Inquiry­based investigation of ectothermic metabolism University of California, Irvine Nancy Aguilar­Roca ​
(​
[email protected]​
) Over the course a traditional physiology course, discussions of metabolism often focus on mammals which are endothermic homeotherms. Students may not be exposed to heterothermic ectotherms, which are the vast majority of all living organisms. In recent years, research on heterothermic ectotherms has surged because of their vulnerability to climate change. The goal of this lab exercise is to add an open­ended inquiry element to a traditional protocol for measuring the effect of temperature on an ectotherm (the Madagascar Hissing Cockroach, ​
Gromphadorhina portentosa​
). ​
Gromphadorhina​
colonies are easily maintained in small spaces, genders are easily distinguished, adults are large enough to be individually marked for tracking, and it’s possible to create custom diets with controlled quantities of supplements. Prior to this lab exercise, students will learn about metabolism and the differences between ectotherms and endotherms, and they will design their experiment. This system is flexible so that a multitude of different experimental designs are possible. The experimental treatments that have been used so far are the effects of chronic caffeine, green tea and a sports supplement on O​
consumption and CO​
production. For a class of 227 2​
2​
physiology students (in 11 lab sections), we set­up multiple colonies of Gromphadorhina with 2 replicate colonies of each supplement. Each lab section put the results of their experiments into a common database (a Google spreadsheet), and each TA was given the opportunity to structure a post­experiment activity for their own students. Potential Learning Goals ● compare and contrast endotherm, ectotherm, homeotherm and heterotherm. ● explain the relationship between environmental temperature and the metabolic rate of an ectotherm (Madagascar Hissing Cockroach, ​
Gromphadorhina portentosa​
). ● develop and test a hypothesis about the effect of a dietary or pharmacological substance on metabolic rate Materials ● Madagascar Hissing cockroaches ● Metabolic chambers and gas analyzers or manometers ● Dry dog or cat food ● Coffee grinder or mortar & pestle ● Assorted supplements (liquid or powdered) Cockroaches Madagascar hissing cockroaches are fairly easy to keep. They will thrive (and probably reproduce) in a container with a mix of foods (dog/cat food, small pieces of apples, lettuce, o​
carrots), places to hide (egg cartons work well) and a heating pad or heat lamp (~25­30​
C). http://www.petco.com/caresheets/invertebrates/Cockroach_MadagascarHissing.pdf http://agweb.okstate.edu/fourh/aitc/lessons/extras/cockroach.pdf 1 Cockroaches also need a water source. Water beads are a good option because they don’t spill and you can monitor how fast they are used. Potential suppliers of madagascar hissing cockroaches (and most of these also sell water beads/crystals) New York Worms: ​
http://www.nyworms.com/index.html Rainbow mealworms: ​
http://rainbowmealworms.net Ken the Bug Guy: ​
http://kenthebugguy.com West Coast Roaches: ​
http://westcoastroaches.com Cockroaches are large enough for marking individuals if students want to track weight changes. Students can create their own marking system with dots of acrylic paint on the carapace. Custom food for experiments The exact ratio of dried dog/cat food to liquid will vary with the brand of food so you may need to experiment a little to find a ratio that will work for you. We’ve been using 1g food plus 1 ml water or juice. Put your desired quantity of dried food into the coffee grinder (or mortar) and grind it. Add the appropriate amount of liquid to create a paste (roughly the same consistency as play­doh). Cut the tip off of a 10ml syringe and put the food into the syringe. You can extrude the paste from the syringe into regularly shaped pellets. (A pastry bag with a frosting tip would work too). Let the pellets dry out. If you are adding a powdered supplement, add a known quantity to the ground dog/cat food before making the paste. Students can change the ratio of supplement to ground food to generate dose­response curves. If you are adding a liquid supplement, add it to the water (or juice) for making the paste. Again, you can vary the ratio of water to supplement to generate dose­response curves. Before giving the roaches the supplemented food, they should be starved for 1 to 2 weeks (but with access to water beads). Deciding how long to feed the supplement to the roaches before measuring metabolic rate is a factor that students will have to decide on. Measuring metabolic rate The protocol described below can be accomplished in a 3­hr lab period. Glass jars are best for metabolic rate measurements (many plastics are oxygen permeable). I use 500ml glass canning jars. 2 In this set­up the lid of the jar has 2 holes: 1 has a tubing connector to attach to the gas analyzer and the second one is to allow for air­flow to the gas analyzer. (I put a magnet on top to prevent it from floating in the water bath). I put 2­3 similar sized cockroaches into the chamber, close the jar, and cover the openings with tape, so that the chamber is completely sealed and no fresh air can enter. Record the time at which the chamber was first sealed shut and put the chamber into a water bath. Let the sealed chambers o​
incubate for at least 60 minutes at temperatures above 23​
C, o​
and at least 90 minutes at temperatures below 23​
C. When ready, stop the timer and record the time to the nearest minute. Uncover both openings and attach the tube from the gas analyzers to the connector on the lid of the chamber. The other opening must be uncovered, to allow fresh air into the chamber as the analyzers remove the experimental air. Record the data from the gas analyzers. It should take about 2 minutes get these readings. Record the lowest %O​
and the highest %CO​
. Disconnect your chamber, remove the lid, and 2​
2​
return the roaches to their cages. Calculations Vo​
(ml O​
/min) = 0.9 * (Fo​
_Starting ­ Fo​
_Ending) * (Chamber Volume) / min 2​
2​
2​
2​
Fo​
_Starting = fraction of oxygen in the air = 0.2095 2​
Fo​
_Ending = what you measured (percent divided by 100). 2​
o​
0.9 is to convert from ATPS to STPD (assuming room temperature is 21​
C and atmospheric pressure is 760mmHg) Relative­Vo​
= Vo​
/ mass 2​
2​
Vco​
(ml O​
/min) = 0.9 * (Fco​
_Ending) * (Chamber Volume) / min 2​
2​
2​
Fco​
_Ending = what you measured (percent divided by 100). 2​
If you don’t have gas analyzers, you can use a manometer. Manometers can be made with varying levels of sophistication, but they all consist of a closed chamber with a piece of water filled­tubing attached. As the animal inside the chamber respires, Soda lime or (potassium hydroxide) absorbs CO​
and the volume of oxygen inside the chamber decreases. The water 2​
in the tubing will move as the gas volume in the chamber changes. The simplest version is below. It consists of a jar on its side and with a one­hole stopper with a 1­ml pipet (Hiebert and Noveral, 2007). The organism can be kept away from the soda lime with wire mesh. The volume of oxygen consumed is read directly from the movement of the 3 water. By removing the soda lime, you can measure CO​
produced (Vco​
). This apparatus 2​
2​
works best in an incubator because the volume of air in the chamber will change with variations in ambient temperature. In the second manometer set­up shown below, the second chamber corrects for temperature­induced changes in air volume. As the animal consumes oxygen, the fluid in the manometer will shift towards the chamber with the organism. At set­time points, air (or pure oxygen) is injected into the chamber until the fluid in the manometer returns to its original position. The volume of air injected is the volume of oxygen consumed. As with the simple manometer above, you can determine CO​
production by removing the soda lime. 2​
http://sites.sinauer.com/animalphys3e/boxex07.04.html http://www.nuffieldfoundation.org/practical­biology/measuring­rate­metabolism Noveral, J. and S. Hiebert. 2007. Are chicken embryos endotherms or ectotherms? A laboratory exercise integrating concepts in thermoregulation and metabolism. Adv. Physiol. Ed. 31: 97­109. ​
http://advan.physiology.org/content/31/1/97 4