2005 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
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, 2004 through November 30, 2005
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000) RFA Text |  Recipients Lists
Research Category: Water , Ecosystems , Ecological Indicators/Assessment/Restoration

Objective:

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. Macroarrays were constructed using 78 potentially hypoxia-responsive genes, as determined through suppression subtractive hybridization and direct cloning. Arrays were hybridized with 33 P -labeled cDNA from shrimp exposed to hypoxia under controlled laboratory conditions. Analysis of intensity data identified 47 of the 78 genes as the most hypoxia responsive. Grass shrimp exposed to moderate, chronic hypoxia (2.5 ppm dissolved oxygen [DO]) showed minimal changes in gene expression. The response after short-term (3 day) exposure to severe chronic hypoxia (1.5 ppm DO) was upregulation of genes encoding proteins involved in oxygen uptake/transport and energy production, such as hemocyanin and ATP synthases. The major response by day 7 was an increase of transcription of genes in the mitochondrial genome (16S rRNA, cytochrome b, cytochrome coxidase I and III), and upregulation of genes encoding proteins involved in iron metabolism, possibly reflecting the dependency of mitochondria on Fe for heme (cytochrome) biosynthesis and for the biogenesis of [Fe-S] clusters that are present in more than 10 subunits of enzymes in Complex I, II, and III of the respiratory chain. By day 14 a dramatic reversal was seen, with a significant downregulation of both mitochondrial and Fe-metabolism genes. Grass shrimp exposed to cyclic hypoxia (1.5 ppm – 8 ppm DO over a 24- hour cycle) showed changes in gene expression profiles distinct from chronic hypoxia exposures. After 3 day exposure, a dramatic upregulation of the antioxidant enzyme mitochondrial Mn superoxide dismutase (SOD) was observed, a common response to oxidative stress. Hemocyanin expression was not affected. After 7 day exposure, four genes encoding mitochondrial proteins involved in protein synthesis, lipid degradation, ATP synthesis, and electron trans port were downregulated. Yet another mitochondrial enzyme involved in gluconeogenesis (synthesis of glucose from amino acids) was upregulated, together with crustapain, a cytosolic proteolytic enzyme. Taken together these changes in gene expression profiles suggest downregulation of mitochondrial protein synthesis, trichloroacetic acid (TCA) cycle and electron transport with upregulation of proteolysis and gluconeogenesis. After 14 days of exposure to cyclic DO, this pattern has returned to pre-exposure conditions. Genes coding for vitellogenin, an egg yolk protein, were upregulated after 77 day exposure to cyclic hypoxia. Validation of the macroarray results of the chronic hypoxia studies with real-time q-PCR showed similar up- or downregulation at multiple time points for nine genes. Currently, an expanded microarray is being constructed with genes responsive to cyclic hypoxia, which appear to be distinct from the hypoxia-responsive genes, to enable more thorough testing of gene expression in response to cyclic hypoxia, which commonly occurs in Gulf of Mexico estuaries and both the Weeks Bay, Alabama, and East Bay, Pensacola, field sites during the summer. Laboratory experiments suggest grass shrimp from Weeks Bay and East Bay react differently to chronic hypoxia exposure. Shrimp from Weeks Bay had higher mortality and fewer females produced eggs than shrimp from East Bay, suggesting the Weeks Bay shrimp may be more sensitive to hypoxia than the East Bay shrimp. Gene expression data from the two populations are currently being analyzed. A suite of 28 molecular and whole-animal indicators of hypoxia have been identified for field-collected grass shrimp to be used in an ecosystem-wide integration for the Consortium for Estuarine Ecoindicator Research for the Gulf of Mexico (CEER-GOM) research group. Progress during this period has been negatively impacted by H urricane Katrina, which, among others, has resulted in a loss of all 2005 field samples.

The objectives for year 4 were as follows: (1) Identify a suite of molecular indicators for hypoxia 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) Characterize DO and salinity parameters in Weeks Bay and Mobile Bay during the 2005 field season. (5) Validate response of the DO stress indicators in grass shrimp from hypoxic and reference sites in two CEER-GOM targeted estuaries. (6) Determine reproductive status of field- collected grass shrimp. (7 ) Determine if there are population differences between Weeks Bay and Pensacola Bay grass shrimp in response to hypoxia through controlled laboratory experiments. (8 ) Identify indicators of crustacean hypoxia for use in overall integration models.

Progress Summary:

Data analysis continued on chronic hypoxia data collected from laboratory experiments with grass shrimp conducted during years 2, 3, and 4 of this project. Gene expression data were normalized four different ways (using the mean, median, al pha-tubulin, and hemocyanin). Significant up- or downregulation of each of the 77 genes for each laboratory experiment and field collection, based on the four normalization methods, was tabulated. Genes that showed significant changes with three of the four normalization methods at any time point or field collection were considered “responsive” genes. The 47 genes that appear to be responsive to hypoxia are listed in Table 1; only these genes were included in all laboratory and field data analyses.

Table 1. List of Grass Shrimp Hypoxia Responsive Genes Based on Significant Changes in at Least Three of Four Normalization Techniques

GENE

Definition/Function

Peptidyl prolyl cis-trans isomerase

Involved in the folding of proteins.

HSP 70 cognate (heat shock protein)

Hsp70 cognates function in concert with a variety of co-chaperones to facilitate folding of de novo synthesized proteins, assist in transport of precursor proteins into organelles, and to help target damaged proteins for degradation.

TIM 14

Mitochondrial import inner membrane translocase subunit TIM14. Probable component of the PAM complex at least composed of a mitochondrial HSP70 protein, Roe1, TIM44, blp/TIM16, and TIM14.

HSP 70 (heat shock protein)

Stress-induced Hsp70s function to mitigate aggregation of stress-denatured proteins and to refold non-native proteins, restoring their biological function.

HSP 70 (NP)

Same as above.

rprot S6

Ribosomal protein S6. Structural constituent of the ribosome. Involved in translation of mRNA into proteins.

rprot L27A

Ribosomal protein L27A. Structural constituent of the ribosome. Involved in translation of mRNA into proteins.

Mitochondrial rprot S2

Ribosomal protein S2. Structural constituent of the mitochondrial ribosome. Involved in mitochondrial protein synthesis.

rprot L3

Ribosomal protein L3. Structural constituent of the ribosome. Involved in translation of mRNA into proteins.

EF-2 (elongation factor 2)

EF2 regulates mRNA translation through phosphorylation.

rprot S14

Ribosomal protein S14. Structural constituent of the ribosome. Involved in translation of mRNA into proteins.

Cathepsin L

A cysteine proteinase that is involved in intracellular protein catabolism.

Cysteine proteinase

An enzyme that destroys proteins by hydrolysis, turning them back into individual amino acids.

trypsin

Cleaves peptide bonds involving the amino groups of lysine or arginine.

Crustapain (cysteine proteinase)

A cysteine proteinase from a crustacean species.

Acetyl CoA binding protein

Binds medium- and long-chain acyl-CoA esters with very high affinity.

Lipase I

An enzyme capable of breaking down fats into fatty acids and glycerols.

Vitellogenin_1

Egg yolk precursor protein.

apolipoprotein A_I

Plays an important role in lipid transport and metabolism.

fertilization envelope

Protein of the outer layer of the fertilization envelope of fish eggs.

Vitellogenin_2

Egg yolk precursor protein.

cytochrome c oxidase subunit III

Part of the cytochrome c oxidase complex, which mediates the transfer of electrons from reduced cytochrome c to molecular oxygen. Encoded by the mitochondrial genome.

apocytochrome b

Component of the mitochondrial electron transport chain. Encoded by the mitochondrial genome.

ATP synthase d chain

This is one of the chains of the nonenzymatic component (membrane proton channel) of the mitochondrial ATPase complex. This is a mitochondrial protein encoded by the nuclear genome.

cytochrome c oxidase subunit III

Part of the cytochrome c oxidase complex, which mediates the transfer of electrons from reduced cytochrome c to molecular oxygen. Encoded by the mitochondrial genome.

ATP synthase f chain

This is one of the chains of the nonenzymatic component (CF(0) subunit) of the mitochondrial ATPase complex. This is a mitochondrial protein encoded by the nuclear genome.

ATP synthase subunit b

This is one of the chains of the nonenzymatic component (CF(0) subunit) of the mitochondrial ATPase complex. This is a mitochondrial protein encoded by the nuclear genome.

cytochrome oxidase subunit 1

Part of the cytochrome c oxidase complex, which mediates the transfer of electrons from reduced cytochrome c to molecular oxygen. Encoded by the mitochondrial genome.

Hypoxia Inducible Factor

Under hypoxic conditions activates the transcription of over 40 genes, whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia.

HEMOCYANIN

Blue, oxygen transporting, copper containing protein found in the blood of molluscs and crustacea. Hemocyanins are multisubunit proteins with each subunit encoded by a separate gene. The next 2 hemocyanins represent distinct hemocyanin genes.

hemocyanin

See above.

hemocyanin subunit 4

See above.

amylase I

Hydrolyses 1,4-alpha-D-glucosidic linkages in oligosaccharides.

PmAV

PmAV, a novel gene involved in virus resistance of shrimp.

beta_1,3_glucan binding protein

Immune defense protein. Activates prophenoloxidase when combined with fungal 1,3-beta-D-glucans.

Acid beta glucosidase

Lysosomal, membrane bound protein, which functions as a gluco-hydrolase: D-glucosyl-N-acylsphingosine + H2O = D-glucose + N-acylsphingosine.

phosphoenolpyruvate carboxykinase

Phosphoenolpyruvate carboxykinase catalyzes the formation of phosphoenolpyruvate by decarboxylation of oxaloacetate while hydrolyzing GTP, a rate limiting step in gluconeogenesis.

chitinase

An enzyme which breaks down chitin, which is a polysaccharide that forms the hard outer shell of crustaceans.

troponin C gamma

Troponin is the central regulatory protein of striated muscle contraction.

16S rRNA

16S mitochondrial ribosomal RNA gene.

actin

A filamentous protein involved in muscle contraction in both smooth and striated muscle and is also a structural molecule for the cytoskeleton of many eukaryotic cells.

Heme binding protein

Heme binding protein. Physiological function unknown. Possibly involved in transfer of heme to heme proteins or in binding of free heme to protect against heme catalyzed oxidative damage.

ferritin subunit

Iron (ferric) binding protein.

cSOD

Cytosolic form of the antioxidant enzyme manganese superoxide dismutase, which converts superoxide into O2 and H2O2.

mSOD

Mitochondrial form of the antioxidant enzyme manganese super oxide dismutase, which converts superoxide into O2 and H2O2.

Orn decarboxylase antizyme

Binds to, and destabilizes, ornithine decarboxylase (the rate-controlling enzyme in polyamine biosynthesis), which is then degraded by the proteasomes.

glutamine repeat protein_1

GRP-1 may be a transcription factor associated lipopolysaccha-ride-induced activation of macrophages.

Objective 2. Response of Molecular Indicators to Chronic and Cyclic DO

Chronic Hypoxia. There were no significant changes in gene expression after 3 day exposure to moderate, chronic hypoxia. However, after 7 day exposure, there was significant downregulation of 2 HSP70 genes (Figure 1A). After 14 day exposure to moderate, chronic DO, there was a significant 19-fold decrease in expression of the gene encoding the antioxidant enzyme cytosolic Mn s uperoxide d ismutase (cSOD), which in crustacea has replaced the more commonly found cytosolic Cu,Zn s uperoxide d ismutase. Expression of one other gene, which shows weak sequence similarity (E value = 3.83-07) with a mouse gene encoding for glutamine repeat protein, is upregulated (Figure 1B). It appears in general that no genes are robust in dicators of moderate chronic DO exposure, with the possible exception of cSOD.

Fold Changes (log2(hypoxic/normoxic)) in Gene Regulation Measured by Macroarrays in Grass Shrimp Exposed to Chronic, Moderate (2.5 ppm DO) Hypoxia

Figure 1. Fold Changes (log2(hypoxic/normoxic)) in Gene Regulation Measured by Macroarrays in Grass Shrimp Exposed to Chronic, Moderate (2.5 ppm DO) Hypoxia. Data shown are normalized to α-tubulin and all changes are significant (t-test, p < 0.05). Genes are pattern coded by functional group. Data are presented as log2 ratios. Therefore, genes with values of -1, -2 , -3, and -4 are downregulated 2-, 4-, 8-, and 16-fold , respectively. Genes with values of 1 and 2 are upregulated 2- and 4 -fold, respectively. A. 7 day exposure. B. 14 day exposure.

In contrast to moderate hypoxia, grass shrimp exposed to severe (1.5 ppm DO), chronic hypoxia showed significant changes in expression of a number of genes that are potentially robust indicators of hypoxia. Furthermore, gene expression profiles change over the time-course of chronic DO exposure, lending further insight into how the grass shrimp adapt to severe hypoxia. After 3 days exposure to severe hypoxia, significantly upregulated genes include cytochrome b (cytB), ATP synthase d and f chains, three hemocyanin genes, troponin C and I, and ferritin (Figure 2A), suggesting an attempt to increase oxygen uptake/transport (hemocyanin), mitochondrial electron transport and ATP synthesis (cytB and ATP synthase), and locomotion (troponin C and I). After 7 days exposure to severe chronic hypoxia, the adaptation induced by day 3 becomes insufficient, and ATP synthase, hemocyanin, and troponin are no longer upregulated (Figure 2B). The major response by day 7 appears to be an increase of transcription of genes present in the mitochondrial genome (16S mitochondrial rRNA (16SrRNA) and cytochrome c oxidase 1 (Ccox 1; Figure 2B). The upregulation of two genes encoding proteins involved in iron metabolism, heme-binding protein and ferritin (Figure 2B), possibly reflect the dependency of mitochondria on Fe for heme (cytochrome) biosynthesis and for the biogenesis of [Fe-S] clusters that are present in more than 10 sub-units of enzymes in Complex I, II, and III of the respiratory chain. The adaptation seen after 7 days once again becomes insufficient by Day 14, and a dramatic reversal is seen, with a significant downregulation of transcription of genes in the mitochondrial genome (16SrRNA, Ccox I, cytochrome c oxidase III [Ccox III], and cytB) as well as ferritin and heme binding protein (Figure 2C). At day 14, PmAV is also downregulated, suggesting grass shrimp may become more susceptible to viral infections after prolonged hypoxia exposure.

Prolonged 24-61 day exposure to severe hypoxia shows continued downregulation of mitochondrial proteins Ccox III and cytB, as well as upregulation of one of the hemocyanin genes (Figure 2D). Thus, mitochondrial genes such as 16SrRNA, cytB , Ccox I, and Ccox III as well as hemocyanin and Fe-proteins appear to provide promise as indicators of chronic severe hypoxia exposure in grass shrimp.

Fold Changes (log2(hypoxic/normoxic)) in Gene Regulation Measured by Macroarrays in Grass Shrimp Exposed to Chronic, Moderate (2.5 ppm DO) Hypoxia

Figure 2. Fold Changes (log2(hypoxic/normoxic)) in Gene Regulation Measured by Macroarrays in Grass Shrimp Exposed to Chronic, Severe (1.5 ppm DO) Hypoxia. Data shown are normalized to α-tubulin and all changes are significant (t-test, p < 0.05). Genes are pattern coded by functional group. Data are presented as log2 ratios. Therefore, genes with values of -1, -2 , and -3 are downregulated 2-, 4-, and 8-fold, respectively. Genes with values of 1, 2, 3, and 4 are upregulated 2-, 4-, 8-, and 16-fold, respectively. A. 3 day exposure. B. 7 day exposure. C. 14 day exposure. D. 26-61 day exposure.

Cyclic Hypoxia. Grass shrimp exposed to cyclic hypoxia (1.5 ppm – 8 ppm DO over a 24 hour cycle) showed changes in gene expression profiles distinct from chronic hypoxia exposures. After 3 day exposure, a dramatic upregulation of the antioxidant enzyme mitochondrial MnSOD was observed, a common response to oxidative stress (Figure 3A). Hemocyanin expression was not affected. After 7 day exposure, 4 genes encoding mitochondrial proteins were downregulated: mitochondrial ribosomal subunit S2, acylCoA dehydrogenase (which generates acetylCoA from long-chain fatty acids to be used as substrate for the TCA cycle), ATP syn thase d, and cytochrome b. Yet another mitochondrial enzyme (phosphoenolpyruvate carboxykinase, involved in gluconeo genesis: synthesis of glucose from amino acids) was upregulated, together with crustapain, a cytosolic prote olytic enzyme (Figure 3B). Taken together these changes in gene expression profiles suggest downregulation of the mitochondrial protein synthesis, electron transport chain and TCA cycle, with upregulation of prote olysis and gluconeogenesis. This pattern in gene expression reverses after 14 day exposure to cyclic hypoxia, with upregulation of ribosomal protein L13 and ATP synthase b and the appearance of a 120-fold upregulation of cuticle protein gene expression (Figure 3C). Long-term (77 day) continuous exposure of grass shrimp to cyclic hypoxia resulted in upregulation of two genes coding for vitellogenin (Figure 3D), an egg yolk protein.

Work is currently proceeding on the development of microarrays including genes identified as responsive to cyclic hypoxia using suppression subtractive hybridization. The library contained many genes not found in the hypoxia-responsive library. Most striking is the presence of genes that are involved in sulfur redox metabolism, indicative of defense against oxidative stress. Approximately 800 new genes have been amplified and purified in preparation for spotting on expanded microarrays. Grass shrimp have been exposed to cyclic and chronic hypoxic conditions. RNA has been extracted and purified from these samples, which will be hybridized onto the expanded microarrays.

Fold Changes in Gene Regulation in Grass Shrimp Exposed to Cyclic Hypoxia (1.5 ppm DO to 8 ppm DO during a 24 hour cycle).

Figure 3. Fold Changes in Gene Regulation in Grass Shrimp Exposed to Cyclic Hypoxia (1.5 ppm DO to 8 ppm DO during a 24 hour cycle). Data shown are normalized to α-tubulin and changes are significant (t-test, p < 0.05). Genes are pattern coded by functional group. A. 3 day exposure. B. 7 day exposure. C. 14 day exposure. D. 77 day exposure.

Objective 3. Validate Gene Expression With Real-Time qPCR

q-PCR was used to validate the gene expression results from the macroarrays for both the moderate and severe hypoxia experiments, as well as to verify the utility of using α-tubulin as a normalizing gene. Alpha-tubulin did not change significantly in response to 14 day exposure to moderate hypoxia or to 3, 7, or 14 day exposure to severe hypoxia (Figure 4).

These results, in combination with the consistent appearance of α-tubulin with high intensity values on all membranes, justify the use of α-tubulin for normalization of the gene intensities on the arrays.

Moderate hypoxia resulted in few significant changes in gene expression on macroarrays (Figure 1), and q-PCR also showed no significant changes in expression of α-tubulin, Hcy I,or ATPsyn-f, although the direction of gene regulation (up or down) was the same for these genes on the macroarrays and q-PCR (Figure 4A).

In contrast to results for moderate hypoxia, the q-PCR showed a greater, but still not significant, upregulation of 3 separate hemocyanin genes after 3 day exposure to severe hypoxia (Figure 4B) mirroring macroarray results for the same time point (Figure 2A). Additionally, ATPsyn-f was also upregulated in both q-PCR (Fig. 4B) and on macroarrays (Figure 2A). Furthermore, 2 separate groups of shrimp exposed to severe hypoxia for 3 days showed a similar upregulation of the 3 hemocyanin genes and ATPsyn-f measured using q-PCR and macroarray analysis, suggesting this is a robust, consistent response.

There were significant changes in gene expression as measured by q-PCR after both 7 and 14 day exposure to chronic severe hypoxia (Figures 4C and D). Three genes (Ccox I, Ccox III,and ferritin) were significantly upregulated at day 7, and two of these (Ccox I and ferritin) were also significantly upregulated on the macroarrays at the same time point (Figures 2B and 4C). Gene expression changed significantly for 16S rRNA and ferritin after 14 day exposure to severe hypoxia (Figure 4D), and both these genes were significantly downregulated on the macroarrays (Figure 2C). There was only one instance of disagreement among all genes tested using q-PCR and macroarray analysis. Ferritin was upregulated as measured by q-PCR and downregulated as measured by macroarrays after 14 day exposure to severe hy poxia. Overall, the similarity in response of 9 genes at multiple time points using both q-PCR and macroarrays validates the macroarray gene expression results.

Fold Changes (log2(hypoxic/normoxic)) in Gene Regulation Measured by Real-Time qPCR in Grass Shrimp Exposed to Chronic Hypoxia. Data are presented as log2 ratios

Figure 4. Fold Changes (log2(hypoxic/normoxic)) in Gene Regulation Measured by Real-Time qPCR in Grass Shrimp Exposed to Chronic Hypoxia. Data are presented as log2 ratios. Therefore, genes with values of -1 are downregulated 2-fold. Genes with values of 1 and 2 are upregulated 2- and 4-fold, respectively. Striped bars indicate the direction of the fold change was the same for both real-time PCR and macroar rays. Data shown are normalized to 18S rRNA. Significant (t-test, p < 0.05) real-time qPCR changes in dicated by *. A. 14 day exposure to moderate (2.5 ppm DO) hypoxia. B. 3 day exposure to severe (1.5 ppm DO) hypoxia. C. 7 day exposure to severe hypoxia. D. 14 day exposure to severe hypoxia.

Objective 4. Characterize DO and Salinity in Weeks Bay and Pensacola Bay

The field study site selection for 2005 included returns to East Bay in Pensacola, Florida, and Mobile Bay/Weeks Bay in Mobile, A labama. The sites of sonde deployment in the Mobile Bay system included Weeks Creek (WC), Weeks Bay Mouth (WBM), and OBS 2, all of which were also monitored in 2004. The WC and WBM sites are lo cated within Weeks Bay. The OBS2 site is located in the lower center of Mobile Bay. The water quality of the Weeks Bay sites (WC and WBM) was continuously monitored from July 15 to 22, 2005 (Summer), August 7 to 16, 2006 (late summer), and November 2 to 9, 2005 (Fall) using YSI data sondes. The Mobile Bay site (OBS2) was monitored with a data sonde from July 15 to 23, 2005 (Summer). During the summer, DO exhibited a cyclic pattern in both WC and WBM (Figure 5). However, DO often fell below 2 ppm in WC and rarely went into the hypoxic range at WBM during summer. By November, DO never fell below 6 ppm at any site.

DO Profiles From Weeks Bay Sites During July and August 2006

Figure 5. DO Profiles From Weeks Bay Sites During July and August 2006

Three sites were monitored in East Bay, F lorida, during the summer and fall of 2005, which included GP1, Marsh Creek (MC), and Marsh Pond (MP). A YSI data sonde was deployed at each site during the month of August (Summer) and November (Fall). All three sites exhibited cyclic DO during the summer, although none consistently reached 2 ppm DO on a daily basis (Figure 6). The MP site exhibited the great est daily DO variations, while changes were more moderate at the GP1 site. During November, DO variations were evident at all sites, but no site had DO values lower than 6 ppm in the fall.

DO and Salinity Profiles From East Bay (Pensacola) Sites During August 2005. DO indicated by dark blue line, salinity by pink line.

Figure 6. DO and Salinity Profiles From East Bay (Pensacola) Sites During August 2005. DO indicated by dark blue line, salinity by pink line.

Objective 5. Gene Expression and Reproduction in Field-Captured Grass Shrimp

Limited gene expression data are available for the 2005 field season due to loss of samples collected during July and August as a result of Hurricane Katrina (see below for a discussion of Hurricane Katrina effects). Samples collected from Weeks Bay and Pensacola during November 2005 are currently being processed for gene expression results.

There were no significant differences in relative fecundity or condition factor between sites at either Weeks Bay or East Bay during summer (July or August) or fall (November) 2005. However, it is interesting to note that condition decreased for Weeks Bay shrimp between summer and fall, while condition increased for East Bay shrimp during the same time period. This may be related to Hurricane Katrina, which impacted Mobile Bay, but not East Bay, in late summer. A greater percentage of females from the WC site in Weeks Bay were carrying eggs (48.9%) compared to the WBM site (17.6%) in July. The sex ratio was skewed towards females at WC (3.2:1) and was almost equal at WBM (1.1:1). These data are unavailable from the East Bay sites due to Hurricane Katrina. These results were unexpected, as in 2004 a greater percentage of WBM females were carrying eggs compared with WC females.

Objective 6. Differences Between Two Grass Shrimp Populations During Hypoxia Exposure

Grass shrimp were field collected from Weeks Bay (WC) of the Mobile Bay system and MP of the East Bay Pensacola system to examine potential differences between populations in the response to hypoxia during a 14 day laboratory exposure. Shrimp collected from the two study systems were returned to the laboratory and gravid females isolated from males and non- gravid females. Shrimp were held for a minimum of 10 days to allow any gravid females to release hatched larvae from eggs, which the females had on the day collected.

The 2 week exposure of grass shrimp involved 20 female shrimp isolated individually into each of six aquaria from each of the two study sites. Each of the six aquaria for each site evaluation was stocked with 20 shrimp on day 0 of the exposure. Three aquaria were maintained under nominal hypoxic conditions of 1 to 2 ppm DO, and three were maintained at a nominal 6 to 8 ppm DO. D O as measured continuously in one hypoxic treatment aquarium during the 14 day evaluation was 1.50 ± 0.21 ppm. Shrimp were isolated individually in the test aquaria into retention chambers covered with a disposable petri lid to prevent escape. Retention chambers were maintained in a minimum depth of approximately 7 cm dilution water in all glass aquaria. Shrimp were fed brine shrimp nauplii once daily and commercial flake food once daily, and survival and molt incidence were monitored daily from day 0 to study termination.

None of the shrimp died in the normoxic treatments. Mobile Bay ( WC) grass shrimp responded somewhat differently to hypoxia than the Pensacola Bay ( MP) grass shrimp. Fifteen of the Weeks Creek shrimp died under hypoxic conditions, whereas only 3 died in the Pensacola Bay (Marsh Pond) population. The Weeks Creek population shrimp produced 8 egged females under hypoxic conditions whereas the Marsh Pond shrimp population produced 45 egged females. This suggests the Pensacola Bay ( MP) shrimp may be less sensitive to hypoxia than the Mobile Bay ( WC) shrimp. At day 3, week 1, and week 2 shrimp were removed from the hypoxic and the normoxic aquaria for gene expression analysis. RNA has been extracted from these samples and macroarray analy sis is currently underway.

Objective 7. Crustacean Hypoxia Indicators For Overall CEER-GOM Integration

A suite of potential hypoxia indicators have been identified for grass shrimp to be used in overall integration and model development for the CEER-GOM project. Data from field collections in the Pensacola Bay system during 2003 and 2004 and in the Mobile system for 2004 have been compiled for these indicators. Whole animal indicators of importance are individual values for condition factor and relative fecundity, and the percent age of females carrying eggs at each site during each month.

Twenty-five genes have been identified as biologically relevant indicators of hypoxia (Table 2). These genes are based on laboratory data from chronic and cyclic exposures; five genes are important in both sets. Mean changes in gene expression for each field site relative to normoxic laboratory control gene expression values have been assigned a rank value of upregulated (1), downregulated (-1), or no change (0) for purposes of data integration. These rank values are based on 20 percent “noise” on control membranes for individual genes; any fold change values within 40 percent are considered “noise” and are thus considered to have no change (i.e., fold change values < 1.4 or > 0.6).

Table 2. Biologically Relevant Hypoxia-Indicator Genes Used for CEER-GOM Data Integration. Presented as mean upregulated (1), downregulated (-1), or no change (0) gene expression for each field collection. Genes with * occur in both the chronic and cyclic lists

Chronic DO Genes (abbreviation)

Pensacola 2003

Pensacola 2004

Mobile 2004

 

July

August

November

August

July

Sept

November

 

MC

MP

MC

MP

MC

MP

GP1

MC

MP

WC

WBM

WC

WBM

WC

WBM

TIM 14*

-1

1

1

1

1

1

1

1

1

-1

1

1

1

1

1

Mitochondrial rprot S2 (mprot S2)

-1

1

0

-1

1

1

1

1

1

-1

1

0

-1

1

1

apocytochrome b (cytb)

0

1

-1

0

-1

1

0

-1

-1

0

1

-1

0

-1

1

ATP synthase d chain (ATPd) 

-1

1

0

-1

0

0

0

1

0

-1

1

0

-1

0

0

cytochrome c oxidase subunit III (ccoxIII)

-1

1

0

0

-1

1

0

0

-1

-1

1

0

0

-1

1

ATP synthase f chain (ATPf)

0

1

1

1

1

1

1

1

0

0

1

1

1

1

1

ATP synthase subunit b (ATPb)

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

cytochrome oxidase sub-unit 1 (ccoxI)*

0

1

1

0

1

1

-1

-1

-1

0

1

1

0

1

1

HEMOCYANIN (HEMO)

-1

1

-1

-1

0

0

0

0

0

-1

1

-1

-1

0

0

hemocyanin (hcy)

-1

1

-1

-1

1

1

0

-1

-1

-1

1

-1

-1

1

1

hemocyanin subunit 4 (hcy4)*

0

1

-1

0

0

0

0

1

0

0

1

-1

0

0

0

phosphoenolpyruvate carboxykinase (PEP)*

0

1

-1

-1

1

1

-1

1

0

0

1

-1

-1

1

1

16S rRNA 

-1

1

1

0

1

1

-1

-1

-1

-1

1

1

0

1

1

Heme binding protein (heme)

1

1

1

1

1

1

1

-1

0

1

1

1

1

1

1

ferritin subunit (ferritin)

1

1

1

0

1

1

1

-1

1

1

1

1

0

1

1

HSP 70 *

-1

0

-1

-1

0

-1

0

0

1

-1

0

-1

-1

0

-1

rprot S14

0

-1

0

-1

1

1

0

-1

-1

0

-1

0

-1

1

1

Crustapain (cysteine proteinase) (crustp)

0

1

1

0

1

1

1

-1

0

0

1

1

0

1

1

3beta-hydroxysterol delta 24 reductase (hydroxyster)

-1

-1

1

0

1

1

-1

-1

-1

-1

-1

1

0

1

1

Vitellogenin-2 (Vtg2)

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

amylase I 

1

1

1

1

1

0

1

0

1

1

1

1

1

1

0

actin

0

1

1

0

0

-1

0

-1

0

0

1

1

0

0

-1

mSOD

-1

-1

1

0

0

1

-1

-1

-1

-1

-1

1

0

0

1

Cuticle protein AMP4 (cuticle)

1

1

1

0

0

-1

1

1

1

1

1

1

0

0

-1

glutamine repeat protein-1 (gln rpt)

0

1

-1

0

-1

-1

-1

-1

0

0

1

-1

0

-1

-1

Hurricane Katrina Impacts Hurricane Katrina made landfall on the Mississippi Gulf Coast on August 29, 2005, causing wide spread damage and destruction The Gulf Coast Research Laboratory lost five buildings and sustained ap proximately $50 million in damages to buildings, contents, collections, research, and intellectual property as a result of storm surge and winds The loss of power from Hurricane Katrina resulted in the thawing of all tissue and RNA samples. Furthermore, these samples sat at room temperatures (or higher) for 2+ weeks This represents all field and laboratory RNA samples collected during the course of this project, as all the samples had been returned from the University of Florida after macroarray analysis. Surprisingly, the extracted and purified total RNA, stored in RNAsecure at -70°C seemed to maintain its integrity under these harsh conditions Random samples were checked using Agilent gel chip analysis and Nanodrop RNA scans, and degradation of 28S and 18S ribosomal RNA degradation in most, but not all samples, was minimal Whether that also applies to mRNA is still not clear Many of these samples have been used in q-PCR analysis, and while the results are a little more variable than pre-Katrina, it appears that most, but not all, data can be used. RNA was extracted from the majority of the laboratory experiment samples prior to Hurricane Katrina, and these samples were determined to be satisfactory and were sent to the University of Florida for macroarray analysis Unfortunately, tissue samples frozen at -20°C in RNALater did not fare as well after 2+ weeks at room temperature These included all of the July and August 2005 field samples as well as a few of the laboratory samples from Weeks Bay shrimp exposed to hypoxia for 14 days Approximately 60 percent of these samples showed massive RNA degradation and were unusable Thus, there will be no gene expression data from Pensacola for August 2005 as almost all these samples were degraded.

All tissue samples stored at -70°C for protein analysis were ruined and discarded after Katrina. Thus, there will be no additional protein data produced during the course of this project Field collections in November 2005 did not include taking grass shrimp for protein samples, as there are no other protein data to compare the results to All eggs collected for lipid analyses were also thawed and sat at room temperature, and many were lost completely since they were stored in the destroyed Toxicology building However, egg samples from Pensacola in August 2004 and 2005 and Weeks Bay in July and September 2004 were recovered and refrozen at -70°C; it remains uncertain if these samples will prove useful for any analysis.

All clones produced during the course of the project have been retained; their viability after thawing, sitting at room temperature, and refreezing is questionable, and some genes have been recloned All primers for making these clones, as well as all primers for q-PCR were discarded; new q-PCR primers have been ordered and are working well All refrigerated and frozen laboratory and analysis supplies/reagents for RNA and DNA extraction, purification and analysis, q-PCR, and cloning were discarded and have been replaced with new supplies All surviving computer and electronic equipment has been thoroughly tested following the storm and determined to function the same as pre-Katrina The exposure rooms in the Toxicology building survived the storm, but all culture and holding facilities were destroyed and have been rebuilt in approximately half the wet laboratory space as pre-Katrina These facilities are currently up and running and exposures have been conducted post-Katrina Overall, project personnel lost 4 months of research time during Katrina recovery and rebuilding.

Conclusions

We have met most of our objectives for Y ear 4, although data processing and analysis has been slowed due to Hurricane Katrina impacts We have identified a suite of 47 hypoxia-relevant genes showing changes in expression under laboratory or field hypoxia conditions (Objective 1). Data have been fully analyzed for chronic and cyclic laboratory exposures and it appears that mitochondrial genes such as 16SrRNA, cytB , Ccox I,and Ccox III as well as hemocyanin and Fe-proteins provide promise as indicators of chronic severe hypoxia exposure in grass shrimp Few indicators of cyclic hypoxia have been identified to date, but ongoing construction of cyclic hypoxia-responsive microarrays should help identify additional important genes (Objective 2). Real-time qPCR analysis has verified the gene expression results demonstrated using macroarrays for a variety of genes, and supports the use of α-tubulin as a normalization gene (Objective 3) Characterization of salinity and oxygen profiles at the Weeks Bay and East Bay, Pensacola field sites has shown that sites in both estuaries exhibit cyclic hypoxia during the summer, with some sites experiencing DO values < 2.0 ppm on a daily basis (Objective 4) Analysis of 2005 field data was severely impacted by Hurricane Katrina; much of the gene expression data were lost and analysis of November samples delayed Whole animal data (reproduction and condition factor) from 2005 field samples are similar to data reported for the 2004 and 2003 seasons, showing no differences between field sites within an estuary (Objective 5) We added a new objective this year, to determine if there are any population level differences in hypoxia responses between grass shrimp from Weeks Bay and East Bay (Objective 6) Completion of this objective has been delayed by Hurricane Katrina, al though results from the response of the grass shrimp in the laboratory suggest Mobile Bay shrimp may be more hypoxia-sensitive than Pensacola shrimp Finally, we have identified a potential suite of 28 molecular and whole-animal indicators of hypoxia (Objective 7) that can be used in overall CEER-GOM integration to help understand the effects of hypoxia from molecular to landscape scales.


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, Brown-Peterson NJ, Larkin P, Patel V, Denslow N, Manning S, Brouwer TH. Molecular and whole animal responses of grass shrimp, Palaemonetes pugio, exposed to chronic hypoxia. Journal of Experimental Marine Biology and Ecology 2007;341(1):16-31. R829458C003 (2005)
  • Full-text: Science Direct Full Text
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  • Abstract: Science Direct Abstract
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  • 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
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  • Supplemental Keywords:

    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
  • 2004 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