INTRODUCTION

1. MALES EGGS LARVAE PUPAE FEMALES SUCCESSFUL FEMALES MATED FEMALES OTHER MALES Educational material integrating math and biology for middle school, high school, and introductory college sciences classes: Population dynamics and physiology of Drosophila. SAMUEL POTTER1, PHILIP H. CROWLEY1, REBECCA M. KRALL2, SUSAN MAYO3, DIANE JOHNSON3, KIM ZEIDLER-WATTERS2,3, RICHARD COOPER1 and ROBIN L. COOPER1. Module 3: Pupa and adult behaviors in response to diet as a larvae . 1Dept. of Biol., Univ. of KY.; 2Dept. of STEM, Univ. of KY and 3P-12 Math and Science Outreach Unit of PIMSER. Module 2: Food choice of adults and effects on larvae Before starting the modules a pretest will given to examine the knowledge base of the students on population dynamics and thoughts on the effect of nutrition and amount of resources on the population survival. INTRODUCTION The driving principle is to develop educational material which integrates math, biology and environmental factors for middle and high school sciences classes as well as introductory college courses. Here we outline an approach to modeling the life cycle and its implications for population growth in laboratory Drosophila populations. The goal is to develop a viable example of methodology that can be implemented in high schools and middle schools to introduce modeling that links with empirical studies in a way that is accessible to students. The approach is intended to encourage conceptual thinking and open a range of student-driven explorations at the intersection of biology and mathematics. Activity without understanding opposes what we know about effective science teaching (Windschitl, Thompson, & Braaten, 2008a,b). In addition, the lack of cross disciplinary training in K-20 grades compartmentalizes learning and hampers educational integration. This is even evident within the sciences such as with math and biology. We are developing modules which will be cross-disciplinary and can be used for middle school through college level courses. We are developing modules for measuring fecundity with various numbers of females and males, various environmental factors such as temperature and types of food. With the data collected we will develop a means for teachers and students to use mathematical models to explain the collected observations. The mathematical models can be used to predict the impact of environmental disturbances which could influence fecundity (egg laying). Then experiments can be performed to test if the predictions in the mathematical model hold or if adjustments in the model need to be performed. The module begins with students observing Drosophila from nymphs through the adult stage. In time we plan to have students use the parameters and data collected from their studies and use Simulink (The MathWorks, Natick, MA) to simulate the population dynamics over time. In this study we also examined how diet can effect larval development and behavior as well as adult behaviors. The food choices were lemon, banana, and peaches as well as corn meal and sugar water. Adult flies did not show any real difference for food type to eat but tended to lay more eggs on the lemon; however, all the larvae died in lemon due to mold/fungal growth more common on lemon than the other food choices. Larvae only fed sugar water delayed pupation for up to 14 days as compared to 4 days with a healthy diet. The nutrient deprived larvae were smaller in body length for the same instar stage. Behavioral assays in these larvae were carried out as a measure of health. Module 1: Rate of population growth depending on #of females and elclosin rate An example of 2 sets of conditions, repeated 3 times. 1 female :1 male vs 3 females :1 male 1 female :1 male 3 females :1 male The aquarium and the fruits under them. Peach, lemon, and banana . Ever 5 minutes count the number of adults on the various food s. Repeat trails at various time intervals. Graph data (left) Mark each pupa as they form. Take out adults at a particular day before pups elcose. Then count the rate of eclosion by releasing the flies each day. These graphs are showing the number of flies that landed on each fruit. The left panel is the raw data of the total number of flies for the 5, 10, 20 and 50 min time points for the five different trials. The right side panel is the converted data set when transferred to percent of total number of flies. Time to pupation and then the time for the pupa to become an adult with larvae raised on 2, 4 or 6 days on sugar water before being placed in corn meal. 1 female :1 male Righting response after being vortexed. This type of test is used in adult Drosophila to examine if the brain and sensory neurons are functioning well. The results did not show any difference in the adults from the larvae fed sugar water 2, 4, or 6 days. All the flies quickly got up after being vortexed. They were all able to do this in less than 10 seconds. Another example of 2 sets of conditions, repeated 3 times. 1 female :1 male vs 3 females :1 male These large vials one can keep the adults which form and count continuously the number of pupa which form until it become too crowded. Number of eggs 3 females :1 male METHODS Determining pupation rates: Adults at one of two ratios (one male to one female or one male to three females) were placed into food containing vials. They were allowed to lay eggs and were monitored daily. Once larvae began pupating, they were circled and numbered on the exterior of the vial. Each pupa was then documented with the time at which it was first seen. This continued as pupa continued to form. Determining eclosion rates: Using the same vials used for calculation pupation rates, they were monitored for the emergence of new adults. When the first fly eclosed in a vial, it was documented, and all adult flies were removed. The vials were then monitored daily for newly eclosed flies. Adults were continuously removed, and records were kept as to how many flies were removed in each session. This continued until no new flies eclosed in a given vial. Closed system adult counts: Three large containers were setup with ample water and a different amounts of food. A male and female fruit fly were placed in each. They were set aside and allowed to grow indefinitely. The population continued to thrive until the food ran out. Once all of the flies had died, the containers were opened and adults counted. Food choices and effects in quality of food: Adults laid eggs on apple juice containing agar. The eggs were taken off and placed in the various environments containing sugar or a corn meal diet at different developmental times. The developmental growth was examined every 12 hours. The shapes of the mouth hooks were used to determine the larvae stages. To conduct this part of experiments, mouth hooks had to be removed from the larvae and placed on microscope slides to be viewed with a compound microscope at 100x. The adults were tested for various behavioral assays based on sensory function. The 3rd instar larvae were tested for behaviors. Egg Lying In this experiment around 100 adults were placed in a glass cage, adult taste preference and egg laying were examined together in this assay. 3 Petri dishes containing lemon, peach and banana as food were placed inside the cage. These foods were also used to test survival of the larvae. After the adults have had 4 hours on these dishes they were removed and how many eggs were laid on each dish were counted. This showed egg laying preference. Then every day larva developed and formation of pupa in the different foods were examined. Body Wall Movements (BWM) for larvae Early 3rd instar larvae were used for behavioral assays after they fed on sugar water for 2, 4 and 6 days. Feeding and locomotory behavior were performed as described in Neckameyer (1996) and Li et al., (2001). In brief, single animals were placed on a 2% agar surface and the number of body wall contractions counted for 1 min. The 1st instar larvae were placed in sugar water and examined after the set number of days. Mouth Hook Movements (MHM) for larvae Larvae were placed in a 2% yeast solution on an agar plate after they fed on sugar water for 2, 4 and 6 days. In this condition, Drosophila larvae immediately fed, causing a pattern of repetitive mouth hook movements. The number of full mouth hook contractions in 1 minute was counted (Li et al., 2001). The results of these behaviors are plotted as body wall contractions or mouth hook movements per minute. Behaviors for the newly elcosed adults A plastic tube on a wooden stick with a small flashlight on top of the tube was used. The light was placed on the top of the tube to examine the locomotive ability of the flies to go to the top of the tube. The time it took flies to reach the 12 and 24 cm marks was measured. A righting response of the adults was also tested after they were shaken for 20 seconds. This was done by placing the flies in a plastic vial and vortexed and then quickly examined . Animals that had 2, 4, or 6 days in sugar water as a larvae and then placed in cornmeal for pupation were the experimental conditions for this set of experiments. The adult test with light responses with larvae fed sugar water for various days. CONCLUSIONS The number of eggs on each dish for the five trails. Since there was not an equal number of eggs in each trial, calculate a percent of eggs on each dish related to counting the number of eggs on the banana as 100%. 1. Modules examining population dynamics can be successfully implemented in biology classes by altering several factors. Sex ratios in the parental generation and food availability measurably affects how to population grows. 2. Students can use mathematical modeling, along with an understanding of the fruit fly life cycle, to describe how the population changes. This can be accomplished by using a flow diagram or with the aid of computer modeling software. 3. Lemon was the fruit that fruit flies liked the most to lay their eggs, but it turned out that lemon was the worst fruit for the larvae to grow on. 4. Sugar water alone was one of the foods that increased their life span by nearly 2 weeks. Behavioral tests showed no huge changes in larvae fed only sugar water. Only mouth hook movements and body wall movements showed some minor decreases then the ones fed normal food. In the future it would be interesting to test the effects of different types of amino acids on larval development and behavior. TABLE 1: Survival: Peach Lemon Banana Egg laying Many pupa Larvae died Many pupa & adults & adults 1st instar 28 pupa All larvae died 30 pupa 25 adults (fungus) 28 adults TABLE 2: These are showing the survival rate of the different diets. Sugar water Corn meal Peach Banana Form Pupa: 15 days (3/60) 5 days 5 days 5 days (14 day larvae but sick) Adults: All died as larvae Day 1015% 18% 23% The 3 pupa died Day 11 78% 81% 77% Day 12 98% 98% 98% The three fruits, cornmeal, and sugar water diets all in their containers. Closed system 1 male: 1 female but each container with different amounts of food. Let the chamber go until populations die out and then count total number of adults. References Now one can start to express the population dynamics in a flow chart. And even graph the data gathered and construct mathematical expressions. Students collaborate on developing a flow diagram expressing these ideas. This should start simple (e.g. the four boxes linked horizontally in the diagram below) and then take on more realistic detail. The result should be something approximating the figure on the left. Aceves-Piña EO, Booker R, Duerr JS, Livingstone MS, Quinn WG, Smith RF, Sziber PP, Tempel BL, Tully TP (1983) Learning and memory in Drosophila, studied with mutants. Cold Spring Harb Symp Quant Biol 48 Pt 2: 831–840. Alpatov, W.: Growth and Variation of the Larbae of Drosophila Melanogaster. J. Exp. Zoology, Vol.52(No.3) Hendel T, Michels B, Neuser K, Schipanski A, Kaun K, Sokolowski MB, Marohn F, Michel R, Heisenberg M, Gerber B (2005) The carrot, not the stick: appetitive rather than aversive gustatory stimuli support associative olfactory learning in individually assayed Drosophila larvae. J Comp Physiol A. 191:265–279. Kaznowski, E.C. Schneiderman, H.A., and Bryant, J.P. (1985) Cuticle secretion during larval growth in drosophila melanogaster. J. Insect Physiology. Vol 31(No.10), 801-813 (1985) Li, H., Harrison, D., Jones, G., Jones, D., and Cooper, R.L. (2001) Alterations in development, behavior, and physiology in Drosophila larva that have reduced ecdysone production. Journal of Neurophysiology 85:98-104. Watts, Thomas., Woods, Arthur., Hargand, Sarah., Elser, James., Markow, Therese: Biological stoichiometry of growth in Drosophila melanogaster. J. Insect Physiology, 52 (2006), 187-193 White LA, Ringo JM, Dowse HB (1992) Effects of deuterium oxide and temperature on heart rate in Drosophila melanogaster. J Comp Physiol B 162:278–283 Websites: Larva Food http://flystocks.boi.indiana.edu/german-food.htm These graphs show the body wall and the mouth hook movement results with the different days of larvae eating sugar water.