Soluble protein changes in pollution-adapted Gammarus duebeni.

The euryhaline amphipod Gummarus dueheni colonized the saline sewage treatment plant at Looe, Cornwall, in 1973 and has established a thriving population [I] . After full operation was started, the primary settlement tanks became too anoxic for amphipod survival, recruitment ceased and the population became confined to the filter beds and humus tanks. I t is from the latter that individuals are now collected. The presence of Gummarus ducheni in this unusual environment, together with a precise date for colonisation, offers a unique opportunity to study the long-term effects of sewage on a range of physiological, biochemical and genetic parameters. The resident population has undergone several changes since colonisation. The proportion of males is greater in the sewage population and the individual amphipods are smaller. Relative gill areas have increased as has the ability to survive periods of anoxia, with decreased rates of lactic acid accumulation [2]. The tolerance to heavy metals, including zinc and copper, has also increased in the sewage population [ 3 ] . Further work is in progress to elucidate the biochemical adaptations underlying the above observations and this paper reports some of the phenomena associated with the response to hypoxic stress. Gummarus duebeni were collected from the settlement tanks at the sewage works in West Looe, Cornwall, and from the Looe river estuary upstream of the works. In the laboratory, specimens were kept in tanks in clean, well aerated sea water until required. Specimens for extraction were used either within 24 hours of collection or after acclimation in the laboratory for a period of 2 months. Decapitated animals were extracted immediately in cold SO mM Tris/HCI buffer, pH 6.8 containing 1 mM PMSF 5mM 2 mercaptoethanol, and 10% glycerol, using a pre-cooled mortar and pestle. Extracts were transferred to Eppendorf tubes and spun at 13,000 rpm for 2 minutes. Clear supernatants were removed and stored in ice until required. Samples for SDS-PAGE were denatured immediately and stored at -20°C until required. Assays for lactate dehydrogenase (LDH) activity were carried out with pyruvate and NADH as substrates using SO mM Na phosphate buffers in the pH range 5.5-8.5. Substrate affinities were determined from direct linear plots [4]. Samples of extracts were subjected to electrophoresis under non-denaturing conditions on 7% polyacrylamide gels or under denaturing conditions on 916% gradient polyacrylamide gels [ S ] . Gels were stained for LDH activity at pH 8.5 using D,L sodium lactate as substrate [6]. SDS-PAGE gels were stained for proteins with Coomassie Brilliant Blue (3250 [7]. The LDH from Gummarus of sewage works, estuarine or acclimated conditions showed identical kinetic characteristics in respect of Km values for pyruvate (0.120 niM), and NADH (0.073 mM) and susceptibility to excess substrate inhibition by pyruvate (> 1.5 mM). V,,,, values were also identical. The levels of LDH, calculated as either units per gram fresh weight Estuary