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Chapter 6 Digestive Enzymes of the Cockroach C. Leon Harris Department of Biological Sciences State University College of Arts and Science Plattsburgh, New York 12901 C. Leon Harris is Associate Professor at the State University Col- lege of Arts and Science at Plattsburgh, New York, where he has taught Principles of Animal Physiology for nine years. He earned degrees in physics and in biophysics at Virginia Polytechnic Insti- tute and Pennsylvania State University. His major area of research is the neural control of behavior in the cockroach. He is now writing a book on historical and philosophical aspects of evolution. 112 Digestive Enzymes Introduction This experiment on digestion in cockroaches is an important part of my senior-level course in animal physiology, not only because it reinforces the lectures in digestion and enzymes, but also because it clarifies the fundamen- tals of statistics and introduces students to the critical thinking required in transforming data into a scientifically valid conclusion. The experiment also has the advantage of employing current research techniques. (See for example Bland and House 197 1.) Yet, even freshmen in a general biology laboratory can complete the experiment in three hours, except for the protease test which requires brief observations overnight. Not the least advantage, from the in- structor's point of view, is the study's inexpensiveness and ease of preparation. It uses common reagents and inexpensive cockroaches (see Appendix A), and requires less than three hours to prepare if the materials are at hand. My upper-level undergraduates work in pairs, with each pair preparing en7yme extracts from homogenized salivary glands, crop, midgut and hindgut of a single cockroach, and also a control consisting of a heated mixture from the other extracts. Each pair of students then tests each extract and the control for either sucrase or lipase or amylase and protease. (Since the last two assays are so simple, the same students do both, using the same extracts.) Students rank the enzyme activities for each section on a scale from 0 to 3, with the control defined as 0. The instructor compiles the data and returns them to students to calculate the means and standard errors (optional) for each section. They then use the sign test to determine whether activity in each section is significantly greater than that in the control, or greater in one section than in another section with significant activity. I? the sign test, P is determined simply from the proportion of cases in which the activity from one section was greater than in the control or in another section. (See Appendix B.) This non- parametric test is so transparent that students find that it clarifies basic principles of statistics, even (or especially!) if the students have already had a statistics course. Because the conclusions are based on compiled data, the results are immune to the effects of an occasional aberrant cockroach or student. From the data, students can deduce which sections of the cockroach digestive system contain the enzymes. However, inferring from this which section actually secretes the enzyme is not always straightforward. Enzymes may travel from one section to another and not be rinsed off the tissue before the extract is prepared. Thus, even though all the enzymes are supposed to be secreted only by the midgut, except for amylase which is also secreted by the salivary glands, the crop sometimes shows significant activity. Students should be able to infer that this activity could be due to the much greater mass of tissue adsorbing enzymes from other sections. Students should also be able to suggest that significant enzyme activity in the hindgut might be due to its being "downstream" and hard to clean. Secretion of enzymes by Digestive Enzymes 113 the hindgut would make little sense, because nutrients could not be absorbed from the hindgut into the hemolymph. Initially students are apt to view these deviations from what "the book says" as failures. Perhaps the most valuable lesson from the experiment is that whoever wrote the book was smart enough not to accept data at face value. Of course it is necessary te emphasize the difference between fudging data to get the "right" conclusion, and interpreting data in the discussion of a report. In addition to interpreting data, I also . suggest that in report discussions students compare functions of the sections of the cockroach digestive system with analogous organs of mammals. Student Material The strategy of digestion in cockroaches is similar to that in humans and in other vertebrates: food passes from mouth to anus through a tube where it is mixed with digestive enzymes. A cockroach's meal, like yours, is first mixed with saliva. It then enters the crop (foregut), which can store up to a two month's supply of food. The contents of the crop eventually pass through the proventriculus (gizzard), which crushes solids and acts as a valve. The food then enters the midgut, which includes eight ceca at the anterior end. Almost all of the absorption of nutrients takes place in the midgut. The remaining material goes into the hindgut, which is joined to the midgut at the site of attachment of the Malpighian tubules, which are analogous to the vertebrate kidney. Finally, feces are formed in the rectum. Although the overall process of digestion in cockroaches is similar to that in vertebrates, there are important differences in detail. For example, nutrients are not absorbed into capillaries in cockroaches, which lack circulatory vessels. Instead, the nutrients are absorbed directly into the body fluid (hemolymph) surrounding the digestive system. A second major difference is the absence of mucus to protect the inner lining of the cockroach gut. Instead, there is a continuously formed semi-permeable sac--the peritrophic membrane--which contains the food in the midgut, and a layer of impermeable chitin to protect the crop and hindgut. (Chitin is the material which also forms the exoskele- ton.) Cockroaches are notoriously omnivorous. We therefore expect them to employ a variety of digestive enzymes. From exp&ience we know that they digest sucrose, proteins, starch and fats, so we Gould expect that somewhere in their digestive systems are secreted sucrase (invertase), protease, amylase and lipase. The question we wish to answer is "Where in the cockroach digestive system are these four kinds of enzymes secreted?". The first step in answering that question is to test the ability of extracts of the salivary glands, crop, midgut, and hindgut to digest sucrose, gelatin, starch and olive oil. High digestive activity in a section suggests, but does not prove that the enzyme is produced and secreted in that section. 114 Digestive Enzymes Malpighian tubules Figure 6.1. A. Adult American cockroaches, Periplaneta americana. Male, ventral view; female, dorsal view. Males may be distinguished from females by their more slender body and by the pair of styles on the anal segment. One style is visible between the hind legs of the male. The length of each cockroach, excluding antennae, is approximately 2 cm. B. Digestive system of the American cockroach. The gut is brown, the salivary glands are white, and the reservoir is transparent. The Malpighian tubules are yellow, and the tracheae are white. Digestive Enzymes 115 Instructor's Materials I. Preparation of enzyme extracts A. Obtain a large cockroach which has been immobilized by CO,, by cooling in a freezer for 15 min, or by holding it under warm water. B. Cut the anus transversely to free the rectum, and remove the digestive tract by carefully pulling on the head. The entire digestive tract should come out with the attached head. Often the tract breaks at the proventriculus; if this happens cut open the abdomen and pull out the midgut and hindgut. Place the digestive tract into a watch- glass containing insect saline. C. Using the dissecting microscope, identify and separate the salivary glands, crop, midgut and hindgut. The salivary glands are particu- larly difficult; they look like small white globules of fat just behind the head. Often the salivary glands are lost. If the glands are not present, leave a blank under S in the data. Discard the proventriculus and large tracheae. Also discard the part of the gut where the Mal- pighian tubules are attached. Small trachae may be included since they do not secrete digestive enzymes. D. Cut open the crop and rinse. If possible, gently squeeze solids out of the midgut and hindgut, since these may have absorbed enzymes from other parts of the digestive tract. E. Label 4 depressions of a spot plate S, C, M and H. Add 5 drops of water to each of the 4 depressions. Place the sections isolated in step C above into the appropriate depressions: S salivary glands (with reservoirs) C crop M midgut and ceca (hereafter referred to as midgut) H hindgut F. Grind each section with the blunt glass rod to release enzymes from the cells. IMPORTANT: rinse the glass rod thoroughly when going from one section to another. G. Use a separate Pasteur pipette to withdraw the liquid extract from each depression. Combine 1 drop of each extract into a fifth 10-ml test tube labelled K; the rest of the extract in-each pipette goes into the appropriate 10-ml test tube labelled S, 'C, M and H. IMPORTANT: keep the pipettes in correct order. They will be used in step I and for the protease and amylase tests. H. Each extract should just fill the rounded bottom of the test tube (.2 ml). If necessary, add water to increase volume. I. Place test tube K, containing the mixture from the four sections, into a boiling water bath for 15 min.

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