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SUMMARYThree acclimation groups [i.e. non-diapause (LD), diapause (SD) and diapause, cold-acclimated (SDA)] of the adult bugs Pyrrhocoris apterus differed markedly in their levels of chill tolerance. Survival time at a sub-zero, but non-freezing, temperature of –5°C (Lt50)extended from 7.6 days, through 35.6 days, to >60 days in the LD, SD and SDA insects, respectively. The time necessary for recovery after chill-coma increased linearly with the increasing time of exposure to –5°C, and the steepness of the slope of linear regression decreased in the order LD>SD>SDA. The capacity to prevent/counteract leakage of Na+ down the electrochemical gradient (from haemolymph to tissues) during the exposure to –5°C increased in the order LD<SD<SDA. As a result, the rates of counteractive outward movement of K+, and of the EK dissipation, decreased in the same order. The least chill-tolerant insects (LD) showed the highest rate of body-water loss. Most of the water was lost from the haemolymph compartment. The ability to regulate a certain fraction of ion pools into the hindgut fluid was the highest in the SDA group, medium in the SD group and missing in the LD group. The adenylate energy charge in the fat body cells was constant in all three groups. The total pools of ATP, ADP and AMP, however, decreased in the SD and SDA groups but remained constant in the LD group. The inability of insects to maintain ion gradients at sub-zero temperature is discussed as an important cause of pre-freeze mortality.

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A small urinary bladder attached to the seminal duct in caudal part of the abdominal cavity was registered for the first time in dissected males of tench. The urinary bladder wall was of whitish color and the bladder contained 0.5-2 ml of urine. When collected in the experiment, the tench sperm was whitecolored. Spermatozoa density is highly variable due to contamination by urine, and the latter additionally activates spontaneous motility of the spermatozoa. Seminal fluid contains ions such as Na + (18.4 ± 1.3 mM M), K + (1.9 ± 0.6 mM M), Ca 2+ (0.6 ± 0.2 mM M) and Mg 2+ (0.5 ± 0.1 mM M), leading to osmolality of 230 ± 82 mOsmol kg )1 depending on the dilution by urine. Urea was detected in urine samples uncontaminated by sperm with an osmolality of 85 ± 58 mOsmol kg )1 . Urine also contained high concentrations of ions such as Na + (30.9 ± 8.9 mM M), K + (4.3 ± 2.9 mM M), Ca 2+ (0.9 ± 0.5 mM M) and Mg 2+ (0.6 ± 0.2 mM M). The spontaneous sperm activation by urine was up to 100%, but could be prevented by collection in an immobilizing solution. Motility was observed for 90-100% spermatozoa just after their transfer to distilled water or in a swimming medium (SM, 30-45 mM M KCl) with a velocity of 120-140 lm s )1 . A flagellar beat frequency of 60-70 Hz and forward motility lasted up to 80 s in distilled water, and up to 180 s in SM at room temperature.

Laboratory-reared cultures of Enchytraeus crypticus were used in a reproduction toxicity test to evaluate the toxicity of 46 spoil substrates collected in four brown coal mining areas in the Czech Republic and Germany. A set of substrate parameters (pH, conductivity, Na, Ca, K, Al, Fe, loss of ignition and polyphenol contents) were measured for each spoil and correlated with spoil toxicity for E. crypticus. Toxicity increased with decreasing pH and increasing Al, Fe content. Spoil with a pH below 3 did not support the survival of E. crypticus. However, some alkaline spoils with high conductivity and ion concentration were also toxic. Toxicity was positively correlated with the loss of ignition and polyphenol content. The results indicate that the toxicity of post-mining spoil substrates have multiple origins. Most frequently the toxicity of post-mining substrates corresponds with low pH and consequent toxicity of Al and Fe. However some substrates may be toxic due to high ion concentrations. The potential role of fossil organic matter (namely polyphenols) in toxicity of post-mining substrates requires further research.