine integrity, ecosystem assessment, crustaceans, hypoxia, ecological assessment, estuarine ecoindicator, environmental indicators, environmental stress, water quality, aquatic ecosystem restoration, dissolved oxygen

    Relevant Websites:

    http://www.usm.edu/gcrl/ceer_gom/ Exit

    Progress and Final Reports:

    Original Abstract
  • 2002 Progress Report
  • 2003 Progress Report
  • 2005 Progress Report
  • 2006
  • Final

  • Main Center Abstract and Reports:

    R829458    EAGLES - Consortium for Estuarine Ecoindicator Research for the Gulf of Mexico

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R829458C001 Remote Sensing of Water Quality
    R829458C002 Microbial Biofilms as Indicators of Estuarine Ecosystem Condition
    R829458C003 Individual Level Indicators: Molecular Indicators of Dissolved Oxygen Stress in Crustaceans
    R829458C004 Data Management and Analysis
    R829458C005 Individual Level Indicators: Reproductive Function in Estuarine Fishes
    R829458C006 Collaborative Efforts Between CEER-GOM and U.S. Environmental Protection Agency (EPA)-Gulf Ecology Division (GED)
    R829458C007 GIS and Terrestrial Remote Sensing
    R829458C008 Macrobenthic Process Indicators of Estuarine Condition for the Northern Gulf of Mexico
    R829458C009 Modeling and Integration