THE EFFECTS OF CO2 AND HYPOXIA ON THE PHYSICAL BEHAVIOR AND HEART RATE IN DROSOPHILA LARVAE

1. THE EFFECTS OF CO2 AND HYPOXIA ON THE PHYSICAL BEHAVIOR AND HEART RATE IN DROSOPHILA LARVAE NICOLAS H. BADRE AND ROBIN L. COOPER DEPARTMENT OF BIOLOGY, UNIVERSITY OF KENTUCKY, LEXINGTON, KY 40506-0225 • CONCLUSION • Under a high concentration of N2, the larvae have a decreased locomotion which seems to increase with time. • Under a high concentration of CO2, the larvae have a even stronger decrease in locomotion which leads to a stopping of movements. • Under a high concentration of N2, the larvae does not react by stopping its BWM. • Under a high concentration of CO2, the larvae cease movement in less than minute. • Under a high concentration of N2 or with air, the larvae do not have any kind of cardiac arrest. • Under a high concentration of CO2, the larvae cease cardiac activity. • The larvae needs a recovery time close to a minute to regain cardiac activity after a being in an environment high in CO2. • CO2 provokes to the larvae behaviors, which are different to the one caused by hypoxia and, which are similar to the one of anesthesia. • In 67 % of the CO2 exposure, the larvae will go in shell position before stopping its cardiac activity. • In 100 % of the CO2 exposure, the larvae will have go in elongated position once the cardiac activity is stopped. • 4.63 minutes is the time needed on average by a larvae to regain its natural shape once it has been exposed to CO2. • 8.8 minutes is the time needed on average by a larvae to regain its natural mobility once it has been exposed to CO2. Heart Beats RESULTS Body Wall Movements INTRODUCTION AND BACKGROUND Carbon dioxide is commonly used as an anesthesia for adult Drosophila melanogaster, however, the mechanism of its actions is unknown. This is important, as it might lead to the discovery of new types of insecticides that would be innocuous to plants and plant eaters. Since mosquitoes have been shown to have sensory structures that detect carbon dioxide, we postulated that Drosophila must also contain similar types of receptors because they share the same kind of environment. Laval insects have never been examined for carbon dioxide sensory neurons. Previous experiment supposed that carbon dioxide affected larvae the same way than humans: a increase in body fluid acidity causing different behaviors such as anesthesia (Sillans and Biston, 1979). Those experiments also showed that carbon dioxide had different effects to hypoxia as a high concentration of carbon dioxide and oxygen could also cause anesthesia (Sillians et al., 1969). However, this current research has an objective to find sensory neurons on the larvae capable of detecting the CO2. The results of the HB test show difference between the CO2, and Air and N2. However, under a high concentration of CO2, the larvae cease cardiac activity.. The average time for the movement ending under high concentration of CO2 is 57 sec. Even under a high concentration of N2 or with air, the heart beat does not stop as with CO2. Another figure determined by this experiment is the time needed for the larvae to recover its cardiac activity once the CO2 injection is stopped and that the container is opened. The average for the recovery time of the larvae is: 59.6 sec. (This figure is only available for the CO2 as the larvae would only stop their cardiac activity under high concentrations of CO2). METHODOLOGY We tested Canton S, the common ‘wild-type’ laboratory strain of Drosophila melanogaster. This experiment focused on larvae at the beginning of the “wandering” phase of the third instar. Many of the techniques used in this experiment have already used in Cooper and Neckameyer (1999). Each larva was in a sealed agar plate with carbon dioxide injected into the container. Part I – Body wall movements (bwm) & Heart Beats (HB) We injected CO2 in the sealed container for a period of 10 minutes, after which the container was opened. We recorded the bwm for the first and last two minutes. If at any time bwm or the HB stopped, the time would be recorded. If the HB stopped, the time when the HB started again once the container was open, would be recorded. The objective of this test is to quantify the difference between CO2 and hypoxia in the larvae using common features of the animal. Part II – The reaction of the larvae with the CO2 In our effort to identify particular characteristics of the larval response to carbon dioxide, we designed several terms to quantify those responses. Shell positiondesignates larvae which are in a curved position. Elongated positiondesignates larvae which are flaccid and which look longer than usual. Contracted position designated larvae which had returned to their normal shape after being in elongated position. Those response were tested by placing the larvae under anesthesia for approximately 5 minutes and recording the different behaviors of the larvae during the first minutes and the minutes following the end of the CO2 injection. This test has for objective to understand and detail the reaction of the larvae to CO2. We repeated the experiment with N2 to make sure that the results were specific to CO2. We also had a control, recording the natural bwm and HB of the larvae, without the injection of any gas. Here is a model of the experiment. CO2 and N2 are represented on the left and the control on the right. The results of those bwm tests show difference in all three conditions. The normal (air) average number of body wall movements for the first two minutes is 76 bwm. However, with the N2, the results were twice smaller with an average of 35.4 bwm for the first two minutes. Furthermore, with CO2, the results were very small with an average of 10 bwm which is 7 times smaller. The normal (air) average number of body wall movements for the last two minutes is 98.2 bwm. However, with the N2, the results were five times smaller with an average of 19.4 bwm for the first two minutes. Furthermore, with CO2, the larvae stopped moving so the average for the last two minutes is 0 bwm. The trend is that, under a high concentration of N2, the larvae have a decreased locomotion which seems to increase with time. For CO2, the result seem comparable to N2, except that the effects are stronger as the results are smaller for the first two minutes and inexistent for the last two. Physical Behavior Current Research Objectives Determining where the CO2 receptors are located on the larvae by inhibiting certain part of the body at detecting the presence of CO2. Understanding which parts of the larvae are still active/responding while the animal is under anesthesia. Finding out how the larvae knows when to stop its anesthesia mode to return to a normal activity. Explaining the benefit of having such quick responses to CO2. Recording the activity of the sensory neurons by electrophysiology. shell position elongated position contracted position The physical behavior test showed that in 67 % of time, the larvae would go in shell position before stopping its cardiac activity. During the other cases, the larvae would directly go in elongated position. In all of the trials (100%), the larvae would go in elongated position once the HB was stopped. When the injection of CO2 was stopped, the larvae would have many different behaviors which did appears in the same order every time. However, slight head turns and mouth hooks movements were very common behaviors in the first three to four minutes. It took on average 4.63 minutes for the larvae to gain back their common size: contracted position. Once the larvae is in contracted position, it usually does a couple of non-motional bwm. Before starting its natural motion, the larvae often turns its head left and right and then starts its motion. The average time for the larvae to come back to a normal behavior after five minutes of exposition to CO2 is 8.8 minutes. REFERENCES Biston J, Sillans D (1979) Studies on the anesthetic mechanism of carbon dioxide by using Bombyx mori larvae. Biochimie 61, nº2:153-156 Sillans D, Esteve J, Legay JM (1969) C.R. Acad. Sci., 269: 1209-1212 Cooper RL, Neckameyer WS (1999) Dopaminergic neuromodulation of motor neuron activity and neuromuscular function in Drosophila melanogaster. Comp Biochem Physiol [B] 122:199-210. The results of this bwm test show difference for the CO2. Under a high concentration of N2, the larvae does not react by stopping its bwm. However, under a high concentration of CO2, the larvae cease movement in less than minute. The average time for the movement ending under high concentration of CO2 is 40 sec.