This project aims to understand how changes in temperature and salinity affect the way American oysters take in, store and metabolize harmful substances called brevetoxins, which are produced by harmful algal blooms (HABs). Scientists will experiment with different temperature and salinities to find the best conditions for oysters to reduce accumulation of brevetoxins and most effectively remove them. By studying the genes in oyster gonad tissue to see which ones are affected by brevetoxins, the research team will learn how exposure to them can impact oyster reproduction and how temperature and salinity affect reproduction. Research results from this project will provide oyster fishers, oyster farmers and environmental managers with rapid and accurate tools for more effective reef management during HAB exposure.

Abstract

Shellfish, including the American oyster Crassostrea virginica, accumulate and metabolize brevetoxins (BTXs) produced by the dinoflagellate Karenia brevis. Human health impacts result from inhaling aerosolized BTXs, and ingesting seafood, particularly shellfish, that is contaminated with BTXs. These health impacts manifest in many different ways, but exposure has been shown to result in neurotoxic effect, known as neurotoxic shellfish poisoning. BTX contamination also causes economic impacts, as reopening oyster reefs once they have been closed to harvesting is a time-consuming process. Understanding how, when, and why BTXs are removed from contaminated shellfish is therefore a critical human health concern. While many studies have examined the rate of BTX depuration, no studies have examined the impact of altered temperature and salinity on the uptake and depuration kinetics of BTXs, even as K. brevis has been demonstrated to bloom and produce toxins at salinities as low as low salinities. Understanding and predicting how brevetoxins interact with oysters and what factors influence brevetoxins' uptake, accumulation and depuration are critical to effectively managing healthy oyster reefs under periods of heightened environmental and economic uncertainty. In this proposal, we will examine how altering temperature and salinity alter uptake and depuration of BTXs in oysters, as well as examining the biological impacts of BTX contamination on the oysters themselves, with an eye towards assessing potential impacts on reproductive activity.

Our overall hypothesis is that uptake and depuration of BTXs are influenced by temperature and salinity, that there exists a combination of these variables that maximizes the depuration (thus minimizing tissue concentration), and that exposure to BTXs under different environmental conditions causes identifiable adverse effects to oyster health and reproduction. To test these hypotheses, we will conduct a series of controlled exposures of oysters to a K. brevis culture for 96 hours under varying salinity and temperature conditions. The oysters will then be continued in clean water for 24 days. By sampling the oysters and quantifying BTX concentrations in the tissue throughout the experiment, we can accurately identify the relationship between temperature, salinity and BTX exposure duration on tissue loads. By assessing global transcriptional profiles under different exposure conditions, we can identify the precise impacts of BTX uptake on critical biological pathways in oysters.

At the successful conclusion of this project, we will have new information that will be highly important to oyster fishers, farmers, and environmental managers responsible for opening and closing reefs in the event of red tide blooms.  While this will not result in an immediate shift in management decisions, it will provide the information necessary to manage oyster reefs more effectively in a highly uncertain environment. We will know the uptake and depuration kinetics of brevetoxins in oysters under conditions of varying temperature and salinity, including quantification of brevetoxin metabolites as a function of time under these conditions. We will also have a better understanding of how BTX exposure affects the oysters themselves, through an in-depth examination of global transcriptional profiles following BTX exposure.

Objectives

1. To characterize the impact of varying temperature and salinity on the uptake, accumulation, and depuration of brevetoxins in American oysters. We will expose oysters to cultured K. brevis under differing temperatures and salinity, characterize the uptake of BTX in the tissue, and identify the combination that maximizes the removal rate. By assessing BTX concentrations as a function of salinity, temperature, and time, we will be able to identify 1) which factor or combination of factors is most important in determining rates of BTX depuration in oysters and 2) the optimal conditions under which loss of BTXs occur.
2. To assess the biological implications of exposure to BTX in oyster tissue. At the end of the BTX loading phase (96 hours), we will remove one oyster per tank (N = 4 per treatment) and excise the gonads. We will use RNAseq to perform global transcriptional profiling and standard bioinformatic approaches with which we are well experienced to identify altered gene pathways that BTX exposure affects. By quantifying alterations on gene transcriptional patterns and pathways analysis in gonad tissue, we will be able to identify 1) which reproductively relevant pathways are affected by BTX exposure, 2) which pathways are potentially responsible for biotransforming BTXs, and 3) how salinity and temperature interact to modify this transcriptional response. We will focus primarily on gonads as we hypothesize that BTX exposure impacts the proper development of that tissue, with concomitant impacts on later reproductive activity.

Methodology

Organism culture.  Eastern oysters (Crassostrea virginica) will be obtained from USM's Thad Cochrane Marine Aquaculture Center as juveniles/young adults and transported to the Toxicology Laboratory 96h before experiment initiation.

BTX production. Active K. brevis cultures producing BTX will be grown, harvested, and centrifuged to concentrate the cell culture. The cells will be lysed, and the BTX (~10 mg total BTX) will be isolated at Mote Marine Laboratory, then transported to USM.  Initial analyses of the BTX will be conducted to determine concentrations.

BTX Exposures. Chronic exposures will be conducted as static-renewal assays with quadruplicate replicate tanks per treatment and 10 individuals per tank. A fully factorial experimental design will evaluate two temperatures (25C and 30C) and three salinities (20, 25, and 30 PSU). BTX exposures will be conducted for 96 hours at a concentration replicating previous bloom events (5 parts per million), followed by clean water only for 24 days. Tissue samples will be taken on days 4 and 5 during uptake and days 7, 10, 14, 21, and 28 during depuration. Samples will be stored at -80C until analyses of BTX constituents. 
BTX Analysis. BTXs in the tissue will be analyzed following Wetzel et al. (in prep). Briefly, the homogenized oyster tissue will be BTX surrogated, and then acetone extracted. The extracts will be purified using both C-18 and Agilent Captiva EMR-Lipid SPE. Samples will be evaporated, redissolved in acetone and a mobile water phase, then spiked with a BTX internal standard. Samples will be analyzed by liquid chromatography-mass spectrometry (LC-MS) using an Agilent 1100 LC coupled to an Agilent G1956B single quad MS for qualitative and quantitative identification of brevetoxins. Calibration curves for each toxin have been established, and surrogate and internal standards are used to determine recoveri
 

Rationale

Increased episodes of harmful algal blooms (HABs) threaten the Gulf of Mexico.  While multiple species cause HAB events, the most common, and the one with the most significant impact on humans, is commonly referred to as "red tide" (Kirkpatrick et al., 2004). Red tide, produced by blooms of the dinoflagellate Karenia brevis, severely impacts human communities through the potential for neurotoxicity from ingesting contaminated shellfish and the economic harm resulting from the closures of productive harvest areas once a red tide event has been established. As a result, much research has been aimed at understanding how red tide blooms affect human health and how to assess that a shellfish harvest area may safely be reopened for human consumption. Understanding how to balance human health and safety with the economic considerations of a large and active industry is critical to manage shellfish harvest areas during red tide exposures effectively. This proposal will address one component of this by examining the rate of depuration of red tide toxins from American oyster, Crassotrea virginica, and assessing the biological impacts of BTX loading on oyster health.

The overall goal of this proposal is to examine the impacts of varying temperature and salinity on depuration rates of BTX from oyster tissues and use that information to provide farmers and managers with a mitigation strategy to minimize the economic losses incurred during a red tide bloom and providing an additional tool for more effective oversight of oyster reefs. There are several aspects to this. First, there is little information about the relative depuration rates of BTX.  Second, there is little knowledge about the impacts of BTX loading on the health status of the oysters themselves, and what impact transient exposure to BTXs has on the oyster. This project will produce significant new knowledge about this.