The standard fungiflex bioassay. Treatment of vertical stipes of Coprinopsis cinerea with crude stipe extract. A drop (5 μl, = 2.5 mg) of extract was placed on the left-hand side (LHS) of stipe A and on the right-hand side (RHS) of stipe C. A drop of distilled water (control) was placed on the LHS of stipe B. Treated stipes (A and C) bent towards the extract drop within 1 h of application whereas the control stipe did not bend. By 2 h after application, the treated stipes were bent 30-40° away from the vertical (stipe A to the LHS and stipe C to the RHS) and towards the drop. Bending towards the applied drop was called Fungiflex 1 activity. The control stipe was bent only slightly (no more than 10°) from the vertical. Fungiflex 1-type bending persisted but by 3 h the apices of the treated stipes had started to return to the vertical in response to gravity. By 6 h stipes B and C were more or less vertical; stipe A had not only returned to the vertical but had continued bending past by 20°. After 18 h, the control stipe (B) was still vertical but stipes A and C were bent away from the applied drop by more than 180° and 140°, respectively. Bending away from the applied drop was called Fungiflex 2 activity. Control stipes often swayed slightly from one side to another during the observation period but they did not bend by more than 10° and in 90% of the bioassays did not bend by more than 5° from the vertical. In all of the experiments that follow, the experimental approach shown in this figure was used as a standard bioassay to detect and monitor Fungiflex activity.

Use of the bioassay to assess the effects of enzyme digestion on the Fungiflex extract. Bioassay of cap and mature primordium extracts together with trypsin-digested cap extracts. One drop (5 μl = 1.25 mg extract) was applied to the RHS (right hand side) of each stipe. Numerals at top left of each panel show the hours elapsed since the start. Labelling key: t = trypsin-digested cap extract; c₁ = cap extract control (cap extract treated in the same manner exactly as the trypsin digest, but excluding the enzyme); tb = trypsin-digested and boiled cap extract; cb = boiled cap extract control; c₂ = crude cap extract; p = crude extract from mature, differentiated but pre-meiotic primordia; z = distilled water. Slight Fungiflex 1 activity (bending towards the applied drop) was observed in cap extracts (at t = 1 and 3 h) but none was observed in the primordium extracts, although in all other bioassays, Fungiflex 1 activity was present in primordium extracts as well (data not shown). By 6 h after application, stipes had begun to bend in the other direction (that is, away from the drop), characteristic of Fungiflex 2 activity. After 21 h, stipes treated with cap and primordium extracts were bent away from the applied drop significantly. The control stipe was bent only slightly. Extracts from mature primordia exhibited slightly less bending activity than extracts from mature stipes (shown in the upper figure on this page) and caps (t = 21 h). Extracts from pre-meiotic, undifferentiated primordia contained Fungiflex 1 activity but no Fungiflex 2 activity (data not shown). Fungiflex 1 and 2 activity was observed in cap extracts from all four treatments. Trypsin digestion, with or without boiling, had no effect on the activities of either Fungiflex 1 (t   = 1-3 h) or 2 (t = 21 h).