Thesis announcements

The purpose of this research was to examine the ability of the cold water, green, marine, macro-alga Acrosiphonia coalita to take up and metabolize polycyclic aromatic hydrocarbons (PAH) from seawater. Axenic suspension tissue cultures of the alga were contacted with seawater containing PAH in sealed experimental vessels. Uptake time courses and equilibrium partitioning were examined. To determine if uptake was passive or active, equilibrium partitioning was also examined for uptake by heat-killed A. coalita tissue. Additionally, the seawater and biomass were monitored for the formation of PAH metabolites. The two model PAH compounds used in this study were naphthalene and phenanthrene.

The results of this study indicate that A. coalita did remove PAH from seawater, but that uptake was passive and the PAH was not metabolized. Both phenanthrene and naphthalene were taken up very quickly by the alga. Equilibrium partitioning between the seawater and biomass was achieved within 24 hours. Additionally, both compounds were found to partition linearly between A. coalita tissues and seawater. A. coalita removed significantly more phenanthrene than naphthalene from the seawater. The equilibrium partition coefficients for phenanthrene and naphthalene partitioning into living A. coalita tissue were 0.171 ± 0.0083 L/g FW and 0.0500 ± 0.0025 L/g FW, respectively. Naphthalene partitioning into heat-killed A. coalita tissue was equivalent to naphthalene partitioning into living A. coalita tissue, indicating that uptake was likely passive. No compounds were detected in the seawater or biomass that could be identified as products of PAH degradation. Additionally, all unidentified compounds that were present in the experiments with PAH and biomass were also present in the control experiments without PAH. The absence of detectable products of degradation indicates that the PAH was not metabolized.

The capability of the marine red macroalga Portieria hornemannii to remove and detoxify the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) in a seawater environment was evaluated using an axenic microplantlet suspension culture system. Microplantlets were challenged with TNT dissolved in seawater at concentrations of 1 to 50 mg L-1 in well-mixed photobioreactors under both batch and continuous additions. Photosynthetic activity was monitored by chlorophyll a fluorescence and oxygen evolution rate (OER) to determine the effects of TNT on the microplantlet photosynthetic viability.

Microplantlets in 1.1 gFW L-1 suspension effectively removed 100% of TNT from seawater medium at concentrations under 10 mg L-1 within approximately 72 h of exposure. First-order rate constants for TNT uptake were 0.025 to 0.037 L g FW-1 h-1 under both illuminated and dark conditions. Biotransformation products of TNT, 2-ADNT and 4-ADNT, were identified as the immediate transformation products. However, these products only accounted for 29% of initial TNT. A mass balance of 14C-labeled TNT in radioisotope tracer studies however, indicated that over 60% of 14C-label taken up by microplantlets was released back into the liquid medium, primarily in the form of polar and ionic metabolites. Polar metabolites were responsible for a change in liquid medium to a yellow color. These metabolites were tentatively identified by mass spectrometry to be Meisenheimer TNT complex (H-TNT) and tetranitro-hydrazotoluene (TN-HydrazoT) dimerization products. In contrast, only 26% of the 14C-label, primarily as solvent-extractable compounds, accumulated within the biomass tissue. Furthermore, bound residues of TNT metabolites accounted for less than 5% of initial TNT added.

Exposure to TNT inhibited photosynthetic activity in microplantlets by a rapid reversible mechanism. The extent of inhibition was dependent on TNT concentration, with higher concentrations causing permanent damage to the photosynthetic apparatus. A flow-recirculation bioreactor was developed to monitor real-time oxygen OER of microplantlets exposed to pulse and continuous additions of TNT. Pulse additions of TNT caused a rapid decrease in oxygen evolution by microplantlets, with the recovery of OER following TNT uptake dependent on the initial concentration. Microplantlets were able to continuously take up and transform TNT during 20 d perfusion additions of 0.35 and 2.5 mg TNT d-1, even though at the higher concentration photosynthesis was significantly suppressed.

Pacific oyster Crassostrea gigas aquaculture is an important industry in the Pacific Northwest region and is worth over $110 million annually. Sporadic summer mortality outbreaks have caused severe financial hardship for oyster aquaculturists for several decades. These outbreaks appear to result from a complex interaction of deleterious environmental conditions with spawning-related energy metabolism and opportunistic infection.

Selective breeding programs have been used successfully to produce oyster stocks that survive summer mortality syndrome. These programs are expensive to maintain and require substantial investments of time and effort. The efficiency of breeding programs can be increased by selecting broodstock based upon molecular markers associated with candidate genes for survival and also by using laboratory assays in the hatchery to identify and exclude poor-performing families prior to field evaluation. However, at this time candidate genes have not been identified for use in breeding programs and laboratory assays have not been fully developed. Therefore, the goal of this research was to identify potential candidate genes for improved survival by using cDNA microarrays to compare transcriptome-level responses between oyster families with low or high survival after heat shock, and to determine whether transcription of these candidate genes in juveniles was correlated with subsequent family-level field performance of adults.

Overall, the observed patterns of gene transcription were consistent with current literature concerning summer mortality in oysters and marine mussels Mytilus edulis. In two different cohorts of oyster families, transcription levels of the adhesive protein galectin before or after heat shock were higher in families with low (< 30%) survival of heat shock, whereas those of the protease inhibitor cystatin B before or after heat shock were greater in families with high (> 65%) survival of heat shock. We found that transcription levels of a number of genes including galectin, heat shock protein 27, and glutathione peroxidase in juveniles were significantly correlated with subsequent family-level survival or growth in the field.

We conclude that galectin and cystatin B could be used for marker-aided broodstock selection and that the relationships between gene transcription in juveniles and subsequent family-level performance of adults could be exploited to identify and exclude potentially poor-performing families in the hatchery. These results should facilitate the improvement of selective breeding programs by providing a method of selecting broodstock for traits that are otherwise impractical to measure, and by reducing the costs associated with rearing and evaluating oyster families in the field.