Active Transport in Insect Malpighian Tubules
Jonathan S. Harris
Anderson High School, Austin, TX 78759
AHBIOTEACH@AOL.COM
with extreme gratitude to Dr. Dee Silverthorn at Univ of Texas, Austin

Introduction
This laboratory exercise uses insect Malpighian tubules to demonstrate transport processes in a living tissue. The procedure 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. Because 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.
In a lower level laboratory, 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 10 X stocks. Setup time for the laboratory is 2-3 hours and consists mainly of making solutions. The laboratory exercise itself works best with a 3-hour block of time, although it can be done in 2 hours if the lecture is done separately. When the exercise is done as an investigative project, students repeat the project 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.

Background
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, located at the junction of the midgut and hindgut. 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. 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. Because the individual tubules are not bound together, they make an excellent study system in which to examine the properties of a transporting epithelium. The transport systems of the Malpighian tubule include several species of indirect active transporters that utilize the energy of one molecule moving down its concentration gradient in order to push a second molecule against its concentration gradient. The sodium-glucose transporter of mammalian kidney is a typical example. Direct active transport uses energy stored in the high-energy bond of ATP. Two direct active transporters are the Na+/K+-ATPase (sodium-potassium pump), located on the basolateral membrane facing the ECF and a proton pump (H+-ATPase) on the apical membrane.
Renal transport systems for organic anions depend on indirect active transport proteins. Although the system has not been well studied in insects, the membrane proteins appear to be very similar to those involved in transepithelial organic anion transport in mammals. Some compounds transported by the organic anion system of the vertebrate kidney include penicillin, saccharin, salicylate, benzoate (used as a preservative), amino acids, and bile salts.

The figure shows a model for organic anion transport. The transepithelial transport of organic anions from the ECF into the lumen of the kidney is a two-step process: import of the anion into the tubule cell in exchange for a dicarboxylate such as a-ketoglutarate (aKG) or glutamate, followed by export into the lumen in exchange for another anion such as chloride, hydroxyl, or urate. The cell maintains a supply of aKG by importing it from the ECF along with Na+ ions. The concentration gradient for Na+ is maintained by the ATP-dependent sodium-potassium pump (inhibited by ouabain) and apparently also by an apical H+/Na+ exchanger.
In this experiment, chlorophenol red (CPR), a colored organic anion, is actively transported into the tubule lumen against a concentration gradient. The amount of dye being transported can be estimated visually. Because 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. p-Aminohippurate (PAH) is a colorless organic anion that is transported on the same membrane proteins as chlorophenol red and therefore competes with CPR for transport.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

In the absence of ATP or the presence of ouabain, a specific inhibitor of the sodium-potassium pump, the Na+ concentration gradient that drives CPR secretion gradually disappears.

Materials
Large adult crickets (Acheta domestica)* or cockroaches (Periplaneta or Biaberus) (g- 10/group)
Test tubes large enough to hold insects (1 -2/gr~up)
Ice buckets, tubs or 1 liter beakers to use as ice containers (1/group)
Dissecting microscope with light (1/group)
Petri dishes, 35-mm, that fit under microscope (1/group)
Fine point forceps, fine point scissors and blunt glass or metal probes (1-2/group)
White paper to put behind petri dishes or white ceramic spot plates (1/group)
Pasteur pipettes and bulbs (24/group)
Latex surgical gloves (1 pair/student)
Kimwipes (1/group)
Plastic wrap or foil to cover test tubes (1/class)
 

* Crickets are cheap and easy to obtain from local bait shops or from Fluker Farms, 1-800-735-8537. P.O. Box 378, Baton Rouge LA 70821-0378. Crickets must be kept warm and dry. We use an old aquarium with a screen top. Fluker Farms will send care instructions. The fine powder left at the bottom of a dog food or rat chow bag is excellent for feeding them.

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

Solutions (Instructors need not use all of these)
Insect saline, 1 mM chlorophenol red in saline. Inhibitors/competitors: 0.1 mM 2,4-DNP (inhibits ATP
production); 0.1 mM 2,4-DNP + 10 mM ATP; 10 mM ouabain (inhibits Na+/K+-ATPase); 10 mM PAH (competes with CPR); 1 mM chlorophenol red in 0 K+ (substitute NaCl) or 0 Na+ saline (substitute choline Cl).
The table below gaves simple -recipes for normal saline and sodium- and potassium-free saline.
                                             Normal saline                      Sodium-free                          Potassium-free         NaCl                                          l60mM                       _____________                             170mM
KCl                                             l0mM                               l0mM                                       __________
CaCl2                                           4mM                               4mM                                           4mM
choline chloride                          _________                       160mM                                      __________
 

Because our students use large volumes of saline while doing repetitions of the experiment, we make up concentrated stocks: 1M NaCl and choline chloride, 0~ 1 M KCl and CaCl2. To make 1 liter of solution, use 1 mL of 1 M stock for each millimole/liter in the final solution. Use 10 mL of 0.1 M stock for each millimole/liter in the final solution

chemicals

Sigma #D-7004 2,4-dinitrophenol Sigma #A-1422 p-aminohippurate (PAH)
Sigma #A-2383 ATP Sigma #0-3125 ouabain
Sigma #C-1268  chlorophenol red Solubility 30 mg/mL in water; 80 mg/mL in alcohol.
Sigma #C-1879 choline chloride
 

Procedure
 

All participants should wear gloves when handling insects and chemicals.
 

1. Anesthetize the insect by placing it in a large test tube packed in an ice bucket. Cap the tube or drop a little ice into the tube on top of the insect. Don't let it drown in melted ice! Five minutes is usually long enough.
2. Inject 0.1 mL of plain insect saline (control) or 0.1 mM CPR into the insect's abdomen by sliding the needle between two of the segments. Angle the needle toward the insect's head and keep the tip close to the cuticle. 3. Allow solution to circulate for 30 seconds-1 minute. Cut off insect's head and legs, then cut off last two segments with scissors or forceps. Squeeze out guts into the petri dish and squirt lightly with plain saline. For crickets, the Malpighian tubules will look like yellow (or red) spaghetti. Tease out the tubules and view under high power on a dissecting microscope. Grade their color on a 0 (no red color) to ++++ (intense red) scale.
Female crickets are usually full of eggs that look like yellow rice. Male crickets tend to be smaller and are easier to assess.
4. Dispose of CPR and other chemical inhibitors as directed by your institution's safety office. Plain saline can go down the sink.

Hints
1. Assessment of Dye Accumulation: This is a subjective measurement and therefore is a little tricky. You may want to decide in advance which sample you predict will show the most uptake and arbitrarily assign that sample a maximal value. You should also consider whether the person making the assessment should know which test solution was used. This experiment is a good place to introduce students to the concept of blind and double-blind experiments. Should the people doing the scoring on the tubules know what the incubation solution was or will that impair their judgment? To create a double blind experiment, the instructor can provide solutions identified only by a number or letter.
2. Timing We encourage students to run incubations starting 1 minute apart so that they can compare samples. One difficulty in holding specimens for comparison is that the tubules move fluid into the gut quite rapidly. If the tissues are held in clear insect saline, the tubules will clear themselves of dye within a few minutes. This experiment is a good opportunity for students to develop group protocols where each member of the team has a particular task.
3. Tubule appearance. The cells of the tubule wall appear as a translucent/transparent band separating the dye from the external solution. Sometimes dye stains the cells as well. CPR is a pH indicator, turning from red at pH 6.4 to yellow at pH 4.8. Occasionally CPR will turn orange to yellow in the lumen of the tubule, making the
results a bit more difficult to interpret
4. Tubule movement. Students will often be able to see what looks like peristaltic movement of the tubules. This is not true peristaltic contraction but results from the contraction of spiral bands of muscle that surround the tubules. The movement helps expose the tubules to the hemolymph in the open circulatory system and also helps move the luminal fluid into the gut.

Expected Results
The solutions with added inhibitors, except the one with ATP, should show a marked decrease in dye concentration within the tubules compared to those in CPR-only saline. Transport should be noticeably inhibited in the presence of the mitochondrial uncoupler dinitrophenol (DNP) but may not be completely inhibited. The lack of total inhibition means that there is either some ATP production in the mitochondria (incomplete uncoupling) or that the cells have an additional source of ATP such as glycolysis. When ATP is added to the solution with DNP, transport should be restored.
Results with ouabain may be variable. Although ouabain inhibits the Na+/K+-ATPase, the apical H+-Na+ exchanger can help maintain the low intracellular Na+ concentration that drives the basolateral transporter.
Transport is noticeably decreased in 0 mM potassium and increases with potassium concentrations up to 20 mM. There is a slight decline in transport when K+ is 30 mM, probably due to depolarization of the cell by the high extracellular potassium level. Transport should be almost totally inhibited in 0 mM sodium.
p-Aminohippurate (PAH) is a colorless compound that transports on the same transporter as chlorophenol red. When PAH competes with CPR, the amount of CPR concentrating in the tubule will decrease.