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Application of flow cytometry to assess deepwater horizon oil toxicity on the eastern oyster Crassostrea Virginica spermatozoa.

ABSTRACT The Deepwater Horizon (DWH) oil spill in 2010 resulted in the release of millions of liters of oil into the Gulf of Mexico. Dispersants such as Corexit 9500A were used to disperse oil both at the well-head and at the surface. Polycyclic aromatic hydrocarbons and dispersants have been shown to deleteriously affect early life stages of organisms including oysters. This study examined the impacts of chemically enhanced water-accommodated fractions [CEWAF; 1.29-26.14 [micro]g/1 [tPAH5.sub.0] (a sum of 50 different polycyclic aromatic hydrocarbons)], high-energy water-accommodated fractions (HEWAF: 16.53-248.89 [micro]g/l [tPAH5.sub.0]), and dispersants (0.625-10 mg/l) on the cellular functions (viability, mitochondrial membrane potential (MMP), reactive oxygen species production (ROS), and acrosomal integrity) and resulting fertilization success of eastern oyster Crassostrea virginica spermatozoa. While viability of spermatozoa was not affected by CEWAF and HEWAF at concentrations tested, dispersant exposure caused significant decrease in viability at the highest concentration tested. Fertilization success as well as MMP and ROS production were significantly decreased upon exposure to CEWAF, HEWAF, and dispersants. Also, although not affected by HEWAF exposure, acrosomal integrity decreased upon exposure to CEWAF and dispersants at concentrations tested. The results of this study suggest that impaired fertilization and reduced viability observed after exposure to DWH oil spill contaminants may result, at least partially, from alterations of cellular functions of spermatozoa and contribute to negative effects on oyster populations, and thus the ecology and economy of the Gulf of Mexico.

KEY WORDS: oysters, Deepwater Horizon oil, PAH, dispersant, cellular impacts, Crassostrea virginica

INTRODUCTION

In April 2010, after the explosion of the Deepwater Horizon (DWH) drilling rig operated by BP off the coast of Louisiana, millions of barrels of crude oil were released in the Gulf of Mexico, being the largest marine oil spill in U.S. history (National Commission on the BP Deep Ocean Horizon Oil Spill Offshore Drilling 2011). After almost 3 mo and various attempts to stop the leak, the well was finally cemented on July 15, 2010 (Crone & Tolstoy 2010). In the meantime, chemical dispersants, including Corexit 9500A, were used both underwater and on surface to dissipate oil slicks (Kujawinski et al. 2011); however, despite these efforts, released crude oil contaminated Louisiana, Mississippi, Alabama, and Florida coasts (Kelley 2012).

The eastern oyster Crassostrea virginica is an economically and ecologically important benthic species that propagates along the coast of the United States from Maine to the Gulf of Mexico (Galtsoff 1964, Buresson & Ragone-Calvo 1996, Ford 1996). Eastern oysters are highly valued as food, but their ecological significance is as great or even more important (Grizzle et al. 2008, Volety et al. 2014). Up to 70% of the organic matter filtered is assimilated by the oysters and the remainder is deposited on the bottom where it provides food for benthic organisms. This secondary production, combined with a complex three-dimensional reef structure serving as nesting habitat and/or refuge, attracts numerous species of invertebrates and fishes (Tolley & Volety 2005, Volety et al. 2014). It is this larger ecological function that constitutes a valued ecosystem component. Depending upon size, individual oysters filter 4-34 1 of water per hour, removing phytoplankton, particulate organic carbon, sediments, pollutants, and microorganisms from the water column (Coen et al. 1999, Grabowski & Peterson 2007, Abeels et al. 2012, Volety et al. 2014).

The DWH oil spill occurred at the beginning of Crassostrea virginica spawning season. Exposure of spermatozoa and oocytes to toxicants was inherently likely for eastern oysters that broadcast spawn their unprotected gametes directly into the surrounding water, where external fertilization occurs. Therefore, the fertilization success of C. virginica had the potential to be adversely affected by the petroleum hydrocarbons and dispersants found in the Gulf of Mexico at that time. Previous studies have shown that spermatozoa and oocytes can be affected by exposure to polycyclic aromatic hydrocarbons (PAH), major constituents of crude oil. For example, exposure of gametes of Pacific oysters Crassostrea gigas, and C. virginica to PAH and other sediment-associated chemicals negatively impacted spermatozoa motility and fertilization success as well as embryonic and larval development (Pelletier et al. 2000, Geffard et al. 2001, Lyons et al. 2002, Jeong & Cho 2005, Nice 2005, Wessel et al. 2007, Laramore et al. 2014, Vignier et al. 2015); however, although most ecotoxicological studies on response of gametes to toxins use fertilization success as the assay end point (Dinnel et al. 1989, Warnau et al. 1996, Mwatibo & Green 1997, Vaschenko et al. 1999, Ghirardini et al. 2001, Arslan et al. 2007), various cellular defects of gametes can alter fertilization. Success of fertilization depends on various cellular characteristics of the spermatozoa such as membrane and acrosome integrity, mitochondrial activity, and production of reactive oxygen species (ROS) (Lu & Wu 2005, Espinoza et al. 2009, Kazama et al. 2012). In the Pacific oyster C. gigas, fertilization process requires acrosomal reaction to bind and penetrate the oocyte (Akcha et al. 2012), and the spermatozoa capacity to successfully fertilize the oocyte is positively related to the intracellular ATP content, percentage of live spermatozoa and negatively correlated to the percentage of dying spermatozoa (Boulais 2014). Ecotoxicological investigations rarely use cellular biomarkers to test spermiotoxicity of exogenous compounds. These parameters have already proven valuable in flow cytometric seminal analysis for assessing human infertility (Espinoza et al. 2009), quality of cryopreserved spermatozoa used in large animal assisted reproduction programs (Guthrie & Welch 2006, Selvaraju et al. 2009), as well as the impact of harmful algae (Haberkorn et al. 2010, Le Goic et al. 2013, 2014) and of herbicides (Akcha et al. 2012) on reproduction of the Pacific oyster.

The aim of the present study was to determine, using flow cytometry (FCM), the cellular effects of DWH oil spill contaminants, including both oil and dispersant, on the spermatozoa of the eastern oyster and to relate them to the fertilization success of exposed gametes.

MATERIALS AND METHODS

Conditioning of Brood Stocks

Adult specimens of Crassostrea virginica (average weight of 75 [+ or -] 20 g) were collected between April and September 2011 from natural populations in Estero Bay, FL (latitude 26[degrees] 19' 50" N, longitude 81[degrees] 50' 15" W). Estero Bay, an aquatic buffer preserve, has extensive, healthy oyster reefs. Adult oysters were held in the experimental hatchery at 23[degrees]C [+ or -] 1[degrees]C, in a flow-through system supplied with coarsely filtered (30-[micro]m sand filter) seawater, at ambient salinity [20-30 practical salinity unit (PSU)], under natural light conditions, and fed ad libitum with a mixture of freshly cultured microalgae (Tetraselmis chuii, Chaetoceros sp., and Tisochrysis lutea) at a daily ration of 3% of oyster dry body weight for a period of 2-5 wk of conditioning to develop ripe oysters (Utting & Millican 1997).

Gamete Collection

Ripe oysters were thermally induced to spawn by alternating an immersion in seawater at 18[degrees]C and 30[degrees]C for 30 min each time. Once spawning occurred, spawning oysters were individually isolated in 200 ml of ultraviolet-sterilized and 0.1-[micro]m-filtered seawater (FSW) in 1-1 beakers.

For each oyster, oocytes or spermatozoa were examined under a microscope to select individuals with ripe gametes (i.e., males showing the highest percentages of motile spermatozoa and females showing absence of oocyte atresia). Gametes were filtered and pooled following the methodology described in the work of Vignier et al. (2015). Briefly, spermatozoa were filtered through a 55-[micro]m mesh and pooled in a 1-1 beaker and oocytes were sieved through 55-pm and 20-[micro]m sieve and transferred into 2 1 of FSW. Four replicate groups of between three and six individuals were then prepared. Subsamples of oocytes were stained with 1% Lugol's solution and counted using a Sedgwick-Rafter cell and a dissecting microscope. The concentration of spermatozoa was determined in duplicate by flow cytometry (FCM) according to Le Goic et al. (2013) and adjusted to 5 X [10.sup.6] spermatozoa/ml by further dilution in FSW.

High Energy Water-Accommodated Fraction, Chemically Enhanced Water-Accommodated Fraction, and Dispersant Solutions

Deepwater Horizon surface oil (slick oil A) and dispersant were provided by the trustees under chain of custody during the Deepwater Horizon NRDA efforts. Slick oil was collected near the source on July 29, 2010, from the hold of barge number CTC02404, which received slick oil from various skimmer vessels near the Macondo Well (sample CTC02404-02). The dispersant, Corexit 9500A, was manufactured by NALCO. Solutions were prepared at 25[degrees]C under fluorescent lights to avoid photoreactivity (Landrum et al. 1987). For all solutions, slick oil and/or dispersant was added to FSW at a salinity of 20-30 PSU.

High-energy water-accommodated fraction (HEWAF), chemically enhanced water-accommodated fraction (CEWAF), and dispersant solutions were prepared following methodology described in Vignier et al. (2015). The oil only solution or HEWAF is representative of slick oil that has been dispersed mechanically by wind, currents, and waves. The action of waves and currents was recreated by adding 2 1 of FSW and 4 g of slick oil, with a gastight syringe, to a stainless steel blender pitcher (Waring CB15; Waring Commercial, Torrington, CT). After 30 sec at the lowest blending speed, the solution was transferred to a 2-1 aspirator bottle and left to settle for at least 1 h to separate the residual floating oil. The bottom layer of the mixture was then carefully drained from the aspirator bottle, and FSW was added to this stock solution to prepare different concentrations of HEWAF for exposure treatments. To prepare the stock solution of the oil and dispersant or CEWAF, 2 g of slick oil and 200 mg of dispersant (10:1 w:w) were added to an aspirator bottle filled with 2 1 of FSW. Slick oil and dispersant were added with a gastight syringe and stirred at a vortex adjusted to 25% using a stirring rod and a magnetic stirrer for 18 h. To allow for the separation of the solution from the residual floating oil, the oil and dispersant mixture was left to stand for 3 h prior to use, and the stock solution was carefully drained and diluted with FSW to obtain the different CEWAF concentrations. Dispersant only solution was prepared as described for CEWAF above, except that no oil was added and the mixture was not settled. The dispersant stock solution was collected by draining the aspirator bottle. To obtain different solution concentrations, the stock solution was diluted with FSW. For all exposure assays, the control was composed of FSW only.

Water Quality and Analytical Chemistry

Temperature, dissolved oxygen, salinity, and pH were measured daily using a ProODO optic probe (YS1 Incorporated, Yellow Springs, OH), a refractometer, and a "Pinpoint" pH monitor (American Marine Inc., Ridgefield, CT), respectively. Total ammonia was measured at the start and the end of each exposure experiment using a Seal Analytical AutoAnalyzer 3 HR and the G-171-96 method (Koroleff 1983). Chemical analyses of the stock solutions, HEWAF, CEWAF, dispersant concentrations, and the FSW control were performed by ALS Environmental (ALS; formerly Columbia Analytical Services) and validated by Ecochem (Kelso, WA). Polycyclic aromatic hydrocarbons, including alkylated homologs, were determined by gas chromatography with low-resolution mass spectrometry using selective ion monitoring and a sum of 50 different PAH (tPAH50) were quantified (Vignier et al. 2015). The analytical procedure was based on EPA Method 8270D with the GC and M S operating conditions optimized for separation and sensitivity of the targeted analytes.

Experimental Design

Concentrations of HEWAF, CEWAF, and dispersant used in this study were based on preliminary experiments. Nominal concentrations used for exposure to HEWAF, CEWAF, and dispersant, as well as corresponding [tPAH.sub.50] contents, are listed in Table 1. Exposure designs were based on standardized protocols described in "U.S. EPA. 1996. Ecological effects test guidelines: OPPTS 850.1055 bivalve acute toxicity test (embryo larval)."

Spermatozoa and oocytes were exposed separately to the different concentrations of HEWAF, CEWAF, and dispersant (Table 1) for 30 min prior to FCM analyses of spermatozoa and fertilization success assay: 10 ml of spermatozoa mixture at 5 X 106 spermatozoa/ml and 4,000 oocytes were incubated separately in 40 ml and 200 ml, respectively, of HEWAF, CEWAF, or dispersant (four replicates for each concentration).

Fertilization Success

After the 30-min incubation, oocytes from each exposure replicate were fertilized with 10 ml of spermatozoa mixture from corresponding spermatozoa exposure replicates, and fertilization success was determined I h post fertilization following the methodology described in the work of Vignier et al. (2015). At least 100 embryos per treatment were examined under a microscope for cell cleavage to assess the fertilization success (% fertilization).

Cellular Flow Cytometric Characteristics of Spermatozoa

Flow cytometry analyses were performed on a Cytomics FC 500 (Beckman Coulter) equipped with a 480-nm argon laser. Collected data were analyzed with WinMDI 2.9 software. Analysis of membrane integrity, mitochondrial membrane potential (MMP), and ROS production of spermatozoa were measured according to Rolton et al. (2015).

Viability

Viability of spermatozoa was evaluated using a double staining procedure including SYBR-14 and propidium iodide (PI) (Life/dead Sperm Viability Kit; Molecular Probes). SYBR-14 permeates cells with preserved membrane integrity, binds to double-stranded DNA, and then emits green fluorescence. Detection of this fluorescence allows distinction between single cells and aggregates as well as debris. Contrastingly with SYBR-14, PI permeates only spermatozoa that lost membrane integrity. In brief, an aliquot of 200 [micro]l of spermatozoa diluted at 2 X [10.sup.5] cell/ml in FSW was stained with both SYBR-14 and PI (final concentrations of 1 [micro]M and 10 [micro]g/ml, respectively, for 10 min). SYBR-14 and PI fluorescences were measured at 500-530 nm (green) and at 550-600 nm (orange), respectively.

Mitochondrial Membrane Potential

The MMP of spermatozoa was measured using the potential-dependent JC-10. This probe enters selectively into mitochondria and exists as two forms, monomeric or aggregate, depending upon membrane potential. The JC-10 monomer form predominates in cells with low MMP and emits light in the green wavelength (525-530 nm). The JC-10 aggregate form accumulates in mitochondria with high membrane potential and emits light in the high orange wavelength (590 nm). JC-10 forms can change reversibly. In brief, an aliquot of 200 ml spermatozoa mixture at 2 X [10.sup.5] cell/ml were stained with JC-10 (final concentration 5 uM, for 5 min) and then diluted at 1:10 to stop the reaction prior to FCM analysis. Membrane potential of active cells was estimated by the ratio of aggregate:monomer (i.e., orange:green fluorescence ratio).

ROS Production

Determination of oxidative activity was performed using 2',7'-dichlorofluorescein diacetate (DCFH-DA), a membrane permeable, nonfluorescent probe. Inside cells, the DA radical is first hydrolyzed by esterase enzymes. Intracellular hydrogen peroxide ([H.sub.2][O.sub.2]), as well as superoxide ion ([O.sub.2.sup.-*]), then oxidizes DCFH to the fluorescent DCF molecule. Oxidation of DCFH can also be mediated by nitrite radicals (N[O.sub.2] or [N.sub.2][O.sub.3]) and various oxidase and peroxidase enzymes. The DCF green fluorescence, detected on the FL1 detector of the flow cytometer, is proportional to the total oxidative activity of spermatozoa, including ROS production. To a 200 [micro]l suspension of [10.sup.6] spermatozoa/ml, DCFH-DA (10 [micro]M final) was added for 30 min.

Acrosomal Integrity

Analysis of acrosomal integrity of spermatozoa was adapted from the work of Thomas et al. (1997) and Donaghy et al. (2010). The acrosome is an acidified organelle that can be specifically stained by LysoTracker probes, which spontaneously enter acrosome. Once the acrosome is no longer intact, the dye then diffuses out of the organelle, resulting in a decrease of associated fluorescence. An increase of the volume of the acrosome meanwhile results in higher detected fluorescence intensity. After exposure to dispersant, CEWAF and F1EWAF, 200 ml of spermatozoa at 2 X [10.sup.5] cells/ml was incubated with LysoTracker Red DND-99 (Molecular Probes, Invitrogen; final concentration 1 [micro]M, 10 min). Acrosomal activity is expressed as the level of red fluorescence detected on the FL3 detector of the flow cytometer.

Statistical Analysis

For statistical analyses, results were modeled using one-way analysis of variance to test the null hypothesis of equality of means between treatment groups. If an overall F-test rejected the null hypothesis (P < 0.05), pairwise comparisons of treatment group means were conducted using Tukey's honestly significant difference method to account for multiple comparisons. Statistical calculations were performed using Tibco Spotfire S + 8.2 (TIBCO Software Inc.).

RESULTS

Water Quality and Analytical Chemistry

Temperature and salinity throughout the experiments ranged from 24.6[degrees]C to 26.0[degrees]C and from 21 to 25 PSU, respectively. The pH averaged 7.8 [+ or -] 0.5 and dissolved oxygen never decreased below 6.9 mg/l or 90% saturation. For each tested concentration of HEWAF, CEWAF, and dispersant, total ammonia concentrations remained at safe levels (0.084 [+ or -] 0.075 mg/l).

Fertilization Success

In all experiments, fertilization in control conditions was very high, reaching more than 97% of success (Fig. 1). Incubation of spermatozoa with concentrations of dispersant and CEWAF higher than 10 mg/l and 14.24 [micro]g/1 [tPAH.sub.50], respectively, resulted in a statistically significant decrease of fertilization success. In both conditions, a major decrease occurred at 10 mg/l [tPAH.sub.50] of dispersant and 26.14 [micro]g/l [tPAH.sub.50] of dispersant/ oil mixture. Fertilization success showed a significant decline with HEWAF concentrations above 57.5 [micro]g/l [tPAH.sub.50], down to 47% of fertilization success (Fig. 1).

Cellular Flow Cytometric Characteristics of Spermatozoa

In control conditions, the percentage of intact spermatozoa was always higher than 93% (data not shown). In the range of concentrations tested, CEWAF and HEWAF showed no effect on spermatozoa viability; however, dispersant at the highest dose tested decreased spermatozoa viability (Fig. 2).

The MMP of spermatozoa incubated with dispersant from 5 to 10 mg/l [tPAH.sub.50] was statistically decreased (Fig. 3). All concentrations of CEWAF induced a decrease of spermatozoa MMP, and incubation of spermatozoa with concentrations of HEWAF at 36.94 pg/1 [tPAH.sub.50] and above also resulted in a statistically significant decrease of MMP, in a dose-dependent manner (Fig. 3).

Incubation of spermatozoa with dispersant resulted in a regular decrease of ROS production (Fig. 4). The lowest concentrations of dispersant and CEWAF resulted in a 50% decrease of ROS production.

Incubation of spermatozoa with dispersant resulted in a dose-dependent increase of acrosome-associated fluorescence from 2.5 to 10 mg/l [tPAH.sub.50] of dispersant, up to 200% of control condition (Fig. 5). The CEWAF also induced an increase of acrosome-associated fluorescence with concentration of 14.24 and 26.14 [micro]g/1 [tPAH.sub.50] (Fig. 5). Spermatozoa incubated with HEWAF did not induce any change of spermatozoa acrosome-associated fluorescence.

DISCUSSION

The DWH oil spill, the largest marine oil spill in the U.S. history, occurred at the beginning of the spawning season of the eastern oyster Crassostrea virginica. Because of its very high ecological and economic significance for the Gulf of Mexico, the effect of the oil spill on the cellular responses of spermatozoa of C. virginica was studied under laboratory conditions. The work described here aimed at determining the influence of the DWH-released oil as well as the dispersant (Corexit 9500A) on the reproductive output of C. virginica.

In the preliminary range finding experiments of this study, fertilization was inhibited by HEWAF, with an EC50 around 500 mg/l (Table 1), and the experiments showed that the DWH oil prepared as low-energy water-accommodated fraction had no effect on the fertilization success of Crassostrea virginica even with concentrations as high as 1,000 mg/l (nominal concentration; data not shown). Similarly, crude oil WAF did not inhibit fertilization of gametes from coral species (Negri & Heyward 2000). In the present study, HEWAF from 57.50 [micro]g/l [tPAH.sub.50] (125 mg/l) negatively impacted the fertilization success of C. virginica spermatozoa, demonstrating that the blending of the oil might then have increased the bioavailability of petroleum compounds, as observed with weathered crude oil that significantly reduced fertilization rates in polychaete species (Lewis et al. 2008). Indeed, the DWH oil is mainly composed of PAH: naphthalenes, fluorenes, and phenanthrenes (Forth et al. 2015), and these molecules are nonsoluble and less volatile, making them potentially long to spontaneously diffuse into seawater and become bioavailable. Thus, HEWAF might then be a good proxy for slicks of oil submitted to current, wind, and waves. Chemically enhanced water-accommodated fraction as well as dispersant alone (Corexit 9500A) impaired fertilization success, as observed with the coral species Acropora millepora (Negri & Heyward 2000). While CEWAF at 5/50 (dispersant/ oil) mg/l (14.24 [micro]g/l [tPAH.sub.50]) negatively impacted spermatozoa fertilization success, no change in this parameter was observed with 5 mg/l dispersant. Overall, the results of the present study suggest that toxic effects might mostly be attributed to Corexit 9500A, with an EC50 of about 10 mg/l and that combining oil and dispersant is even more toxic for the success of oyster fertilization than oil or dispersant alone. Toxicity of Corexit dispersants on the fertilization success of marine invertebrates has been studied and noted for a long time (Lonning & Hagstrom 1976, Law 1995). Similar results were found by Vignier et al. (2015). They demonstrated that exposure of eastern oyster gametes and embryos to oil preparations and dispersant induced larval death and that CEWAF exposure resulted in a higher percentage of abnormal larvae than Corexit alone (at 5 mg/l). Stefansson et al. (2016) showed that the addition of Corexit 9500A to oil in the preparation of CEWAF resulted in higher concentrations of oil components in the test media (tPAH and saturated hydrocarbon compounds) and suggested that additive and/or synergistic toxic effects of Corexit combined with the higher concentrations of PAHs present in fresh oil CEWAF resulted in this increased toxicity.

The results of the present study showed that the reproductive output of eastern oysters may have been highly impaired by the DWH oil spill and, most of all, by adding dispersant in the effort to protect coastline and marine environments.

Fertilization success depends on numerous cellular mechanisms occurring both in spermatozoa and oocytes. Until now, male toxicity effects have rarely been studied compared with maternal effects (Lewis & Galloway 2009).

In the present work, neither CEWAF nor HEWAF altered viability of oyster spermatozoa. This parameter was, however, highly reduced at 10 mg/l of dispersant (Fig. 2), suggesting that observed impairment of fertilization ability of exposed spermatozoa with dispersant may be related to reduced spermatozoa viability. For the analysis of plasma membrane integrity, the double staining procedure SYBR 14/PI is used, which stains the same intracellular target (DNA).

Considering that PI stains DNA of spermatozoa with compromised membranes, potential DNA damage may have occurred in oyster spermatozoa. In marine invertebrates, the integrity of gamete DNA is crucial for the development of the resulting embryo (Lewis & Ford 2012). Gwo et al. (2003) reported a close relationship between spermatozoa with increased DNA damage and lower percentages of fertilization rate in the Pacific oyster.

Analogous to mammalian spermatozoa, oyster sperm cells have an acrosome (Paniagua-Chavez et al. 2006). The acrosome is a large acidic secretory organelle of the spermatozoa head filled with hydrolytic enzymes allowing spermatozoa to bind to the extracellular vestment of the oocytes and penetrate the oocytes (Hylander & Summers 1977, Akcha et al. 2012). Spermatozoa with a damaged acrosome are likely not capable of fertilizing oocytes (Silva & Gadella 2006). Most fluorescent dyes used to evaluate acrosome only provide information on its membrane integrity: probes only penetrate acrosomes with nonintact membranes. Contrastingly, the dye used in this work, LysoTracker, freely diffuses into acrosomes, and impairment of acrosomal membrane integrity results in a leak of the dye and thus a loss of fluorescence. Interestingly, in the present experiments, dispersant and CEWAF induced an increase of acrosome-associated fluorescence. This might reflect an increase of acrosomal volume, which could be related to the acrosomal reaction process. These results suggest that dispersant and CEWAF have increased acrosomal volume and/or triggered acrosomal exocytosis in oyster spermatozoa; however, further studies are needed to determine if the effect of dispersant and CEWAF on spermatozoa acrosome is rather positive (i.e., triggering acrosomal reaction) or negative (i.e., impacting the acrosome).

Mitochondria are organelles that generate most of the supply of adenosine triphosphate (ATP), the energy transfer nucleotide, during oxidative phosphorylation in oyster spermatozoa (Boulais et al. 2015). Information on mitochondrial functionality can be obtained from the MMP assay as any change in the MMP will cause variation in ATP synthesis (Stendardi et al. 2011, Piomboni et al. 2012). The results of the present study show a dose-dependent decrease of MMP during dispersant, CEWAF and HEWAF exposures, with CEWAF impacting MMP at a lower dose than the respective concentration of dispersant alone. The decrease of spermatozoa MMP suggests the stimulation in mitochondrial ATP synthesis, as the ATP synthase uses this proton gradient for the synthesis of ATP and might be partly responsible for the observed dose-dependent decrease of the fertilization success. Indeed, it has been demonstrated that a decrease in spermatozoa ATP content reduces their capacity to successfully fertilize the oocytes of the Pacific oyster (Boulais 2014), the acrosomal reaction (Christen et al. 1983, 1986), and the stability of spermatozoa binding to the oocyte surface in the sea urchin Strongylocentrotus purpuratus (Hirohashi & Lennarz 1998). Furthermore, in the Pacific oyster, spermatozoa with a high intracellular ATP content seem to be more likely to successfully fertilize oocyte as trochophore yield was positively correlated to intracellular ATP content (Boulais 2014). These results reflect an increase in energy demand, which may be needed to face decreased viability of spermatozoa exposed to oil contaminants.

In oyster, another function of mitochondria is the production of ROS (Turrens 2003). During mitochondrial respiration, it is estimated that about 1 %-2% of oxygen consumed is not completely reduced to water but instead partially reduced to [O.sub.2.sup.-*], which can be converted to [H.sub.2][O.sub.2] and the highly reactive O[H.sup.*] (Poyton et al. 2009). The results of the present study show that spermatozoa exposure to dispersant, CEWAF, and HEWAF highly decrease intracellular ROS production from the lowest doses tested. This is partly due to the reduced MMP as the mitochondrial respiration is involved in ROS generation in spermatozoa (Koppers et al. 2008, Kazarna et al. 2012, Boulais 2014). Although high intracellular ROS concentrations have been reported as dangerous for spermatozoa, they are by-products of normal cellular respiration (Han et al. 2001), and low and controlled concentrations of ROS yet play an primordial role in signal transduction mechanisms for spermatozoa physiology, such as spermatozoa hyperactivation and acrosomal reaction (de Lamirande & Gagnon 1993, de Lamirande et al. 1997, Griveau & Lannou 1997). The dose-dependent decrease of ROS production observed in spermatozoa incubated with dispersant, CEWAF, and HEWAF may be related to decrease MMP, as these two parameters are generated in mitochondria by the mitochondrial respiratory chain. As suggested for the MMP, the diminished ROS production could be linked to an increase in mitochondrial ATP synthesis. It has been suggested that mitochondrial respiratory chain complexes display higher affinity for electrons, reducing the potential for production of ROS, during need of mitochondrial ATP synthesis (Gnaiger et al. 1998, Sussarellu et al. 2013).

In summary, results presented here show that impaired fertilization observed after exposure to DWH oil spill contaminants may result, at least partially, from alterations of spermatozoa viability, acrosomal integrity, and mitochondrial functionalities (MMP and ROS production). Toxic effects might mostly be attributed to Corexit 9500A, with an EC50 of about 10 mg/l. The DWH oil spill occurred at the beginning of the spawning season of the eastern oyster, and its impaired fertilization may result in negative effects on oyster populations and thus the ecology and economy of the Gulf of Mexico.

ACKNOWLEDGMENTS

This work was supported by funds provided as part of the natural resources damage assessment for the Deepwater Horizon oil spill. We thank Jeff Morris, Claire Lay, Michelle Krasnec, and Dave Cacela for their help and input on experimental design and data analyses. Data presented here are a subset of a larger toxicological database that is being generated as part of the Deepwater Horizon Natural Resource Damage Assessment; therefore, these data will be subject to additional analysis and interpretation, which may include interpretation in the context of additional data not presented here.

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ASWANI VOLETY, (1,3) * MYRINA BOULAIS, (3) LUDOVIC DONAGHY, (1) JULIEN VIGNIER, (1) AI NING LOH (1,3) AND PHILIPPE SOUDANT (2)

(1) Department of Marine and Ecological Sciences, College of Arts and Sciences, Florida Gulf Coast University, 10501 FGCU Boulevard, Fort Myers, FL; (2) Laboratoire des Sciences de l'Environnement Marin, UMR 6539, Institut Universitaire Europeen de la Mer, Universite de Bretagne Occidentale, Technopole Brest-Iroise, 29280 Plouzane, France; (3) University of North Carolina Wilmington, 601 South College Road, Wilmington, NC 28403

* Corresponding author. E-mail: voletya@uncw.edu

DOI: 10.2983/035.035.0111

TABLE 1.
Range of nominal concentrations used for HEWAF, CEWAF,
and dispersant exposures and corresponding PAH content.

          HEWAF                           CEWAF              Dispersant

                                Nominal                       Nominal
 Nominal     [tPAH.sub.50]    dispersant/    [tPAH.sub.50]   dispersant
oil (mg/l)   ([micro]g/1)     oil (mg/l)     ([micro]g/1)      (mg/l)

0                  0         0                     0         0
31.25            16.53       0.625/6.25          1.29        0.625
62.5             36.94       1.25/12.5           3.32        1.25
125              57.50       2.5/25              6.43        2.5
250              94.47       5/50                14.24       5
500 (EC50)      248.89       10/100 (EC50)       26.14       10 (EC50)

[tPAH.sub.50]] is the sum of 50 PAHs analyzed by gas chromatography
with low-resolution mass spectrometry using selective ion monitoring.
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Author:Volety, Aswani; Boulais, Myrina; Donaghy, Ludovic; Vignier, Julien; Loh, Ai Ning; Soudant, Philippe
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:0GULF
Date:Apr 1, 2016
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