Contamination of bivalve haemolymph samples by adductor muscle components: implications for biomarker studies

Abstract

Haemolymph samples and haemocytes collected via the adductor muscles of bivalve molluscs are extensively used in ecotoxicological studies. Withdrawal of haemolymph from mussels, Mytilus edulis, via the posterior adductor muscle, may lead to contamination with the intracellular contents of adductor myocytes. Lysopine dehydrogenase (LyDH) activity, an adductor myocyte marker, was used to investigate the impact of this potential contamination on levels of total glutathione, glutathione peroxidase (GPx) and acetylcholinesterase (AChE) measured in cell-free haemolymph. The mean glutathione content of cell-free haemolymph from 28 mussels was 3.2 +/- 1.8 microM (mean +/- SD). There was a linear relationship (slope = 0.28 +/- 0.03 min; mean +/- SE; P < 0.0001, n = 28) with haemolymph LyDH levels suggesting that at least some of the glutathione measured in cell-free haemolymph had arisen from contamination. Haemolymph LyDH activity was significantly higher in samples extracted using larger diameter needles, and also in samples where there had been some difficulty in the extraction. Exposure of mussels to oxidative stress using 40 microg l(-1) Cu for 5 days resulted in a 1.7 fold increase in glutathione (P = 0.033), but no increase (P = 0.810) in LyDH activity in adductor muscle. This was reflected in a similar increase in the slope of a plot of cell-free haemolymph glutathione versus LyDH activity (P = 0.011), consistent with both of these having originated from the adductor muscle. Cell-free haemolymph GPx and AChE activities also correlated with LyDH activity (Spearman rank correlation coefficients of 0.531 (P = 0.0068) and 0.537 (P = 0.0062), respectively, n = 27) suggesting that these also arise from contamination of the haemolymph. For GPx there was a significant linear relationship (P = 0.025) with haemolymph LyDH levels consistent with both enzymes originating from the myocytes. However, there was hyperbolic relationship (P = 0.0004) between haemolymph AChE and LyDH activities. It appears that this is because the AChE originates from a different compartment to the LyDH, i.e. cholinergic neuromuscular junctions in the adductor muscle. We conclude that it would be prudent, when considering the possibility of using a biomarker in cell-free haemolymph from bivalve molluscs, to check whether contamination could be an issue.

Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes

“…Aerobic mitochondrial metabolism, occurring in free-living and juvenile parasitic stages of F. hepatica in the mammalian host and leading to the reduction of oxygen and water production, is shaded in gray (and in all following metabolic figures as well) to distinguish it from anaerobic respiration and fermentation pathways, drawn in black. Abbreviations: CI to CIV, respiratory complexes I to IV; UQ, ubiquinone; RQ, rhodoquinone; C, cytochrome c; A, ATPase; FRD, fumarate reductase; [2B], acetate:succinate CoA-transferase (subfamily 1B); [3], succinyl-CoA synthetase; [5], malic enzyme; [6], pyruvate dehydrogenase complex; [8], fumarase; [10], phosphoenolpyruvate carboxykinase (ATP dependent); [11], malate dehydrogenase; [14], methylmalonyl-CoA mutase; [15], methylmalonyl-CoA epimerase; [16], propionyl-CoA carboxylase. The photograph shows Fasciola hepatica at the adult stage, with a length of ca.…”

“…These short branched-chain fatty acids are typical end products for Ascaris (249,251,298). Abbreviations: MOP-CoA, 2-methyl-3-oxo-pentanoyl-CoA; MOB-CoA, 2-methyl-3-oxo-butanoyl-CoA; CI to CIV, respiratory complexes I to IV; UQ, ubiquinone; RQ, rhodoquinone; C, cytochrome c; A, ATPase; FRD, fumarate reductase; [2A, B], acetate:succinate CoA-transferase (subfamilies A and B); [3], succinyl-CoA synthetase; [5], malic enzyme; [6], pyruvate dehydrogenase complex; [8], fumarase; [11], malate dehydrogenase; [14], methylmalonyl-CoA mutase; [15], methylmalonyl-CoA epimerase; [16], propionyl-CoA carboxylase; [17], phosphoenolpyruvate carboxykinase (ITP/GTP dependent); [44], condensing enzyme; [45], 2-methyl acetoacyl-CoA reductase; [46] can be converted to strombine and to alanine. In the case of Arenicola, however, the L-alanine formed can be converted to D-alanine by alanine racemase (445).…”

“…Living attached to hard substrates, for example, rocks in intertidal habitats, the bivalve has to face anaerobiosis periodically. Oxygen-independent cytosolic energy metabolism produces ATP via substrate-level phosphorylation accompanied by the formation of various end products, including alanopine, strombine, and alanine (11). Under conditions of prolonged anaerobiosis, propionate is preferentially formed instead of succinate in mitochondria.…”

“…6. Abbreviations: CI to CIV, respiratory complexes I to IV; UQ, ubiquinone; RQ, rhodoquinone; C, cytochrome c; A, ATPase; FRD, fumarate reductase; SQR, sulfide:quinone oxidoreductase; ST, sulfur transferase; SD, sulfur dioxygenase; [2B], acetate:succinate CoA-transferase (subfamily 1B); [3], succinyl-CoA synthetase; [5], malic enzyme; [6], pyruvate dehydrogenase complex; [8], fumarase; [9], pyruvate kinase; [10], phosphoenolpyruvate carboxykinase (ATP dependent); [11], malate dehydrogenase; [14], methylmalonyl-CoA mutase; [15], methylmalonyl-CoA epimerase; [16], propionyl-CoA carboxylase; [18], alanine aminotransferase; [19], aspartate aminotransferase; [20], alanopine dehydrogenase; [26] A strictly anoxic animal among the loriciferans. All animals with anaerobic metabolism discussed above do encounter oxygen permanently or from time to time.…”

“…4. Abbreviations: CI to CIV, respiratory complexes I to IV; UQ, ubiquinone; RQ, rhodoquinone; C, cytochrome c; A, ATPase; FRD, fumarate reductase; [2B], acetate:succinate CoA-transferase (subfamily 1B); [3], succinylCoA thiokinase; [5], malic enzyme; [6], pyruvate dehydrogenase complex; [8], fumarase; [9], pyruvate kinase; [11], malate dehydrogenase; [14], methylmalonylCoA mutase; [15], methylmalonyl-CoA epimerase; [16], propionyl-CoA carboxylase; [17], phosphoenolpyruvate carboxykinase (ITP/GTP dependent); [18], glutamate:pyruvate transaminase; [19], glutamate:oxaloacetate transaminase; [26], strombine dehydrogenase; [27] Fig. 9 is based.…”