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2003 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
Current Investigators: Brouwer, Marius , Denslow, Nancy
Institution:
Current Institution: University of Southern Mississippi
EPA Project Officer: Levinson, Barbara
Project Period: December 1, 2001 through November 30, 2005 (Extended to May 20, 2007)
Project Period Covered by this Report: December 1, 2002 through November 30, 2003
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000)
Research Category: Ecological Indicators/Assessment/Restoration

Description:

Objective:

The objectives for Year 2 of this research project were to: (1) develop DNA macroarrays and antibodies for use in detection of expression of dissolved oxygen (DO) stress genes and proteins in blue crabs and grass shrimp; (2) test the response of the molecular indicators to chronic hypoxia and diurnal DO cycles in blue crabs and grass shrimp under controlled laboratory conditions; (3) determine the variability in gene expression as it relates to endogenous, biological cycles in blue crab; (4) determine if molecular signals can be used as predictive indicators of reduced fitness (molting and reproduction) in grass shrimp in response to DO stress; and (5) validate response of the DO stress indicators in blue crab and grass shrimp from hypoxic and reference sites in Center for Estuarine Ecoindicator Research (CEER)-targeted estuaries and determine reproductive status of field-collected grass shrimp.

Progress Summary:

We have examined the use of hypoxia-responsive gene and protein expression profiles in the blue crab, Callinectes sapidus, and grass shrimp, Palaemonetes pugio, as early warning signals of impacts of hypoxia. We cloned 23 and 74 potential hypoxia-responsive genes of the blue crab and grass shrimp, respectively, which were used to construct gene macroarrays. Blue crabs exposed to chronic hypoxia (2.5 ppm DO) for 15 d showed significant (p < 0.05) changes in gene expression of heat shock protein 70 (Hsp70), copper metallothionein (CuMt3), cytosolic MnSOD (cyt-MnSOD), and ribosomal proteins S15 and L23. In all cases except for CuMt3, gene expression decreased after 5 d exposure to hypoxia. Expression of Hsp70, CuMt3, and cyt-MnSOD also increased (p < 0.05) in normoxic crabs held for 15 d, suggesting confounding effects from confinement stress. Hemocyanin protein concentrations changed significantly (p = 0.006) across the 15 d chronic hypoxia exposure. Hemocyanin in crabs exposed to 10 d intermittent hypoxia (2.5 ppm DO to 8 ppm DO over a 24-hour cycle) did not change, but cyt-MnSOD gene expression increased significantly (p = 0.037), whereas cytochrome c oxidase subunit 1 (ccox1) showed a 2.2-fold downregulation. Blue crabs collected from Pensacola Bay, FL, showed significant (p < 0.006) downregulation of ccox1 and cyt-MnSOD gene expression as well as hemocyanin protein levels at a diurnally hypoxic marsh site. Several hypoxia-responsive genes (elongation factor 2, cryptocyanin and hemocyanin) also were significantly elevated (p < 0.006) in intermolt versus premolt normoxic blue crabs. Grass shrimp fecundity was significantly elevated in animals exposed to chronic moderate (2-3 ppm DO, 6 weeks) or severe (1.5 ppm DO, 8 weeks) hypoxia compared to normoxic grass shrimp, and eggs from the 1.5 ppm DO females had significantly higher triglyceride levels than eggs from control females. Both quantity and quality of eggs produced under hypoxic conditions appear superior to eggs from normoxic conditions. Analysis of the gene expression profiles of the laboratory-exposed grass shrimp is in progress. The identification of hypoxia-responsive genes and proteins in the blue crab and grass shrimp is an important first step toward development of sensitive molecular tools for rapid detection of sublethal effects of hypoxia in estuarine-resident species. Studies with the grass shrimp suggest 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.

Objective 1: DNA Macroarrays and Antibodies

Blue Crab Gene Arrays. Thirteen additional hypoxia-responsive genes were identified through subtractive suppressive hybridization (SSH) using mRNA from normoxic crabs and crabs exposed to moderate hypoxia (2.5 ppm DO) for 5 days. Subtractions were run in both the forward and reverse directions to obtain both upregulated and downregulated genes. The genes obtained through SSH were sequenced and included cytochrome c oxidase subunit1 (ccox1), alpha-amylase, oxygenase, apolipoprotein A1, a 14 kDa apoliprotein, chitinase, chymotrypsin, trypsin, cathepsin, three additional hemocyanin genes, cryptocyanin, and elongation factor 2. The new genes as well as the 10 genes cloned during Year 1 of the project were spotted in duplicate onto expanded macroarrays for analysis of crabs from the diurnal hypoxia exposure study and crabs collected from the field.

Grass Shrimp Gene Arrays. Seventy-four potentially hypoxia-responsive genes have been identified from grass shrimp through SSH using mRNA from normoxic grass shrimp and grass shrimp exposed to moderate (2-3 ppm) and severe (1.5 ppm) DO. These genes represent a variety of specific biological functions that are impacted by hypoxic conditions, such as ATP metabolism, oxygen transport, protein synthesis, and gluconeogenesis. These genes, along with genes cloned in our laboratory during Year 1 of the project (beta-actin, ribosomal proteins S14 and S20, heat shock protein 70 (Hsp70), Cu metallothionein (CuMt3), mitochondrial and cytosolic manganese superoxide dismutase (mit-MnSOD, cyt-MnSOD), 2 hemocyanin subunits) and hypoxia inducible factor cloned in our laboratory this year, have been spotted in duplicate onto 500 macroarrays that will be used for analysis of the shrimp from laboratory and field exposures.

Cloning, Sequencing, and Antibody Preparation of Hypoxia Inducible Factor (HIF-1a) From Grass Shrimp. We have identified 625 amino acids of grass shrimp HIF-1a. The protein contains the classical H-L-H domain, two PAS domains, and a proline hydroxylation motif found in vertebrate HIF. We have selected the peptide that contains the hydroxylation motif as an antigen for antibody production. We expect to have antibodies by August 2004.

Objective 2: Effects of Chronic Hypoxia and Hypoxic Normoxic Cycles on Gene Expression in the Blue Crab and Grass Shrimp

Blue Crabs. Data analysis of the blue crab gene expression studies is complete. Ten genes were analyzed during the 15 days of chronic hypoxia (2.5 ppm DO), and 6 of those genes (Hsp70, CuMt3, mit-MnSOD, cyt-MnSOD, and ribosomal proteins S15 and L23) showed significant differences in regulation over the 15-day experiment (see Figure 1). Expression of all genes, except CuMt3, was lowest after 5 days of exposure to hypoxia.

Figure 1. Changes in Expression of Blue Crab Genes, Relative to Day 0, in Response to Chronic Hypoxia (2.5 ppm DO) for 15 Days Under Laboratory Conditions. Day 0 and day 15 controls represent crabs exposed to normoxia (8 ppm DO) at day 0 and day 15, respectively. Expression data were normalized to actin. Hsp70 not pictured due to large changes in relative expression. *Changes in ribosomal proteins S15 and L23, Hsp70, CuMT3, mit-MnSOD, and cyt-MnSOD were statistically significant across the time course of the experiment as determined by ANOVA (p < 0.05).

The significant differences in gene expression of Hsp70, CuMT3, and cyt-MnSOD may be partially due to confinement stress during the 15-day experiment because the expression of these genes also was upregulated significantly in the 15-day normoxic crabs compared to the 0 day normoxic crabs. Changes in expression of ribosomal S15 and L23 and mit-MnSOD appear to be a response to the hypoxia treatment only as there was not a significant difference in gene expression between normoxic crabs at day 0 and day 15. Real time PCR validated the use of actin for normalizing the arrays and validated the changes in gene expression of ribosomal S15 and mit-MnSOD.

As described in Objective 1, expanded macroarrays were constructed for analysis of the diurnal hypoxia data. The ccox1 gene appeared to be responsive to diurnal changes in DO and was downregulated 2.2-fold in crabs exposed to 10 days of diurnal hypoxia when compared to 10 day normoxic crabs, although this difference was not significant (t16=1.432; p=0.171). Levels of expression of cyt-MnSOD were significantly increased 1.6-fold (t14=2.296; p=0.037) and expression of both hemocyanin genes 1 and 2 was slightly, but not significantly, higher in crabs exposed to 10 days of diurnal hypoxia fluctuations when compared to normoxic controls. The results of the two laboratory studies suggest that several genes change in response to chronic as well as diurnally fluctuating DO, indicating that hypoxia-responsive macroarrays will be useful tools for monitoring effects of hypoxia in estuarine crustacea.

Grass Shrimp. Two separate exposures of grass shrimp to chronic hypoxia (2.5 ppm and 1.5 ppm DO) have been completed. For each study, 310 shrimp were isolated individually in 10 mm diameter mesh cages in a total of 12 aquaria. Eight aquaria (25 shrimp/aquaria) were to maintain male (N = 50) or female (N = 150) shrimp at low DO while four were to maintain male (N = 35) or female (N = 75) shrimp between 6 and 8 ppm DO. Molts of individual shrimp were recorded. Female shrimp (N = 16) were sampled at day 3, 1 week, and 2 weeks from the hypoxic and normoxic aquaria during the course of the study for analysis of DO stress proteins (N = 8 shrimp/exposure/sample time) and gene expression (N = 8 shrimp/exposure/sample time). Control shrimp (N = 20) from the normoxic population also were sampled at day 0. Shrimp with egg masses were preferentially sampled, and eggs were removed, counted, and frozen (-70ºC) for lipid analysis at a later date. Hepatopancreas tissues were archived in RNA later to be processed for macroarray analysis and frozen at -70ºC for Western blot analysis. At the end of 2 weeks of exposure, males and females were paired into reproductive groups (N = 16 pairs/group) to determine fecundity and survival of the F1 generation (see Objective 4). Samples from the moderate chronic hypoxia laboratory exposure have been run on the arrays; array normalization and analysis is in progress.

Western and Slot Blot Analysis for Blue Crab. Data analysis is complete for blue crab protein expression. There were significant (p < 0.05) differences in hemocyanin concentrations for the 15-day chronic hypoxia exposure (see Figure 2). However, there were no differences in hemocyanin concentration after 10-day exposure to diurnally fluctuating DO. Crabs exposed to chronic hypoxia at days 5, 10, and 15 had a significantly higher percentage of high molecular weight, cross-linked MnSOD bands observed on Western blots. However, the majority of crabs exposed to diurnal hypoxia for 10 days had only noncrosslinked low molecular weight bands typical of normoxia. Thus, it appears that chronic hypoxia induces changes in protein expression of hemocyanin and crosslinking of MnSOD, but a 10-day exposure to diurnal fluctuations in DO does not result in similar changes.

Figure 2. Effect of Chronic Hypoxia (2.5 ppm DO; 34% Saturation; Salinity15 ppt; 27ºC) on Hemocyanin Protein Levels (mean ± SE) in Blue Crab, Callinectes sapidus, Hepatopancreas. Letters indicate means significantly different from each other based on Bonferroni post-hoc test of ANOVA.

Western and Slot Blot Analysis for Grass Shrimp. During the moderate hypoxia study (2.5 ppm DO), there was no difference in hemocyanin concentration in normoxic shrimp across the 14-day exposure period, but there was a significant difference across the hypoxic exposure, with shrimp at day 7 having significantly lower hemocyanin than those at day 3 (p < 0.001) or day 14 (p < 0.03) (see Figure 3A).


Figure 3. Hemocyanin Protein Concentration of Grass Shrimp Exposed to Chronic Moderate (2.5 ppm DO, Panel A) and Severe (1.5 ppm DO, Panel B) Hypoxia. HH, NN, HN represent reproductive females exposed to hypoxia (H) or normoxia (N) for 14 days prior to mating and held in hypoxia (H) or normoxia (N) during the 4-week (Panel A) or 6-week (Panel B) reproductive period.

There were no differences in hemocyanin concentration for females in the reproductive phase of the experiment. In contrast, the normoxic shrimp from the severe hypoxia study (1.5 ppm DO) showed significant differences in hemocyanin, with day 0 and day 3 significantly higher than days 7 and 14 (p < 0.002). Severe hypoxia appeared to decrease hepatopancreatic hemocyanin concentration over time, as shrimp exposed to 1.5 ppm DO for 14 days had significantly lower hemocyanin than those exposed for 3 or 7 days (p = 0.001; Figure 3B). However, there was once again no difference in hemocyanin concentration of females in the reproductive phase of the experiment, regardless of DO exposure (see Figure 3B).

Objective 3: Endogenous Variability in Blue Crab Gene Expression

During analysis of macroarray membranes, we noticed substantial variation in several genes within the same treatment and hypothesized this might be due to endogenous factors such as molt cycle. To investigate the hypothesis that molt cycle may differentially affect gene expression, we examined gene expression profiles of five premolt and six intermolt crabs held under normoxic conditions. The results showed that elongation factor 2 expression levels in intermolt crabs were 180-fold greater than in premolt crabs (t8 = -5.233; p = 0.001; see Figure 4). Expression of hemocyanin 1 and 2 and cryptocyanin was 15-fold (t9 = -3.694; p = 0.005), 37-fold (t8 = -4.077; p = 0.004), and 60-fold (t7 = -3.834; p=0.006) higher, respectively, in intermolt crabs compared to premolt crabs. Several other genes appeared to be "molt independent" as there was no difference in gene expression between premolt and intermolt crabs (see Figure 4): ccox1 (p = 0.163), ribosomal proteins S15, S20 and L23 (p = 0.114, p = 0.078 and p = 0.229), cyt-MnSOD (p = 0.101), mit-MnSOD (p = 0.909), and Hsp70 (p = 0.793). Thus, variations in expression of these genes should indicate exogenous, rather than endogenous, changes in the crab's environment.

Figure 4. Comparison of Gene Expression Levels in Intermolt and Premolt Blue Crabs. Elongation factor 2, hemocyanin 1 and 2, and cryptocyanin expression levels were 180-fold, 15-fold, 37-fold, and 60-fold higher, respectively, in intermolt crabs compared to premolt crabs. * p < 0.05.

Objective 4: Effects of DO on Grass Shrimp Reproduction

Sixteen hypoxic males from the 2.5 ppm and 1.5 ppm chronic hypoxia studies were paired with 16 hypoxic females in individual breeding chambers and maintained under continued hypoxia. All other mating pairs (hypoxic male symbol x normoxic female symbol; hypoxic male symbol x hypoxic female symbol; normoxic male symbol x hypoxic female symbol; normoxic male symbol x normoxic female symbol) were kept under normoxia. Pairs were checked daily for egg production and sacrificed after the female was determined gravid for a minimum of 2 days. All eggs were removed from the female, counted, and 20 viable eggs were incubated individually in sterile seawater in 24-well polystyrene culture plates. Percent embryo survival was determined as successful hatch by day 10 postisolation. All remaining eggs from each female were frozen at -70°C for later lipid analysis. Shrimp exposed to 2.5 ppm DO were kept in the reproductive phase of the experiment for 4 weeks; shrimp exposed to 1.5 ppm DO were kept in the reproductive phase of the experiment 6 weeks. Relative fecundity (number of eggs/gram of body weight) was determined for each female that produced egg clutches in the moderate and severe hypoxia exposures. Females were combined into three groups; HH (exposed to hypoxia for the entire 6- or 8-week period), NN (exposed to normoxia for the entire 6- or 8-week period) and HN (exposed to hypoxia for the first 2 weeks and then placed in normoxia for the reproductive portion of the study). There was no significant difference in the number of egg-producing shrimp, embryo survival, or time to embryo hatch among groups for either study. However, in both studies, the HH females had a significantly higher (p < 0.05) relative fecundity than the NN or HN females (see Figure 5). It appears that female shrimp maximize reproductive output prior to death, the occurrence of which increases significantly after 6 weeks of hypoxia exposure. Eggs from normoxic and hypoxic females (1.5 ppm DO for 38-61 days) have been examined for lipid profiles by Chuck McKenney at the EPA Gulf Ecology Division (GED) in Gulf Breeze, FL. There is a significantly (p = 0.005) higher level of the dominant neutral lipid class, triglycerides, in eggs from females exposed to 1.5 ppm DO (115 µg/mg dry weight) compared to eggs from normoxic conditions (56 µg/mg dry weight). Not only is egg production quantitatively higher in low DO levels, but the quality of these eggs appears superior to eggs from normoxic conditions. However, these data are derived from production of the first clutch (brood) under hypoxia conditions. Experiments are underway to determine if hypoxic females produce fewer clutches than normoxic females or if differences in relative fecundity change when shrimp are exposed to chronic hypoxia for 3 months and allowed to produce multiple broods.

Figure 5. Relative Fecundity of Grass Shrimp Exposed to Moderate (2-3 ppm DO, left panel) and Severe (1.5 ppm DO, right panel) Hypoxia. Letters indicate significant differences among groups as determined by ANOVA and Bonferroni post-hoc test.

Objective 5: Field Evaluation

Blue Crab. Analysis is complete for crabs collected in September 2002, from an open bay site and a diurnally hypoxic marsh site in Pensacola Bay, FL. There were significant differences in gene expression for ccox1 and cyt-MnSOD between the open bay and marsh pond sites (p = 0.006 for both, Figure 6), with downregulation of expression of both genes evident at the diurnally hypoxic marsh site. Expression of two distinct hemocyanin genes also was downregulated at the marsh site, although the differences were not significant. Importantly, ccox1 and cyt-MnSOD do not appear to be molt-sensitive genes in blue crab (see Figure 4), suggesting that the observed downregulation is most likely related to hypoxic conditions. Although hemocyanin gene expression was not different in crabs from marsh and open bay sites, protein levels were significantly lower at the marsh site (p < 0.001) when compared to the normoxic open bay site. Laboratory experiments have shown that cyclic DO (2-3 to 8 ppm DO up to 10 days) does not affect hemocyanin mRNA or protein. Therefore, it appears unlikely that the low hemocyanin concentrations in marsh pond crabs are caused by the diurnal DO fluctuations at the field site. We speculate that reduced hemocyanin levels, and reduced gene expression levels, are due to starvation because of reduced food availability and/or foraging at the field sites. This is supported by the low amount of fat observed in the hepatopancreas tissue of field-caught blue crabs.

Figure 6. Gene Expression (mean ± S.E.) of Cytochrome C Oxidase Subunit 1, Hemocyanin 1 and 2, and Cytosolic MnSOD From Blue Crabs Collected From Open Bay and Marsh Sites in Pensacola Bay, FL, in September 2002. * p = 0.006.

Grass Shrimp. Slot blots for hemocyanin protein concentration were performed for field grass shrimp collected concurrently with the blue crabs in September and November 2002. Unfortunately, no grass shrimp could be collected from the open bay site in September because of inclement weather. Whereas there was a significant difference in hemocyanin protein concentration between blue crabs collected from the two sites, no site differences were found for grass shrimp. Hemocyanin protein levels were determined for field grass shrimp collected in Garcon Point Marsh Pond and Marsh Creek in July and August 2003. A week prior to collecting wild grass shrimp, caged shrimp were deployed in the same sites; the caged shrimp were analyzed for hemocyanin as well.

Figure 7. Hemocyanin Protein Concentrations (mean ± SE) of Grass Shrimp Collected in a Marsh Pond and a Marsh Creek and Caged in the Marsh Pond for 1 Week at Garcon Point, Pensacola Bay During July and August 2003. Similar letters indicate means not significantly different from each other.

Marsh pond grass shrimp in August had significantly reduced hemocyanin levels compared to July wild shrimp and to the caged shrimp (see Figure 7). DO profiles of the field sites indicated that neither site experienced chronic nor diurnal hypoxia during the collection events. The low hemocyanin concentrations in August 2003 Marsh Pond shrimp are similar to the low hemocyanin concentrations that were observed in crabs in September 2002.

The relative fecundity of grass shrimp collected from the marsh pond and the marsh creek in July and August 2003 was determined. There were no significant differences in relative fecundity between the marsh pond and the marsh creek site in either July or August, and there were no significant differences between months for either site (p > 0.05 in all cases; see Table 1). Relative fecundity of field-collected shrimp was lower than relative fecundity of laboratory shrimp exposed to chronic moderate (2-3 ppm DO) and severe (1.5 ppm DO) hypoxia, but was similar to relative fecundity of shrimp held under normoxic conditions in the moderate hypoxia experiment (see Figure 5).

Table 1. Relative Fecundity (mean ± SE) of Field-Collected Grass Shrimp, 2003

Collection Site
July
August
Marsh Pond
512 ± 41.1
542 ± 35.2
Marsh Creek
564 ± 26.1
559 ± 39.9

Conclusions

We have met most of our objectives for Year 2 of the project. Novel hypoxia-responsive genes of blue crab have been identified. Potentially hypoxia-responsive genes of grass shrimp have been cloned, and DNA macroarrays have been constructed. Western blot and slot blot analysis of hemocyanin and MnSOD proteins for grass shrimp have been developed. Additionally, antibody production for HIF-1? is ongoing (see Objective 1). Laboratory data have been completely analyzed for blue crab gene and protein expression, and several genes (cytochrome c oxidase subunit 1 and cytosolic MnSOD) have been shown to be hypoxia responsive under laboratory conditions (chronic and diurnal low DO). Additionally, hemocyanin protein expression also appears to be hypoxia responsive in under chronic low DO. Data analysis is continuing for protein and gene expression of grass shrimp under laboratory conditions. Hemocyanin protein concentration does appear to differ under conditions of chronic moderate (2-3 ppm DO) and severe (1.5 ppm DO) hypoxia (see Objective 2). A new objective was added and completed this year. We showed that molt cycle affects the expression of several genes in blue crab, including hemocyanin cryptocyanin and elongation factor 2, suggesting that these genes are not good choices as indicators of hypoxia (see Objective 3). Relative fecundity of grass shrimp is significantly increased under chronic moderate and severe hypoxia; we continue to work on relating these data to gene expression profiles (see Objective 4). Finally, gene and protein expression of field-caught blue crabs shows downregulation of two genes (cytochrome c oxidase subunit 1 and cytosolic MnSOD) and the hemocyanin protein in a diurnally hypoxic marsh. Gene expression analysis for field-caught grass shrimp is ongoing. Analysis of hemocyanin protein concentrations suggests hemocyanin concentrations are lower in a historically hypoxic marsh site. However, there were no differences in fecundity of grass shrimp from a marsh pond and a marsh creek site in July and August (see Objective 5). During this year, we added a laboratory experiment (severe chronic DO in grass shrimp) as well as a new objective (see Objective 3), resulting in more samples to analyze than originally anticipated. Analysis is ongoing for both protein (MnSOD) and gene expression data for laboratory and field samples from this year, and initial results suggest that grass shrimp are a good model for detection of hypoxia on a molecular level. In addition, the ability to quantify changes in reproduction through fecundity in both the field and laboratory, and relate these changes to gene and protein expression, has resulted in a focus on grass shrimp for the duration of this project. Additional laboratory experiments to determine the effect of diurnal variations in DO on grass shrimp and to identify cyclic DO-responsive genes are planned for the upcoming year; these data should greatly aid in interpreting field data that primarily show diurnal variations in DO.

Future Activities:

We will continue using hypoxia-responsive macroarrays as tools for monitoring the effects of hypoxia in estuarine crustacea. In addition, we will prepare antibodies of hypoxia inducible factor (HIF-1alpha) from grass shrimp. We will continue experiments to determine if hypoxic females produce fewer clutches than normoxic females or if differences in relative fecundity change when shrimp are exposed to chronic hypoxia for 3 months and allowed to produce multiple broods. Data analysis will continue for protein and gene expression of grass shrimp under laboratory conditions. Analysis will continue for both protein (MnSOD) and gene expression data for laboratory and field samples. Also, we plan to continue our laboratory experiments to determine the effect of diurnal variations in DO on grass shrimp and to identify cyclic DO-responsive genes during the coming year; these data should greatly aid in interpreting field data that primarily show diurnal variations in DO.


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 161 publications 48 publications in selected types All 44 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 Niemi G, Wardrop D, Brooks R, Anderson S, Brady V, Paerl H , Rakocinski C, Brouwer M, Levinson B, McDonald M. Rationale for a new generation of ecological indicators for coastal waters. Environmental Health Perspectives 2004;112(9):979-986. R829458C003 (2003)
R829458C008 (2003)
R829458C008 (2004)
R828675 (2004)
R828675 (Final)
R828677C001 (Final)
R828684 (Final)
 not available
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, Apalachicola Bay, Consortium for Estuarine Ecoindicator Research for the Gulf of Mexico, CEER-GOM, Environmental Monitoring and Assessment Program, Galveston Bay, Mobile Bay, benthic indicators, ecoindicator, ecological exposure, ecosystem monitoring, environmental indicators, environmental stress, estuarine ecoindicator, estuarine integrity. , Ecosystem Protection/Environmental Exposure & Risk, Geographic Area, Scientific Discipline, RFA, ECOSYSTEMS, Ecosystem/Assessment/Indicators, Biology, Gulf of Mexico, Ecology, Aquatic Ecosystems & Estuarine Research, Ecological Monitoring, Aquatic Ecosystem, Ecological Indicators, Ecological Effects - Environmental Exposure & Risk, Ecosystem Protection, Aquatic Ecosystems, Ecology and Ecosystems, Environmental Monitoring, water quality, nutrient fluxes, environmental indicators, ecoindicator, aquatic ecosystem restoration, monitoring, dissolved oxygen , hypoxia, estuarine integrity, estuarine ecoindicator, ecological exposure, crustaceans, ecosystem assessment, environmental stress, molecular ecology, ecological assessment, estuaries
Relevant Websites:

http://www.usm.edu/gcrl/contacts/view_vitae.php?id=190 exit EPA

Progress and Final Reports:
2002 Progress Report
Original Abstract
2004 Progress Report
2005 Progress Report


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

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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