250 (BRO-1117-841086) Changes in gene expression and reproduction in grass shrimp, Palaemonetes pugio, in response to chronic and intermittent hypoxia.
Start time: 8:00 AM
Brouwer, M¹, Brown-Peterson, N¹, Manning, S¹, Larkin, P², , N, Denslow, N³, ¹ University of Southern Mississippi, Ocean Springs, MS² EcoArray LLC, Alachua, FL³ University of Florida, Gainesville, FL
Despite the ecological importance of hypoxia, little is known about the sublethal effects of chronic hypoxia on estuarine organisms, and there are no indicators for recognizing populations that are suffering from chronic hypoxic stress. Here we examine the use of hypoxia-responsive gene expression profiles in the grass shrimp, Palaemonetes pugio, as early warning signals of impacts of hypoxia. We cloned 78 potential hypoxia-responsive genes using PCR-select subtractive hybridization. The cDNAs were used to construct macroarrays. Next we exposed grass shrimp to chronic and intermittent hypoxia in a flow-through system and examined effects on gene expression and reproduction. Additionally, gene expression profiles and reproductive parameters were evaluated in wild grass shrimp collected from diurnally hypoxic and normoxic field sites across a large range of spatial and temporal scales. Changes in gene expression profiles are dependent on DO levels and length of exposure. When exposed to 1.5 ppm DO, genes encoding proteins involved oxygen transport and ATP synthesis are upregulated on a time scale of days. Transcription of mitochondrial genes (16S rRNA, cytochrome b and cytochrome c oxidase subunits 1 and 3) and genes involved in heme/iron metabolism are upregulated after 7 days, followed by a coordinated downregulation of the same genes after 14 days. Grass shrimp collected in diurnally hypoxic marsh sites in Pensacola Bay show similar downregulation of mitochondrial gene transcription, whereas shrimp from normoxic controls sites do not. Chronic hypoxia increases fecundity. Diurnal hypoxia decreases fecundity and increases brood interval. Percent gravid females are lower in diurnally hypoxic field sites compared to normoxic sites. These results suggest that hypoxia-responsive macroarrays might be useful for monitoring effects of hypoxia in estuarine crustacea and that it might be possible to link these molecular signals to reproductive fitness. Supported by EPA-STAR Grant R 82945801.

251 (CAR-1117-829710) The Response of Sheepshead Minnows to Hypoxia Exposure: Connecting Molecular and Reproductive Endpoints.
Start time: 8:20 AM
Carlson, E¹, Ryan, R¹, Manning, C¹, Karels, A¹, Mitchell, S¹, Ryan, K¹, Brouwer, M¹, ¹ University of Southern Mississippi, Ocean Springs, MS, USA
Inadequate dissolved oxygen (DO) can be a major stressor in estuarine habitats. Hypoxic conditions often result from increasing urban development, agricultural runoff, and industrial pollution. Some estuarine organisms, including the sheepshead minnow (Cyprinodon variegatus), are well adapted for survival in low DO environments. This study sought to examine the effects hypoxia exposure upon sheepshead minnow reproduction and hepatic gene expression. Hypoxia-induced alterations in gene expression were screened using cDNA microarrays containing 700 clones from hypoxia-responsive suppression subtractive hybridization and sheepshead liver cDNA libraries. In addition, several clones representing genes known to be involved in the hypoxic response of other organisms (i.e., VEGF, HIF-1 alpha) were included on the arrays. Fish were exposed to hypoxia (1.5 and 2.5 mg DO /L) or normoxia (7-8 mg DO /L) in flow-through systems and liver was sampled for RNA isolation at 10h and 2, 4, 7, and 14 d. In addition, a reproductive study was performed in which egg yield, fertilization, hatching success, and larval growth /survival were monitored for a month during continuous exposure to 1.5 mg DO /L. Microarray analysis of these exposures has revealed that several hepatic genes display clear changes in response to hypoxia including interferon inducible factor, enolase, and butyrate respiratory factor. In addition, several genes displayed sex-specific responses to hypoxia. Although hypoxia did not alter sheepshead gonado-somatic indices, egg production, or fertilization rate, significant effects were seen upon embryo and larval survival. Hypoxia-exposed larvae took an average 9 d to hatch compared to 4.5 d for normoxic controls. Furthermore, hypoxic larvae were developmentally immature at hatching with most larvae not surviving to 14 d post-hatch. Experiments examining the effects of hypoxia exposure upon embryonic development and gene expression are ongoing. Overall, it is expected that the current study will reveal gene expression patterns important for the maintenance of reproductive success during hypoxic stress. Elucidation of molecular pathways responsible for survival under prolonged hypoxia exposure may prove useful to both biomedical researchers and environmental toxicologists.

252 (CHE-1117-837976) Hypoxia limits estuarine fish reproduction.
Start time: 8:40 AM
Cheek, A¹, Landry, C², Steele, S², Sutton, A², ¹ Univ Texas Health Science Center at Houston, School of Public Health, Houston, TX, USA² Southeastern Louisiana University, Hammond, LA, USA
Resident salt marsh fishes tolerate periodic hypoxia, but its effects on reproduction are poorly understood. Our hypothesis is that dissolved oxygen (DO) stress limits reproduction. Our aim was to identify reproductive indicators that consistently respond to hypoxia. We measured two morphological and two biochemical indicators in Gulf killifish (Fundulus grandis): gonadosomatic index (GSI), condition factor (K = 100,000*length/weight³), sex steroid hormone levels, and vitellogenin (VTG) levels. In the laboratory, wild-caught killifish were exposed to normoxia (7.3 mg/L DO) or chronic hypoxia (1.8 mg/L DO) for one month. Hypoxia suppressed all reproductive parameters in males and all except VTG in females. In the field, F. grandis were collected from normoxic and cyclically hypoxic marsh creeks in Terrebonne Bay, LA; Pensacola Bay, FL; and Weeks Bay, AL in April, July, and November 2002-2004. Indicators varied in temporal sensitivity as demonstrated by the response patterns observed in marshes with different durations of diel DO minima ≤ 2 mg/L. Medium-term (5 weeks) diel hypoxia in a Weeks Bay marsh creek suppressed male and female GSI and steroid hormones, but condition factor and vitellogenin (VTG) were unchanged. Long-term (> 3 mos) diel hypoxia in a Pensacola Bay marsh creek did not alter male or female GSI or steroid hormones, but did reduce condition factor and VTG. Duration of diel hypoxia was unknown for Terrebonne Bay, but was probably between 5 to 12 weeks, based on the pattern of indicator responses. Like Weeks Bay, male GSI and steroid hormones were significantly reduced at the hypoxic Terrebonne Bay site, but condition factor was not. No female indicators were reduced. These morphometric and endocrine indicators respond to DO stress on different time scales, so are potentially useful as retrospective indicators of stress duration and as early warning indicators of reproductive failure.

253 (THO-1117-822334) Neuroendocrine changes associated with reproductive dysfunction in Atlantic croaker after exposure to hypoxia.
Start time: 9:00 AM
Thomas, P¹, Rahman, S¹, Kummer, J¹, Khan, I¹, ¹ University of Texas Marine Science Institute, Port Aransas, Texas, USA
Previously we have shown that chronic laboratory and field exposure to low moderate hypoxia causes a dramatic impairment of reproductive processes and endocrine function in Atlantic croaker (Micropogonias undulatus). In females, decreases in plasma estradiol, hepatic estrogen receptor mRNA, and plasma vitellogenin levels were observed, indicating an impairment of estrogen signaling after hypoxia exposure. Several indices of neuroendocrine function were also measured to determine whether these endocrine disturbances are due to hypoxia-induced changes at higher levels of the hypothalamus-pituitary-gonad axis. Gonadotropin secretion in response to a gonadotropin releasing hormone (GnRH) injection was attenuated after hypoxia exposure. Hypothalamic GnRH mRNA levels were also decreased, indicating a disturbance of neuroendocrine function in hypoxia-exposed fish. The results suggest the neuroendocrine system is a major site of interference by low dissolved oxygen resulting in reproductive dysfunction in fishes.

(58157) Break.
Start time: 9:20 AM

254 (COO-1117-814922) Antioxidant enzyme response to hypoxia-induced oxidative stress in the estuarine fish Leiostomus xanthurus.
Start time: 10:00 AM
Cooper, R¹, Clough, L¹, Farwell, M¹, West, T¹, ¹ East Carolina University, Greenville, NC, USA
The objective of this research was to examine the physiological impact of hypoxia on antioxidant enzyme activity levels in the estuarine fish Leiostomus xanthurus (spot). The research was conducted in three parts: 1) an examination of the response of antioxidant enzymes to varying degrees of hypoxia; 2) an examination of the response of antioxidant enzymes to hypoxic preconditioning, periods of sub-lethal hypoxic exposure followed by reoxygenation; and 3) an examination of the response of antioxidant enzymes to a subsequent hypoxic challenge after hypoxic preconditioning. The antioxidant enzymes assayed included superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). In response to varying degrees of oxygen saturation, SOD activity levels increased after 12 hours of exposure to 10% (0.8 mg/L) relative to 50% (4.0 mg/L) oxygen saturation. A single, variable duration exposure to hypoxic preconditioning was shown to increase activity levels of SOD and decrease activity levels of CAT immediately following exposure. Activity levels continued to steadily change over the normoxic recovery period, with the degree of change roughly proportional to the duration of the hypoxic preconditioning. Hypoxic preconditioning, applied using a cyclic protocol, did not have an immediate impact on activity levels of the antioxidant enzymes. When preconditioned fish were subsequently exposed to a hypoxic challenge, 12 of the 16 significant comparisons (75%) resulted in preconditioned fish showing reduced activity levels when compared to non-preconditioned fish. This suggests that hypoxic preconditioning in spot does not protect against subsequent hypoxic exposure by increasing activity of the antioxidant enzymes as reported with other organisms. Finally, high levels of inter-individual variation in activity levels were observed in these fish. This variation does not appear to be linked to prior exposure history; instead it likely reflects variable genetic expression between individuals.

255 (RIP-1117-660338) Eavesdropping on pipefish dinner conversation - a novel measure to assess hypoxia.
Start time: 10:20 AM
Ripley, Jennifer¹, Foran, Christy¹, ¹ West Virginia University, Morgantown, WV, USA
This research proposes to utilize the acoustical behavior of two sympatric pipefish species from the Chesapeake Bay as an indication of ecosystem activity and health. No environmental variable of such ecological importance to estuarine and coastal marine ecosystems has changed so drastically, in such a short period of time, as dissolved oxygen. Hypoxic, or low dissolved oxygen, events are severe in the Chesapeake Bay. Oxygen concentrations seasonally decline to anoxic levels, impacting populations by decreasing growth rates, altering behavior, disrupting the endocrine system and interfering with physiological activities. Pipefish were selected for study because they are abundant inhabitants of eelgrass beds (Zostera marina) during the summer months. Thus, they occupy the same area used as nursery grounds by many commercially important fish and shellfish species and their residency overlaps with the period of hypoxia problems. We collected Northern, Syngnathus fuscus, and Dusky pipefishes, Syngnathus floridae, from the relatively pristine Chincoteague Bay, Virginia and audiovisually recorded feeding behavior in the laboratory of fish held in normoxic (>5 mg/L O₂) and hypoxic (2 mg/L O₂) conditions for five days. The production of high frequency (0.9-1.6kHz), short duration (5-43msec) sounds by both species indicate sounds are generated by stridulation or the striking together of hard parts such as two bones. Sound production coincides with feeding strikes and wet weight of ingested food. Thus, sound production serves as an accurate measure of feeding activity. Under hypoxic conditions, reduced food intake correlates with decreased sound production. A drop in food intake and acoustical behavior is evident after only 24 hours of hypoxia exposure. Reductions in feeding will ultimately impact growth and health and eventually reproductive output as resources are devoted to survival instead of gamete production. This work suggests a novel technique to acoustically monitor field sites to assess ecosystem changes.

256 (KOD-1117-802611) Hypoxia restricts recruitment of the stomatopod Oratosquilla oratoria in Tokyo Bay, Japan.
Start time: 10:40 AM
Kodama, Keita¹, Horiguchi, Toshihiro¹, Kume, Gen¹, Nagayama, Satoshi², Shimizu, Takamichi³, Shiraishi, Hiroaki¹, Morita, Masatoshi¹, Shimizu, Makoto⁴, ¹ National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan² Chiba Prefectural Fisheries Research Center, Awa, Chiba, Japan³ Kanagawa Prefectural Fisheries Research Institute, Miura, Kanagawa, Japan⁴ The University of Tokyo, Bunkyo, Tokyo, Japan
We used field surveys to investigate the reproduction and recruitment of the mantis shrimp Oratosquilla oratoria and the spatiotemporal pattern of hypoxia in Tokyo Bay. The spawning seasons of large and small females were May-July and July-August, respectively. More larvae were spawned by small females than by large females, suggesting that there was a low spawning stock abundance of large females. Newly settled juveniles appeared in the northeastern shallow coastal area of the bay in September-October, implying larval transportation from the spawning ground in the southern part of the bay and the successful settling of only those larvae derived from spawning from August onward by small females. Juvenile settlement from spawning before July by large females failed completely, probably because hypoxia (dissolved oxygen concentration of < 2 ml l^-1) persisted in the bottom layer of the north-central part of the bay from May to October. After the hypoxia had abated, the distribution of juveniles expanded to the south-central deep area of the bay, most likely by migration. Hypoxia probably restricts the settlement period and distribution of O. oratoria. Conservation of the spawning stock of small females and regulation to decelerate hypoxia-enhancing eutrophication in the bay are needed for recovery of O. oratoria stocks.

257 (MAX-1117-825477) Effects of hypoxia and anoxia on benthic infauna of the deep Caspian Sea.
Start time: 11:00 AM
Tait, R.¹, Maxon, C.², Parr, T.³, Germano, J.⁴, Newton III, F.⁵, ¹ ExxonMobil Production Company, Houston, TX, USA² CH2M Hill, San Diego, CA, USA³ Terry Parr Consulting, San Diego, CA, USA⁴ Germano and Associates, Bellevue, WA, USA⁵ Battelle Memorial Institute, Carlsbad, CA, USA
Deepwater sediment studies were conducted in the south Caspian Sea in support of oil and gas exploration and production over a four year period. Results document the transformation of sediments from hypoxic to possible anoxic conditions with concomitant increases in stratification of the water column, organic carbon concentrations, and laminar banding at the sediment surface independent of exploration or other anthropogenic activities. This study examines the effect of these physical changes on the resident infauna, which was initially dominated by segmented annelid worms, common in oxygen minimum zones. Decreases in abundance, species density, and biomass were attributed to increases in biological oxygen demand at the sediment surface, suggesting that various stages of hypoxia can be identified and used to predict azoic thresholds for deepwater infauna in the Caspian Sea.

258 (BOO-1117-816920) Laboratory Determination of Molybdenum Accumulation Rates as a Measure of Hypoxic Conditions.
Start time: 11:20 AM
Boothman, W¹, Coiro, L¹, ¹ U.S. Environmental Protection Agency, Narragansett, RI, USA
Previous studies indicate that molybdenum (Mo) in marine sediments may serve as a useful surrogate for direct measurement of hypoxic and anoxic conditions in overlying waters. Analyses of field samples show a relationship between accumulation of Mo and the period of time that dissolved oxygen (DO) concentrations in near-bottom waters are below 2.3 mg/L; lab experiments show significant Mo accumulation at 0.6 mg DO/L, but none at 2.9 mg DO/L. Field samples also indicate that accumulated Mo is stable in sediments for at least 3 months when DO levels rise, whereas the lab experiments show possible loss of Mo from reduced sediments when exposed to DO-saturated seawater. To refine the quantitative relationship between Mo accumulation and DO concentrations, homogenized marine sediments were exposed for up to 12 weeks to seawater maintained at 5 concentrations of DO between 0.7 and 3.5 mg/L, as well as a control (saturated), with DO concentrations and temperature monitored daily throughout the experiment. Sediments initially were either anoxic throughout or modified to present a 2-cm deep oxic layer. Sediments were sampled at 2 week intervals, at which times depth profiles of redox potential were measured in one oxic and anoxic core at each DO level. Profiles show a rapid convergence of redox conditions, regardless of starting conditions, but depth of the redox potential discontinuity remained sensitive to the concentration of DO in overlying water. Mo accumulation rates determined for each level of DO are compared to determine the critical concentration of DO in seawater below which Mo accumulation is constant and concentrations are a function of time below the critical concentration. The results, combined with sedimentation rates in field locations, may be used to determine the fraction of time waters overlying the sediments may have suffered from hypoxia.