![[logo] US EPA](https://webarchive.library.unt.edu/eot2008/20081106011437im_/http://www.epa.gov/epafiles/images/logo_epaseal.gif){kind=logo}
2002 Progress Report: Individual Level Indicators: Molecular Indicators of Dissolved Oxygen Stress in Crustaceans
EPA Grant Number: R829458C003Subproject: 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: Levinson, Barbara
Project Period: December 1, 2001 through November 30, 2005 (Extended to May 20, 2007)
Project Period Covered by this Report: December 1, 2001 through November 30, 2002
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000)
Research Category: Ecological Indicators/Assessment/Restoration
Description:
Objective:Increased nutrient loading into estuarine ecosystems may exacerbate naturally occurring diurnal fluctuations of dissolved oxygen (DO) and may result in chronic hypoxic conditions, which are one of the major factors responsible for declines in habitat quality. Despite the ecological importance of hypoxia, little is known about the sublethal effects of chronic hypoxia, or hypoxic → normoxic cycles on estuarine organisms, and we do not have indicators for recognizing populations that are suffering from chronic DO stress. The overall long-term objectives of this research project are to: (1) develop molecular indicators of DO stress (hypoxia and diurnal DO cycles) in blue crabs (Callinectes sapidus), penaeid shrimp, and grass shrimp; and (2) determine if the molecular signals can serve as predictive indicators of reduced fitness (molting and reproduction) in grass shrimp.
The objectives for Year 1 of the project are to: (1) develop DNA macroarrays and antibodies for use in detection of expression of DO stress proteins in C. sapidus, penaeid shrimp, and grass shrimp; (2) test the response of the molecular indicators to chronic hypoxia and diurnal DO cycles in C. sapidus and shrimp under controlled laboratory conditions; and (3) validate response of the DO stress indicators in C. sapidus, shrimp, and grass shrimp collected from DO-stressed and reference sites in the Mississippi Sound and the Grand Bay National Estuarine Research Reserves.
Progress Summary:In Year 1 of the project, we conducted exposures of C. sapidus and brown shrimp to hypoxia and hypoxia → normoxia cycles under controlled laboratory conditions. Survival of shrimp under hypoxic, and especially under cyclic DO conditions, is significantly reduced, whereas the survival of C. sapidus is not affected. This suggests that increases in frequency, intensity, duration, and extent of hypoxic conditions in estuaries may have serious implications for shrimp fisheries by reducing the availability of suitable habitat.
To quantitate the expression of the hypoxia-responsive genes in C.
sapidus and shrimp from laboratory exposures and from field sites
with known DO histories, we cloned and sequenced partial coding sequences
of C. sapidus (10), brown
shrimp (Farfantepenaeus aztecus) (9), and grass shrimp (9) proteins:
Hsp70, CdMT1, CuMT3 (C. sapidus only), cytosolic superoxide
dismutase (SOD), mitochondrial SOD, hemocyanin, actin, ribosomal proteins S15,
S14, and L23. The first six
were selected because their transcription appears to be controlled by the intracellular
redox potential. The last four served as internal standards. The cDNAs were
used to construct a macroarray by robotically spotting polymerase chain reaction
(PCR) products of these clones onto nylon membranes. The macroarrays were hybridized
with [
-33P]
labeled cDNA prepared from the hepatopancreas of control crabs, crabs that
had been kept under hypoxic conditions (2-3 ppm DO, for 5 days),
and crabs that were exposed to diurnal fluctuations of DO (3 to 8 ppm DO for
5 days). Statistically significant decreases (P < 0.05) in transcription
of mitochondrial MnSOD, hemocyanin and ribosomal S15 and L23 genes were observed
in hypoxia-exposed crabs, but there were no significant differences in any
gene transcription from crabs exposed to DO cycles. These observations suggest
that chronic hypoxia constitutes a greater stress for crabs than conditions
that mimic natural diurnal fluctuations of DO, and the results indicate that
the hypoxia-responsive macroarrays might be useful tools for monitoring effects
of hypoxia in estuarine crustacea.
To measure changes in the concentrations of proteins in crabs and shrimp in response to DO stress, we tested a large number of commercially available and custom-made antibodies using Western blots. The results obtained to date suggest that hemocyanin and MnSOD may be a useful indicator of hypoxia-induced stress. Hemocyanin concentrations significantly increase after 5 days of hypoxia. Whereas control crabs show two bands on Western blots corresponding to the cytosolic and mitochondrial forms of MnSOD, crabs exposed to 5 and 10 days of hypoxia show a significant increase of what appears to be a high-molecular weight, cross-linked form of MnSOD.
Crab Hypoxia Study. An exposure of C. sapidus to low oxygen under flow-through conditions has been completed. Six crabs were placed in each of the eight test aquaria. Six aquaria were exposed to nominal oxygen concentrations of 2 to 3 mg/L, while the remaining two aquaria received seawater at normoxic conditions of 6 to 8 mg/L. The mean DO for the study was 2.46 ± 1.02 mg/L. Crabs were kept separated within the aquarium by 1.2 cm2 mesh plastic sheeting cut with compartments to prevent cannibalism. Crabs were sampled after 0, 5, 10, and 15 days from aquaria maintained under hypoxic conditions. Crabs under normal conditions of oxygen concentrations were sampled on days 0 and 15. Hepatopancreas tissues were archived in RNA later to be processed for macroarray analysis and frozen at -70°C for Western blot analysis. Several crabs died during the study, but there was no significant difference in survival between hypoxic and normoxic aquaria.
Shrimp Hypoxia Study. An exposure of F. aztecus to conditions of low DO was conducted in much the same manner as the crab exposure. Twelve shrimp were compartmentalized in each of the six hypoxic and two normoxic aquaria. The DO level remained more stable than in the previous study, with a mean of 2.64 ± 0.56 mg/L. Shrimp were sampled after 0, 5, 10, and 12 days from aquaria maintained under hypoxic conditions. Shrimp under normal conditions of oxygen concentrations were sampled on days 0 and 12. Hepatopancreas tissues from 12 shrimp/treatment were archived for RNA macroarray analysis, and 12 shrimp/treatment for Western blot analysis. Shrimp survival was significantly affected by the low DO, with 40 percent survival after 12 days.
C. sapidus Exposure to Diurnal Cycling of DO. Exposure was conducted by limiting the oxygen addition to the dilution water reservoir during the evening hours, which resulted in a drop in DO in the treatment aquaria from 2 to 3 mg/L by early morning. Test crabs depleted the oxygen within the aquaria at a rate, which made it unnecessary to displace oxygen by nitrogen sparging. In this evaluation, the frequency of the addition of oxygen was limited in the evening to allow for a drop in the DO. In the morning, the frequency of oxygen aeration was increased in the reservoir, which increased the aquaria to normoxic levels by late morning or early afternoon. This cycle was repeated daily for the duration of the exposure. The DO exhibited a consistent cycle of peak DO of 6 to 8 mg/L during the day and a minimum DO from 4 to 2 mg/L during the early morning hours. Crabs from the cyclic DO treatments were sampled after 0, 5, and 10 days. Those maintained under a consistent normal condition of DO were sampled on day 0 and after 10 days. Sampling was discontinued after 10 days due to crab mortalities limiting the number of sample events. There was no significant difference in survival between cyclic DO and normoxic aquaria.
F. aztecus Exposure to Diurnal Cycling of DO. In this exposure, oxygen was
displaced during the evening hours by nitrogen sparging of a water reservoir
supplying the exposure system. This affected a gradual drop in DO to 2 to 3
mg/L in the treatment aquaria by early morning. In the early morning of the
following day, oxygen then was introduced into the reservoir, raising the DO
to near normoxic levels (6 to 8 mg/L) by late morning to early afternoon. The
cycle repeated for the duration of the exposure. There were eight total aquaria;
two aquaria were maintained under normoxia conditions without cycling, and
six were exposed to the cycling DO (2 to 3 mg/L in the morning to concentrations
near or above saturation (6 or greater mg/L) during the day. The cycling DO
exhibited a consistent cycle of peak DO of 8 mg/L or greater during the day
and a minimum DO from 4 to 2 mg/L during the early morning hours. Shrimp maintained
under a normal condition of DO were sampled on day 0 and after 12 days. Shrimp
maintained in aquaria with a cycling DO were sampled after 0, 5, 10, and 12
days. Survival of shrimp exposed to cycling DO differed significantly from
normoxic shrimp beginning as early as Day 3, with only 20 percent survival
after 12 days.
Antibodies
Hemocyanin. These available antibodies work well for Western blot analysis and enzyme-linked immunosorbent assays.
Oxidized Proteins. Antibodies such as Zymed, which detects oxidized proteins labeled with dinitrophenylhydrazine (DNPH), worked well with hypochlorite-oxidized BSA using Western blot analysis. However, hemocyanin, which is the most abundant protein in the hepatopancreas, is a glycoprotein. The aldehyde group of N-acetylglucosamine also reacted with DNPH, resulting in false positives.
Metallothioneins (MTs). A molluscan MT antibody, EnVirtue, claimed to crossreact with crustacean MTs, did not bind with C. sapidus MTs. Antibodies made against C. sapidus MT1 and MT3 peptide recognized their cognate peptides without showing any cross reactivity. MT3 antibody bound with the MT3 native protein, but only weakly. The MT1 antibody did not bind with the MT1 native protein, but reacted with the MT3 native protein.
Heat Shock Protein (Hsp)-70. With Hsp-70 (Ab EnVirtue, StressGen, and Affinity BioReagents), 70 kDa band in heat-shocked crabs and grass shrimp did not exist.
Hsp 60. Hsp-60 (Ab EnVirtue, StressGen, and Affinity BioReagents) did not bind to proteins in hepatopancreas extract; however, StressGen antibody reacted with 60-kDa protein from C. sapidus mitochondrial extract.
Mitochondrial MnSOD. MnSOD2 Ab (StressGen) worked well.
Cytosolic MnSOD. MnSOD2 Ab (StressGen) also worked well.
Extracellular Cu,ZnSOD. With Cu,ZnSOD Ab (StressGen), no bands were observed with C. sapidus hemocyte extract or with purified EC-SOD protein.
Glutathione Peroxidase (GSH-Px). With GSH-Px Ab (Biotrends), GSHPx Ab worked well with purchased purified protein, but not with tissue extracts.
Catalase. Catalase Ab worked well with purchased purified protein, but not with tissue extracts.
Hypoxia Inducible Factor 1 alpha (HIF) Ab (Novus Biologicals). Tissues from hypoxic crab showed bands of 75 kDa and 57 kDa.
Lactate Dehydrogenase (Research Diagnostics). This worked well with purchased purified protein, but not with the hepatopancreas homogenate.
Actin (Chemicon). This reacted with crab muscle extract, but not with the hepatopancreas homogenate.
Archived tissues from hypoxia-exposed crabs are in the process of being analyzed with antibodies against hemocyanin, MnSOD, and HIF". The results obtained to date suggest that MnSOD may be a useful indicator of hypoxia-induced stress. Whereas control crabs show two bands on Western blots corresponding to the cytosolic and mitochondrial forms of MnSOD, crabs exposed to 5 and 10 days of hypoxia show a significant (P=0.033 and P=0.011, respectively) increase of what appears to be a high-molecular weight, cross-linked form of MnSOD.
Field Evaluations. During Project Year 1, the field studies consisted of five events (September 5, September 13, October 1, October 31, and November 14), in which two selected sites were examined to develop the most viable approach for animal collection and field validation of hypoxic conditions. The two study locations were: Bayou Cumbest (BC), located near the mouth of the Escatawpa River in Jackson County, MS, and a small bayou on Garcon Point (GP), Santa Rosa County, Florida. The headwaters of BC are moderately populated and may be subject to frequent introduction of nutrient waste. The headwaters are subject to periods of low DO (less than 2 ppm) at night, rising to saturation during the day. The mouth of BC, which is tidally flushed and does not experience this same low oxygen during the evening hours, was monitored as a reference site of normoxic conditions. The second site, visited on two of the five events this year, was a small bayou on GP in Santa Rosa County near Milton, Florida. GP is the point of a peninsula separating Escambia and East Bay. The bayou has been designated as Brouwer’s Bayou. The bayou generally is only 1 to 3 feet in depth at maximum and is subject to cyclic conditions of oxygen. The outside of the mouth of the bayou opens into the East Bay, which is a sandy bottom bay. The area outside of the mouth and near the shore was selected as the reference or normoxic site. These sites (BB-2 and GP-1) also were monitored by the biofilm unit of CEER and offered the opportunity for data sharing.
The targeted number of grass and brown shrimp for each site is 20 (10 for RNA later and 10 for Westerns blots). For C. sapidus, the number is 10. Sampling trips were projected for times immediately following neap tides, when flushing in days prior has been minimal. Unfortunately, severe weather events including tropical storms Hannah and Isidore resulted in normoxic conditions at the sampling sites. No animals were captured at the hypoxic site in BC. The targeted number of grass shrimp was captured at the GP hypoxic and reference sites. C. sapidus were 80 percent and 70 percent of the desired numbers; F. aztecus were 25 percent and 100 percent, respectively. Hepatopancreas tissues of the animals were processed in the field and kept on dry ice or in RNA later, before being archived at -70°C for later processing.
We have completed most of our objectives for Year 1. Cloning of DO stress genes for the three Crustacean species has been completed and DNA macroarrays for C. sapidus have been constructed. Western blot analysis has been developed (Objective 1). Laboratory exposures of crabs and shrimp to hypoxia and hypoxia → normoxia cycles have been completed. Tissues are being analyzed for gene expression and protein levels (Objective 2). Field studies have been conducted and tissues have been archived for analysis of molecular DO stress parameters (Objective 3). Analysis of many tissues is still ongoing. Because cloning of all the genes has been completed, all efforts are now dedicated to preparing RNA and cDNA from the archived tissues. Similarly, we now are focusing on conducting Western blots of tissue homogenates. We expect that all of the tissues will have been analyzed by late spring. The results obtained with the DNA macroarrays are promising because significant changes in expression of hypoxia-responsive genes can be detected. However, the changes observed under controlled laboratory conditions are rather small. It may be difficult to observe significant changes under uncontrolled conditions in the field. To address this potential problem, we are now in the process of cloning C. sapidus genes that are upregulated or downregulated in response to hypoxia by PCR-select subtractive hybridization. We aim to clone and sequence 96 hypoxia-responsive genes and use the ones that show the greatest fold induction or repression to construct new macroarrays (together with the targeted genes that we already have). We expect to have this project completed by mid-April.
Future Activities:We will continue to: (1) develop molecular indicators of DO stress (hypoxia and diurnal DO cycles) in C. sapidus, penaeid shrimp, and grass shrimp; and (2) determine if the molecular signals can serve as predictive indicators of reduced fitness (molting and reproduction) in grass shrimp.
Journal Articles:No journal articles submitted with this report: View all 27 publications for this subproject
Supplemental Keywords:p>population, community, ecosystem, watersheds, estuary, Gulf of Mexico, nutrients, hypoxia, innovative technology, ecoindicators, biomarkers, water quality, remote sensing, global information system, GIS, integrated assessment, risk assessment, fisheries, conservation, restoration. , 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
Progress and Final Reports:
Original Abstract
2003 Progress Report
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