To ensure an adequate supply of active enzymes, the grasshoppers should be fed with an ample supply of fresh grass one to two hours before killing. When the grasshopper gut has been isolated, rinsing of its inside should only be done very briefly to avoid complete removal of enzymes from the digestive glands which are lining the epithelium.
Extraction of enzymes from living tissues usually involves grinding, filtering and centrifuging. In this experiment, since the insect gut is small in mass, the enzyme extraction procedure is simplified to grinding in cold distilled water with mortar and pestle, thereby minimizing enzyme loss. But forthing should be avoided to prevent enzyme inactivation by surface denaturation.
The photographic film strip used in this experiment is Kodak PlusX Pan B1W film which has been exposed, developed and fixed. Colour films have been tried but it is difficult to obtain good discernable results due to the multcoating of emulsion on the celluloid sheet.
In carrying out the iodine test for starch, it is advisable to use a very dilute solution Of 12KI . 0. 5 CM3 of the stock solution (prepared by dissolving 1 g 12 and 2 g KI in 100 cm3 of distilled water) should be added to 10 CM3 of distilled water prior to use. Do not fill up the cavities of the spot plates with the dilute iodine solution all at the same time because the brown colour gradually fades. Iodine solution should be added to the plate row by row. If a Pasteur pipette is used, two drops of 12KI solution should be added to each cavity.
The above experiments have been tried out by about 200 teachers in a workshop for teachers conducted by the authors and the majority found that the activity of enzymes was in the following order:
Protease M>F>H
Arnylase
Invertase M > H > F
(F = foregut, M = midgut, H = hindgut)
f,
Result
1
i
1
M > F > H (salivary glands not included)
F > M > H (salivary glands included)
Table showing the results of 'gut extract + sucrose', 'gut extract + starch' and 'gut extract + distilled water'.
Conclusion and explanation
(1) The presence of more precipitate in 'gutextract + starch' and 'gut extract + sucrose'
as compared with 'gut extract + distilled water' (as control) indicates the presence
of arnylase and invertase in the gut because arnylase hydrolyses starch to maltose
(reducing sugar) and invertase hydrolyses sucrose to fructose and glucose (both
are reducing sugars) which can be detected by Benediet's test.
(2) 'Gut extract + distilled water' acts as control to indicate the presence of endogenous
reducing sugar for comparison.
(3) Removing adhering structures and content of the gut reduces the possibility of other
sources of enzymes contributing to the arnylase activity.
(4) It is possible that some of the reducing sugars detected from the test may be the
action of maltase on maltose, which is converted to glucose.
(5) The purpose of setting up boiled gut extract as one of the control is to show that
the reaction proceeds only in the presence of active proteins.
(6) The grasshopper is a herbivore so that anrylase and invertase are normally present
in its gut for the digestion of plant materials, but not cellulose.
