Sublethal exposure of eastern oyster Crassostrea virginica to the goniodomin-producing dinoflagellate Alexandrium monilatum: Fate of toxins, histopathology, and gene expression

Abstract

Objective: The dinoflagellate Alexandrium monilatum forms blooms during summer in tributaries of the lower Chesapeake Bay. Questions persist about the potential for A. monilatum to negatively affect aquatic organisms. Its main toxin, goniodomin A (GDA), a polyketide macrolide, has been shown to have adverse effects on animals, for example through cytotoxicity and interaction with actin.

Methods: Eastern oysters Crassostrea virginica were exposed for 96 h to sublethal concentrations of A. monilatum (615 ± 47 cells/mL [average ± SD]; containing mainly intracellular GDA [215 ± 7.15 pg/cell] and to a lesser extent goniodomin B, goniodomin C, and GDA seco-acid as quantified by liquid chromatography coupled to tandem mass spectrometry) or to nontoxic phytoplankton or were unexposed. They were subsequently depurated for 96 h by exposure to nontoxic phytoplankton. Clearance rates were estimated, and oysters were sampled daily and tissue (gill, digestive gland, and remaining tissues) excised for analyses by histopathology, gene expression quantified by quantitative PCR, and goniodomin quantification.

Result: A positive clearance rate, no mortality, and no tissue pathologies were observed in oysters exposed to A. monilatum. Goniodomin A was detected in gill 6 h after exposure (504 ± 329 μg/kg [average ± SE]) and to a lesser extent in the digestive gland and remaining soft tissues. In the digestive gland, a trend of transformation of GDA to GDA seco-acid was observed. The majority of toxins (≥83%) were depurated after 96 h. Expression of genes involved in oxidative response increased 14-fold after 6 h, and those involved in actin synthesis showed a 27-fold change after 24 h, while expression of apoptosis genes increased 6.9-fold after 96 h compared with the control (eastern oysters exposed to nontoxic phytoplankton).

Conclusion: Exposure experiments (nonsublethal or chronic) should be carried out to better assess the threat of this species and toxins for eastern oysters and other marine organisms.

Keywords: Alexandrium monilatum; Crassostrea virginica; apoptosis; bivalve mollusks; dinoflagellates; fate of toxins; goniodomin metabolism; goniodomins; oxidative stress.

FIGURE 1

Concentration (μg/kg) of goniodomin A (GDA), goniodomin B (GDB), goniodomin C (GDC), and goniodomin A seco‐acid (GDA‐sa) in eastern oyster (A) gill, (B) digestive gland, and (C) remaining tissues during 96 h of exposure to an average ± SD of 615 ± 47 cells/mL of Alexandrium monilatum (Amon07) followed by 96 h of depuration (eastern oysters exposed to a mixture of nontoxic phytoplankton). Data (n = 6) are average ± SE. The horizontal plain bar represents the limit of detection of GDA of 1.8 μg/kg. In some cases, the average of the six replicates were less than the limit of detection. Note that there are different scales for the y‐axes and discontinuous scales for panels (A) and (C).

FIGURE 2

Relative quantification of gene expression ($2^{-∆∆C_T}$ method; Livak and Schmittgen 2001) in eastern oyster gill (Gill) and digestive gland (DG) after (A) 6 h, (B) 24 h, and (C) 96 h of exposure to A. monilatum (Amon07; 615 ± 47 cells/mL) or no exposure, followed by (D) 24 h and (E) 96 h of depuration (exposed to a mixture of nontoxic phytoplankton, except for the unexposed treatment that remained unfed). Data (n = 3) are average ± SD, normalized to glyceraldehyde‐3‐phosphate dehydrogenase (gapdh), with the treatment eastern oysters exposed to nontoxic phytoplankton as a control. Expression of each gene was compared between treatments (exposed to A. monilatum or unexposed) and between tissue compartments (gill and digestive gland) two by two, and differences (p‐value ≤ 0.05) were marked with an asterisk or with different letters, respectively. When $C_T$ (threshold cycle) was higher than 40, the relative quantification of gene expression was written as “nd” (not detected). Note the different scales for the y‐axes. Abbreviations are as follows: anti‐apoptotic protein (aapo), actin (actin), allantoicase (allan), cadherin (cad), caspase‐1 (casp1), glutathione peroxidase (gpx), and glutathione reductase (gr).