Laboratory 7 The Effect of Juvenile Hormone on Metamorphosis of the Fruit Fly (Drosophila melanogaster) (portions of this manual were borrowed from Prof. Douglas Facey, Department of Biology, Saint Michael's College, Colchester, VT 05439) Objectives: (1) To determine the optimal concentration of juvenile hormone required to interfere with normal development of Drosophila melanogaster at specific stages. (2) To determine whether fruit flies are more sensitive to Juvenile Hormone during early or late metamorphosis. (3) To learn about hormonal regulation of animal development and the importance of critical periods in the development process. This will be a 2-week lab, we will perform the experiment during the first week, and analyze the data and discuss the lab report during the second week. Introduction: Insects go through distinct life stages as they grow from an egg to a sexually mature adult. Hemimetabolous insects, such as grasshoppers, cockroaches and true bugs, undergo incomplete metamorphosis. Eggs hatch into immature nymphs, which somewhat resemble the adults except that they are much smaller. They then go through several progressively larger stages (instars) before they become adults. This is referred to as incomplete metamorphosis because the immature nymphs somewhat resemble the adults. Holometabolous insects, however, which include flies, beetles, and butterflies, undergo complete metamorphosis, a process which involves four distinct and dramatically different phases. The eggs hatch into worm-like larvae, which we often refer to as maggots, grubs, or caterpillars. These, clearly, do not resemble the adults in any way. After going through several progressively larger larval stages (instars), the larvae become pupae. From the outside, pupae appear dormant, but internally there is major restructuring taking place. Finally, the pupal exoskeleton breaks open and an adult emerges that does not resemble either the larva or the pupa. This is referred to as complete metamorphosis because of the drastic alterations involved development. The entire process of metamorphosis is regulated by various hormones. As in mammals, neurosecretory cells in parts of the insect's brain release hormones that have either a direct or indirect effect on other parts of the animal's body. Your text may include a discussion of this process on pages 343-346. In this laboratory exercise we will investigate the role of juvenile hormone (JH), on the development of the fruit fly, Drosophila melanogaster. Early in an insect's life, JH levels tend to remain fairly high. This promotes the retention of larval traits as the larva goes through several instars. During the last larval instar, the level of JH declines. This results in the development of a pupa during the next molt. JH levels then become very low, allowing the pupa to develop into an adult. The presence of JH, then, promotes the retention of juvenile characteristics. If JH is absent adult development takes place.
There is a "critical period" during metamorphosis when JH applications will be effective. After this period has passed, JH seems to have little, if any, effect and pupae develop into normal adults. Last year the Bio104 students determined that JH has a dramatic effect at several stages of metamorphosis. In earlier stages, the JH caused the death of the pupa, so hatching did not occur. At later stages, the JH interfered with hatching; the adults became stuck during eclosion (hatching). As a class, you will continue this work by testing the effectiveness of different concentrations of JH and examining the periods that are sensitive to JH. Protocol: Each group will collect pupae from the vials that you will be given. These pupae will be staged under the microscope and treated with either JH or a control (acetone). The pupae are then kept at 25 C until eclosion. It is important that all of you keep complete and accurate notes of observations and the timing and concentrations of JH applications throughout the study. You are all working together, so one mistake can affect everyone's results. Because the time of pupariation in Drosophila depends upon the amount of moisture in the food and the temperature, we will not be able to stage pupae according to time. Instead we will stage them according to morphological features. Drosophila proceed through many different steps that have been described morphologically and developmentally. A short description of the stages is given below (taken from Bainbridge and Bownes, 1981). The times given in parentheses are estimates at 25 C. We will concentrate on stage P6-P8 and P 12-P13. A diagram of these stages is on the next page. Color copies will be available in the lab. 1) Third instar larva - stops eating and climbs the vial wall. 2) Pupariation - this is the formation of the structure in which the fly will undergo metamorphosis. Some signs of pupariation include shortening of the body, eversion of the anterior spiracles, and tanning (hardening) of the larval cuticle. 1) Prepupal stages 4) Pupal stages 5) Pharate stages Stage P1 - pupa becomes light brown (0.3-1 hr) Stage P2 - pupa becomes darker brown; gas bubble forms within the abdomen (3 hr) Stage P4 - bubble moves to anterior, displacing pupa posteriorly (12-13.5 hr). Stage P5 - malpighian tubules prominent and green (13-48 hr). Stage P7 - eye color yellow at perimeter (43-47 hr). Stage P8 - eye color bright yellow (49-57 hr). Stage P9 - eye color amber to pink (71-78 hr). Stage P10 - eye color bright red. Stage P11 - tips of folded wings gray (72-77 hr). Stage P12i - wings gray all over 73-78 hr). Stage P12ii - wings darken to black (75-86 hr). Stage P13 - claws become black. Stage P15i - legs begin to twitch 90 hr). Stage P15ii - eclosion.
P1 P2 P4 P5 P7 P8 P9 P1 0 P1 1 P12i P12ii P1 3
So What? 1) Your TA will give each group a collection of vials from which to collect pupae. 2) Wet a brush with water and gently wet the pupa that you want to remove. Let the water sit on the pupae for a minute or two and the gently remove the pupa with the brush. If you accidentally break the pupal case, do not use that pupa; it will not eclose. 3) Place each pupa on a filter disc and examine it under the microscope to determine its stage of development. Transfer the staged pupae to the appropriate petri dish (we will probably combine pupae from different groups). Make sure that the pupae are sitting on filter circles and not on the plastic. 4) Your TA will provide you with a synthetic form of JH which has been dissolved in acetone and an acetone control. Please wear protective gloves when handling the acetone and JH dilutions, and work in the hood. Use pipets to apply 0.5 µl of JH (or acetone) to the exoskeleton of the insects. It is difficult to keep acetone in the pipet tip and it will usually hang as a drop off of the pipet tip. You cannot avoid this, so just work quickly and get the chemical on the animals before the drop dries out. Pay close attention to the labels on the JH containers so that you don't contaminate any stock solutions, and make sure that all of the larva/pupae in a particular dish receive the same dose of hormone. Hormones tend to be effective in very small concentrations, so a seemingly minor contamination event could have a major impact on the results of the study. Treat each animal only once. 5) When you are finished and the acetone has dried on the animals, tape the lid on the dish and make sure that the lid is labeled with the date, the concentration of JH, the stage of the pupa, the number of pupae in the dish, and your initials. Your TA will return the dishes to the 25 C room until the next lab session. 6) During week 2, you will collect the results of the experiment. Metamorphosis takes about 100 hours in the fruit fly, so all of the animals should have completed development (and probably died from dehydration) by the second week of the lab. Locate your group of dishes and examine them under the microscope. Count the number of empty pupal cases and the number of adult flies (they should be equal). Also look at the other pupae and record whether they are unhatched or whether they became stuck during eclosion. If you notice any other interesting abnormalities, make a note of them. Add you results to the class data on the board and add it to the total results on the computer. 7) We will pool the class data at the end of the experiment to see what conclusions we can draw about the role of JH in metamorphosis and the importance of the timing and concentration of the applications. These results will be available Friday afternoon after the lab. Some insects are capable of spreading diseases or causing considerable damage to agricultural crops, forests, lawns, and gardens. Biting insects can also be a significant annoyance when present in large numbers. One approach to fighting the insects has been to treat large areas with poisons that kill the offending insects. Unfortunately the toxic compounds also may kill many non-target species such as insect predators, including other insects, spiders, and birds. The toxins also may kill helpful insects such as those that are important pollinators of flowering plants. Some toxic chemicals also may accumulate in the environment due to their longterm chemical stability. In recent decades, however, other techniques have been developed to try to control "pest" insects without the widespread environmental damage caused by many pesticides. These alternative methods have included "physiological warfare" in which affected areas are treated with synthetic insect hormones to inhibit insect development and subsequent reproduction. Non-target organisms, such as spiders and birds, would presumably not be affected by hormones targeting insect development. How might the lessons learned in this lab exercise be put to use in such a way? Are you aware of any examples of such use?
In addition, studies of hormone receptors has shown that insects and vertebrates respond to hormones in similar ways, at a cellular level. So information gained about hormone response in insects (and other invertebrates) could be useful in the study of hormones in vertebrates.