Active Transport in Insect Malpighian Tubules

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Chapter 11


                    Active Transport
              in Insect Malpighian Tubules
                                    Dee U. Silverthorn

                         Department of Zoology, University of Texas
                                   Austin, Texas 78712
                                 (512) 471-6560, FAX: (512) 471-9651
                                     [email protected]




  Dee Silverthorn began working with insects as an undergraduate at Tulane
  University. She received a Ph.D. from the Belle W. Baruch Institute for Coastal
  and Estuarine Studies at the University of South Carolina. Her research interests
  are focused on crustacean physiology with an emphasis in epithelial transport.
  She is currently a Senior Lecturer in the Department of Zoology at the University
  of Texas and teaches the pre-medical physiology class and is in charge of
  undergraduate physiology laboratories. This experiment was developed in
  conjunction with a National Science Foundation Instrumentation and Laboratory
  Improvement Grant, USE-9251764.

Reprinted from: Silverthorn, D. U. 1995. Active Transport in Insect Malpighian Tubules. Pages
141-154, in Tested studies for laboratory teaching, Volume 16 (C. A. Goldman, Editor). Proceedings
of the 16th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 273
pages.

Although the laboratory exercises in ABLE proceedings volumes have been tested and due
consideration has been given to safety, individuals performing these exercises must assume all
responsibility for risk. The Association for Biology Laboratory Education (ABLE) disclaims any
liability with regards to safety in connection with the use of the exercises in its proceedings volumes.




                                    1995 Dee U. Silverthorn
                                                                                                           141
142        Active Transport

                                                                    Contents

Introduction....................................................................................................................142
Materials ........................................................................................................................142
Student Outline ..............................................................................................................143
Notes for the Instructor ..................................................................................................149
Acknowledgements........................................................................................................152
Literature Cited ..............................................................................................................152
Appendices.....................................................................................................................153


                                                                 Introduction

     This laboratory exercise uses insect Malpighian tubules to demonstrate transport processes in a
living tissue. It does not require complicated equipment and depends on visual assessment of dye
concentration. By varying the solutions in which the Malpighian tubules are incubated students can
show the energy dependence of active transport as well as the properties of specificity, competition,
saturation, and inhibition. Since transport of the dye chlorophenol red across an epithelium is a
complex mechanism involving several different membrane transport proteins, students who have not
been introduced to indirect active transport processes (also known as secondary active transport)
may need additional instruction in this area.
     The accompanying Student Outline is designed for a lower-level laboratory in which the
students are provided with a series of previously prepared solutions. In a more investigative
laboratory students design their own experiments and make up their own solutions from 10X stocks.
     Setup time for the laboratory is 23 hours and consists mainly of making solutions. The
laboratory exercise itself requires a 3-hour block of time. When the exercise is done as an
investigative project students repeat the project in three successive weeks. This allows extra time
for them to make their own solutions and to run replicate experiments for statistical analysis.
     We have used both crickets and roaches successfully for this experiment. Students are less
squeamish about handling crickets. Roaches have a large amount of fat body in the abdomen that
must be removed during the dissection and this slows down the experiment when students are
running duplicates.

                                                                   Materials

Large adult crickets (Acheta domestica) or cockroaches (Periplaneta or Blaberus) (810/group)
Test tubes large enough to hold insects (12/group)
Ice buckets, tubs or 1 liter beakers to use as ice containers (1/group)
Dissecting microscope with light (1/group)
Dissecting pans that fit under microscope (1/group)
Petri dishes, 35 mm (4/group)
Fine insect pins (4/group)
Fine point forceps (12/group)
Spring-handle fine dissecting scissors or other fine point scissors (1/group)
Blunt glass or metal probes
Pipet pumps for up to 10 ml (1/group)
Pipets, 5 or 10 ml (10/group)
White paper to put behind petri dishes or white ceramic spot plates (1/group)
Pasteur pipets and bulbs (24/group)
Latex surgical gloves (1 pair/student)
Grease pencil or marker and tape for labelling (1/group)
                                                                                 Active Transport   143

Kimwipes (1/group)
Plastic wrap or foil to cover test tubes (1/class)
Fine silk thread for ligating roach intestine, optional (1 spool/class)

  For preparation of the solutions:

Balance (1/class unless students make their own solutions)
pH meter (1/class unless students make their own solutions)
Erlenmeyer flasks with stoppers or other storage containers (7/class)
Volumetric flasks

  Solutions

Insect saline in a closed flask (150 ml/group)
1 mM chlorophenol red in saline (25 ml/group)
1 mM chlorophenol red + 0.1 mM 2,4-DNP in saline (25 ml/group)
1 mM chlorophenol red + 10 mM ouabain in saline (25 ml/group)
1 mM chlorophenol red + 0.1 mM 2,4-DNP + 10 mM ATP in saline (25 ml/group)
1 mM chlorophenol red + 10 mM PAH in saline (25 ml/group)
1 mM chlorophenol red in zero potassium saline (25 ml/group)
1 mM chlorophenol red in zero sodium saline (25 ml/group)


                                           Student Outline

                                             Introduction

     In this experiment you will be using the isolated Malpighian tubules of insects to study
secretion of organic molecules across a transporting epithelium. This experiment demonstrates the
effect of metabolic inhibitors, competitors, and ions on the transport of organic dyes such as
chlorophenol red.
     The transporting tubules of excretory systems are remarkably similar in animals as diverse as
insects and mammals. The Malpighian tubules of insects are finger-like extensions of the intestinal
tract. The proximal ends of the tubules are attached to the intestine at the junction of the midgut and
hindgut while the closed distal ends float free in the hemolymph (blood) of the insect. Excretion is
achieved exclusively by secretion of ions and organic molecules from the hemolymph into the
lumen of the tubule (Figure 11.1). This is unlike the mammalian kidney, where most of the contents
of the kidney tubule derive from plasma filtered into the lumen at Bowman's capsule. As in the
mammalian kidney, however, the contents of the lumen are modified as they pass through the
Malpighian tubule and hindgut.
     The walls of the Malpighian tubule are composed of a single layer of cuboidal epithelial cells.
These cells rely heavily on active transport mechanisms to concentrate ions and organic molecules
in the lumen. Since the individual tubules are not bound together, they make an excellent study
system in which you can examine some of the properties of secretion in a transporting epithelium.
You will be using Malpighian tubules from crickets or cockroaches. When incubated with
chlorophenol red, a colored organic anion, the tubules actively transport the dye into the lumen
against a concentration gradient. The amount of dye being transported can be estimated visually.
Since active transport is energy-dependent and uses a protein carrier, the accumulation of dye will
have the same properties as other protein-based mediated transport systems: saturation, competition,
and specificity.
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