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  1. 1. EFFECT OF OCTOPAMINE ON THE ACOUSTIC STARTLE RESPONSE OF THE AFRICAN MIGRATORY LOCUST, Locusta migratoria (L.) A Thesis Submitted to Carleton University By James Salehi In Partial Fulfillment of the Requirements for the Degree of Bachelor of Science (Honours) in the department of Biology *NOTE PDF VERSION, FIGURES are not in LANDSCAPE orientation Ottawa, ON Canada © James Salehi, April, 2014
  2. 2. 1 I. Abstract Locusts are susceptible to predation by bats, as a defensive measure the locusts produce a behavioral response when they detect the echolocation of bats. Octopamine is a stress hormone which is present in locusts and may be released in the hemolymph when the locust detects a bats echolocation. The objective of this study was to see if octopamine modulates the acoustic startle response of the migratory locust (Locusta migratoria). High definition videos of locusts in flight were captured to measure the body positions of the locusts after they responded to artificially generated sounds that sound similar to bats using echolocation. The locusts were measured both without injected octopamine and with injected octopamine in the hemocoel so a pair study could be done. The results show evidence that octopamine does not increase the overall number of responses of the locust but it does have an effect on the different individual components of the acoustic startle response. These results are somewhat surprising due to several previous studies having found increased movement due to octopamine however the behavioral differences found are interesting and more studies focusing on this aspect would be useful in understanding the role of octopamine in the locust.
  3. 3. 2 II. Acknowledgements I would like to thank Dr. Dawson for his support and help throughout this project and always answering my questions when I needed help in certain things. As well as helping me set- up my apparatus that I used for this experiment, and teaching me the methods in which to tether a locust, get it to produce an acoustic startle response and how to analyze the results. I’d also like to thank James Don-Carolis for helping me out when Dr. Dawson was unavailable and answering my constant questions about locusts and looking over my work when he could. Finally I’d like to thank the rest of the Insect Flight Group for being really kind and being generally supportive and helpful if I needed to find or know anything.
  4. 4. 3 Contents I. Abstract...................................................................................................................................... 1 II. Acknowledgements .................................................................................................................. 2 III. List of Tables........................................................................................................................... 4 IV. List of Figures ......................................................................................................................... 5 V. Introduction.............................................................................................................................. 6 A. Introduction............................................................................................................................ 6 B. Acoustic Startle Response in Tethered-Flying Locusts .......................................................... 7 C. Octopamine Overview............................................................................................................ 8 D. Octopamine Pathway and Regulation.................................................................................... 9 E. Octopamine, Flight and the Acoustic Startle Response ....................................................... 11 F. Purpose of the Project.......................................................................................................... 13 VI. Materials and Methods ........................................................................................................ 14 A. Animals................................................................................................................................. 14 B. Apparatus Set-Up ................................................................................................................. 14 C. Flight Recording .................................................................................................................. 16 D. Video Analysis of Tethered Flying Locust ........................................................................... 17 E. Statistical Analysis................................................................................................................ 18 VII. Results.................................................................................................................................. 20 A. Frame by Frame Analysis of the Acoustic Startle Response................................................ 20 B. Response Number ................................................................................................................. 20 C. Components of the Acoustic Startle Response ..................................................................... 23 VIII. Discussion........................................................................................................................... 27 A. Analysis of the Response Number......................................................................................... 27 B. Analysis of the Components of the Acoustic Startle Response............................................. 28 C. Apparatus ............................................................................................................................. 29 D. Possible Sources of Error and Limitations of the Experiment............................................. 29 E. Future Work.......................................................................................................................... 31 F. Conclusions .......................................................................................................................... 33 IX. References ............................................................................................................................. 34 X. Appendix................................................................................................................................. 36
  5. 5. 4 III. List of Tables Table 1: The multiple components of the acoustic startle response that was looked at in a frame by frame analysis. .................................................................................................................................................... 19
  6. 6. 5 IV. List of Figures Figure 1: Synthesis pathway octopamine..................................................................................... 10 Figure 2: Schematic of the apparatus........................................................................................... 15 Figure 3: Example of an acoustic startle response in a locust. .................................................... 21 Figure 4: Number of Locusts that showing an acoustic startle response..................................... 22 Figure 5: Different components of the overall acoustic startle response..................................... 24 Figure 6: Components of the ASR for each of the four different groups tested.......................... 26
  7. 7. 6 V. Introduction A. Introduction Insects have sensitive tympanal organs that can detect a wide range of sound frequencies. In locusts, these ears play a role in a variety of functions but one key function is the detection of the sounds made by a potential predator and avoidance of the predator. Insectivorous bats use echolocation for navigation and often prey on locusts and other insects at night. For an insect to successfully survive an encounter with a bat they must be in a position to both detect the sound and respond by moving themselves away from it (Dawson et al, 1997). Although bats are generally nocturnal and locusts are generally diurnal, there is a period in which both of them are awake which is when the predation occurs. Flying locusts have been demonstrated to evoke a variety of responses when exposed to high frequency bat-like sounds. This response to sound is called the acoustic startle response (ASR) and it is defined in a locust by an increase in head rotation away from stimulus side, abdomen dorsiflexion, leg extension and along with these postural adjustments there are changes in wing kinematics and wing beat frequency (Robert, 1989). Octopamine is a neuromodulator present in the insect’s nervous system that helps promote “dynamic action” and may have an effect on the ASR of an insect (Field et al, 2008; Verlinden et al, 2010).The role of octopamine has yet to be studied in the acoustic startle response of any insect. Octopamine is thought to be a stress hormone in insects so it is possible that it is released in the body when an insect is exposed to predatory sounds (Verlinden et al, 2010). In this study, I looked at the ASR in tethered flying locusts when exposed to two different high frequency bat-like acoustic stimuli and then injected the same locusts with octopamine and induced a response using the same stimuli and measured the resulting differences between the two groups. The results of this experiment showed that there were some behavioral differences in
  8. 8. 7 the locust response between the locusts that were exposed and the locusts that were not exposed to the octopamine. B. Acoustic Startle Response in Tethered-Flying Locusts A flying tethered Locusta migratoria may display an ASR when exposed to frequencies between 10 kHz and 40 kHz and intensities that are above 45 DB (Robert, 1989; Riede, 1992). Echolocation is the biological sonar that is used in many animals. Echolocation is when an animal emits a sound to the environment in order to listen to the echoes of the calls to seek and identify objects. For many animals, echolocation is employed for navigation and for finding food (foraging or hunting). A high frequency echolocation is present in bats, and is typically used by them to find food, as well as for navigation. High frequency sounds are better at reflecting off of surfaces than lower frequency sounds which may be why they are used in the echolocation of the bats. It has been shown that tethered flying locusts will steer away from sources of ultrasonic pulses that resemble bat echolocation cries (Robert, 1989). This is due to the locust likely believing there is a detection of a predator, so the startle response would be the locusts trying to get out of the flight path of the predator. During an ASR locusts are seen to increase wing beat frequencies and deflect both their abdomens and hind legs away from the source of the sound, these postural movements and increase the drag on the side that was deflected and shift the center of mass of which the flight forces act (Dawson et al, 1997). Production of yaw torques also occurs in the ASR of a tethered flying locust, suggesting that if the locust was not tethered it would be trying to change its flight path. In the natural environment, a locust would be free and unrestrained while flying but this cannot be currently replicated in the lab. A free flying locust would be impossible to record and
  9. 9. 8 keep in the same place for every trial so it would be impossible to get significant results. In the lab flying insects are often tethered to an object that allows there to be uniformity in the results and allows flight without positional movement so they would be easily recorded and watched. There are many different tethers used in insects to allow a different range of possible movements. Some tethers are more restrictive than others. When measuring the ASR it is useful to use a flexi-tether as it permits some movement of the insect within a small area, while still keeping the locust in the same area. Tethers however do restrict the overall movement of the insect and is unnatural so this can also have a major influence on the result. It is not generally understood how the tether affects the insect however, but it is generally believed that there is some consequence (Dawson et al, 2004). Tethers have to be attached to the insect in some way; this mechanical interference can also influence the insect flight and ASR, which can have an impact on the results. The tether also removes sensory input seen when the insect is engaging in the behaviour naturally and unrestrained (Dawson et al, 2004). C. Octopamine Overview Octopamine is found in the hemolymph of many arthropods, such as locusts, and it functions as a neurotransmitter, neuromodulator and as a neurohormone, in the insect’s nervous system and helps prompt “dynamic action”(Verlinden et al, 2010). Octopamine was first discovered in the salivary glands of Octopus in the early 1950's, but it has been of real interest since a physiological role it has on insects was discovered more than 20 years later (Roeder, 1999). Octopamine is thought of as the insect equivalent of norepinephrine, and although not identical the octopaminergic systems and the noradrenergic systems present in invertebrates and vertebrates respectively are homologous (Verlinden et al, 2010). There is a similar chemical
  10. 10. 9 structure present in both octopamine and norepinephrine, which give the reason that they function similarly in their respective systems (Figure 1). The neurotransmitters can be easily distinguished since there is an absence of a hydroxyl group in the octopamine (Farooqui, 2007). Octopamine does not possess a physiological effect in vertebrates and norepinephrine does not have a physiological effect in invertebrates, which supports them being homologous (Roeder, 1999). Since they are related, the extensive knowledge already on norepinephrine can help with the understanding of octopamine. Both norepinephrine and octopamine act as stress hormones in their respective systems. In vertebrates the contracting of muscle tissue as well as glycogenolysis is increased when responding to norepinephrine release (Roeder, 1999). This shows how an organism is adaptive to energy demanding situation and is also seen in invertebrates when octopamine is released in the body in times of stress. Octopamine enables the insect to be able to cope with the energy demands of flight as application of the hormone activates fat metabolism in an insect (Roeder, 1999; Field et al, 2008). D. Octopamine Pathway and Regulation Three important regulation mechanisms tightly regulate octopamine concentration in an invertebrate these are the synthesis rate, specific release and re-uptake. Tyrosine is regarded as the starting point of octopamine; tyrosine is transformed to tyramine by tyrosine decarboxylase and from that octopamine is produced from tyramine by tyramine β-hydroxylase (Figure 1). These enzymes potentially regulate the production of octopamine. A study presenting tyrosine decarboxylase knocked out of a Drosophilia mutant shows a lack of octopamine and tyrosine present (Cole et al, 2005; Farooqui, 2012). The mutant flies showed poor locomotory activity as expected in insects with a lack of octopamine, the females were also sterile, while having eggs
  11. 11. 10 Figure 1: The best-known synthesis pathway of tyramine and octopamine. Structural comparison of octopamine with norepinephrine (Verlinden, 2010)
  12. 12. 11 they were unable to deposit them (Cole et al, 2005). While octopamine and tyrosine are related it is generally believed that they act as independent signaling molecules with dedicated receptors (Evans, and Maqueira, 2005) The specific release of the octopamine responds to changes in K+ concentration or electrical stimulation of nerves containing axons of octopamine neurons showing how it may be regulated (Verlinden et al, 2010). When octopamine is released, it elicits a short physiological response to the octopaminergic receptors. This signal is then quickly terminated to allow additional signals to interact with the receptors and reuptake of octopamine then occurs (Farooqui, 2012). Reuptake is also important for the regulation of the octopamine concentration as it stops the octopamine from signaling the octopaminergic receptors. Experiments have looked at octopamine reuptake blockers as well to help understand the pathway more, an example of such a blocker is cocaine (Verlinden et al, 2010). There may be some species-specific reuptake mechanisms however as some reuptake blockers do not function in all insects (Verlinden et al, 2010). However similarities between receptors have been seen in some different insects such as between the Locust and the Honey Bee (Degen et al, 2000).All three mechanisms that regulate octopamine concentration in insects are very imperative, as without them they would either be hyperactive or less active. E. Octopamine, Flight and the Acoustic Startle Response Octopamine is released by dorsal unpaired medial neurons (DUM neurons) and ventral unpaired median neurons (VUM neurons) that make synaptic connections with many flight motor neurons and thus may contribute to the evocation of the acoustic startle response of a
  13. 13. 12 flying locust (Duch et al, 1999). Octopamine is present in high concentrations in both neuronal and non-neuronal tissues of invertebrates and has a plethora of known functions. The peripherally released octopamine from VUM and DUM neurons modulates the activity and energy metabolism of neuromuscular transmission, muscle metabolism, and some sensory activity as well as influences other properties of the target organs (Roeder, 2005). When octopamine is released into circulation in the insect hemocoel it acts as a lipid mobilizing neurohormone during flight and long-lasting motor behaviors (Farooqui, 2012). Since octopamine has been shown to increase motor activity it is predicted that a locust injected with octopamine would be more likely to produce an ASR, or an exaggerated ASR, than one without an injection. A previous study on locust flight and octopamine has shown that the biogenic amine increased the probability that natural releasing stimulus (such as wind) induced a flight motor response in the deafferented locusts (Rillich et al, 2013). A fairly low concentration of octopamine was used to initiate wind induced flight (5 mM). This concentration is likely similar to the physiological concentration in the locust (Rillich et al, 2013). A much higher concentration was needed to initiate flight without the addition of wind (100 mM) although it is likely that the dosage reaching the neurophil was much less (Buhl et al, 2008; Rillich et al, 2013) The insect blood brain barrier is very effective at preventing certain molecules from reaching the neurophil so it likely has an impact of the effectiveness of injected octopamine (Schofield et al, 1984). The conclusion of the study suggested that octopamine selectively promotes the production of flight and that it can exert its overall effect on multiple sites as well as strengthen synaptic connections (Rillich et al, 2013).
  14. 14. 13 F. Purpose of the Project The purpose of this study was to determine if octopamine (50 µl of 10-4 M) injected into the hemocoel of a locust would change the acoustic startle response of the locust to simulated echolocation calls of bats. An analysis of the ASR during flight of the locust when exposed to two different frequencies (30 kHz and 12 kHz) was also part of this study. Octopamine has been shown to increase motor activity in insects so it is predicted that an injection of octopamine in a locust would have an effect on the ASR (Farooqui, 2012). Due to increase in motor activity associated with octopamine a locust that is injected with it may increase may be more likely to produce an ASR and/or produce an exaggerated ASR (Farooqui, 2012). This experiment was modeled on previous studies of locust flight and ASR so optimum results could be found (Dawson et al, 1997; Dawson et al, 2004; Orchard et al, 1981; Orchard et al, 1993). Previous studies on the ASR of a locust have utilized high-speed cameras to pick up the locust movement (Dawson et al, 1997; Dawson et al, 2004). This study will also attempt to prove that a camera of a lower speed (60 FPS) can also be used to study the ASR of a flying insect, in particular Locusta migratoria.
  15. 15. 14 VI. Materials and Methods A. Animals Locusta migratoria L. of male sex in the gregarious phase were selected. The selected locusts were adults and were aged over 2 weeks past their final moult. The animals were reared in fairly crowded wooden containers which maintained between 30-32°C under a 16 h: 8 h light:dark cycle. B. Apparatus Set-Up To be able fly the locust an apparatus was made to allow a locust to be tethered, flown and recorded (Figure 2). A ring stand was used to hold the flexi-tethered locust with a clamp. A mirror was then set up at a 45° degree angle to where the locust was tethered. This allowed the locust to be seen from both the front and the side using a single recording device. A halogen light source was set up above the locust to allow greater light for the locust in order for it to be viewed clearly on the recording device and also help the area maintain a higher temperature of approximately 29°C. The recording device that was used in this experiment was a Toshiba CAMILEO SX900 camera. The camera being used was set to ISO 1600, shutter speed 1/1000, dimensions of 1280 x 720 and 60 FPS and was high definition. An initial test using a timer was performed to ensure that the camera was set to 60 FPS, which it was. The camera was then situated 40 cm away from the tethered locust and pointed directly at the locust. A fan was then placed 10 cm behind the camera to help initiate proper flight of the locust. A speaker was set up 20 cm away from the right side of the locust on a ring stand. The speaker was attached to a switch that allowed it to be quickly turned off and on when it was being used to try and elicit an ASR. The speaker elicited high frequency sounds when turned on. A green LED light was used
  16. 16. 15 Figure 2: Schematic of the apparatus that was made for this experiment to allow a study of locust acoustic startle response.
  17. 17. 16 on the apparatus as well, and was also switched on when the speaker was switched on so a visual signal was present. This visual signal was useful when recording due to some of the high frequency sounds are above the hearing range of humans so the visual signal meant that it was always known when the speaker was on. C. Flight Recording The animals were tethered dorsally with wax on the pronotum to a metal wire that was attached to a flexi-tether. Both fan and light source were then switched on and the locusts were allowed to get into flight position. After they were in flight position the recording device was turned on. After a few seconds recording them flying without an ASR the sound was switched on. A high frequency audio amp was used to elicit bat-like sounds from the speakers. The two frequencies that the locusts were exposed to were 12 kHz and 30 kHz, with the duration of the sound being slightly lower than the period so it was not just one static sound. The volume of the sound used was 80DB and this was checked using a CM-130 SPL Meter. An attenuator was used to make sure that the sound elicited was always 80DB. The sounds were exposed to the locusts when they were in stable flight and lasted between a period of 10ms and 20ms. There was 2 minute period of time between each sound exposure to allow the locust to rest and not become adapted to the sound. Two videos were taken of each of the locusts in the experiment, one video for each of the frequencies that were being used. The locusts were then removed from the flexi- tether and rested for a short period of time. An octopamine solution (50 µl of 10-4 M) was then injected into the hemocoel of the previously flown locusts. Octopamine was at room temperature (23°C) when injected into the locusts to minimize temperature having an effect on the locust flight (as the octopamine was kept
  18. 18. 17 in a fridge). After injection the locusts were allowed to rest for a further 15 minutes, this was to allow the octopamine to circulate around the locust body and possibly be taken up by certain systems. After this waiting period the animals were once again attached to the tether and flown in the same manner as they were previous to the injection of the octopamine. Two additional videos were then taken of the locusts following their exposure to the same two frequencies as before. D. Video Analysis of Tethered Flying Locust The videos that were captured of the locust were in MP4 format but were converted to AVI using MP4Cam2AVI (v. Oleg Mikheev) this allowed the videos to be used in other programs for analysis. The converted videos were then opened in VirtualDub (v. Avery Lee) edited so only three wing beats before and after the stimuli exposure was in the video. This editing was performed to reduce the amount of unneeded analysis of the locust from long before and long after the sound exposure and to allow the focus to be on just prior to and just following the stimuli. The cut down videos were then opened in SkillSpector (v. Video4Coach). This program was used to track the movements of the locust during flight to see if an ASR occurred after the stimuli. The major focus was to track the head of the locust and the end of the abdomen in each of the video frames. This was done in order to show the overall movement of the locust when attached to the flexi-tether as well as possible movements of the abdomen such as dorsiflexion or ventralflexion. The results of the frame by frame video analysis in SkillSpector were then exported to Microsoft Excel to allow further inspection as to whether the locust underwent an ASR or not.
  19. 19. 18 All the videos were also looked at frame by frame in VirtualDub for the components of the ASR. The before and after stimuli parts of the videos were then compared to see if any distinct changes in the locusts occurred due to their exposure to the high frequency bat-like sounds. This meant observing things such as head rotation and tibia extension which were not shown in the SkillSpector frame by frame analysis as well as other components of the ASR (Table 1). E. Statistical Analysis Statistical tests were carried out on the results that obtained from the video analysis. Chi squared tests were performed to observe if the data that was found during this experiment was statistically significant or not.
  20. 20. 19 Table 1: The multiple components of the acoustic startle response that was looked at in a frame by frame analysis. Component Description Ventralflexion A downwards movement of tip of abdomen Dorsiflexion An upwards movement of tip of abdomen Head Rotation Rotation of the head either right or left Tibia Extension An extension of the locust tibia Reduction of Movement Less overall movement in the locust after stimuli Increased movement More overall movement in the locust after stimuli Movement towards Speaker Sideways movement towards the bat-like sounds Movement away from speaker Sideways movement away from the bat-like sounds Forward Movement: Locust moves forward on the flexi-tether after the stimuli Backwards Movement Locust moves backwards on the flexi-tether after stimuli ASR Number of locusts that showed an acoustic startle response
  21. 21. 20 VII. Results A. Frame by Frame Analysis of the Acoustic Startle Response The digitized results from the frame by frame SkillSpector analysis revealed that locusts had a variety of different responses from the sound stimuli, regardless of the sound frequency or whether they were exposed to octopamine or not. There were a total of 18 locusts that were observed overall with 72 different videos depicting the locust ASR. With division into being four groups of videos for study (12 kHz sound exposure with and without octopamine and 30 kHz sound exposure with and without octopamine). A typical response that was seen, regardless of the frequency and octopamine exposure, would see movement of the locust abdomen and movement away from the sound in any direction although many different responses occurred including a lack of response (Figure 3). B. Response Number The digitized SkillSpector analysis led to looking at the overall response number of the locust in regards to the ASR. All 18 locusts were exposed to all 4 different groups and different overall response rates were seen (Figure 4). The number of locusts that showed an ASR was not contingent on sound frequency. Out of the 18 control locusts exposed to 12 kHz at control 6 elicited an ASR and when exposed to 30 kHz 11 of the locusts elicited an ASR, this was not consistent enough to be considered significant however (Χ2 C=1.783, p=0.1817 (Calculated using control data)). A similar result was seen when comparing the different response number to the sound frequencies in the octopamine exposed locusts. The response number between both the octopamine free controls and the octopamine injected locusts was not significantly different based on statistical analysis. When the locusts
  22. 22. 21 Figure 3: A prime example of an acoustic startle response in a locust. A: The abdomen movement of Locust 7 (control) exposed to 30 kHz that was seen when the movement was tracked with SkillSpector. This locust produced a response when exposed to sound. It can be seen that after the stimuli (sound) is produced the locust abdomen moves up (the locust produced a dorsiflexion). The abdomen is also seen moving backwards (the locust tried to move B: The head movement of Locust 7 (control) exposed to 30 kHz that was seen when the movement was tracked with SkillSpector. This locust produced a response when exposed to sound. It can be seen that after the stimuli (sound) is produced the locusts head movement is increased. The head movement indicates that it moves away from the origin of the sound. 1000 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 0 20 40 60 80 100 Pixels Time (ms) Abdome n.Y Abdome n.X Stimuli 480 490 500 510 520 530 540 550 560 0 20 40 60 80 100 Pixels Time (ms) Head. Y Head. X Stimu li B A
  23. 23. 22 Figure 4: The number of Locusts that showed an acoustic startle response was not contingent on sound frequency or octopamine injections. A total of 18 locusts were observed. The number of locusts producing an ASR did not depend upon sound frequency (Χ2 C = 1.783, p=0.1817 (Calculated using control data)). The number of locust responses did not depend on octopamine treatment in either 12kHz or 30kHz sound stimuli (12 kHz: Χ2 C =0.117 p=0.732) (30kHz: Χ2 C =0.524, p=0.469). 6 11 8 14 12 7 10 4 0 2 4 6 8 10 12 14 16 18 20 Control 12 Control 30 Octopamine 12 Octopamine 30 Number No response ASR
  24. 24. 23 were exposed to the 12 kHz sound stimuli the controls had 6 ASRs and the octopamine exposed has 8 ASRs which shows that the response number was not contingent on octopamine exposure (Χ2 C =0.117 p=0.732). The locusts exposed to the 30 kHz sound showed 11 ASRs and 14 ASRs for the control and octopamine injected animals respectively, this also demonstrates that the response number was not contingent on octopamine exposure (Χ2 C =0.524, p=0.469). C. Components of the Acoustic Startle Response Each individual component of the ASR was also looked at frame by frame. The locusts that displayed a response to the stimuli in any of the four distinct groups elicited a variety of different responses (Figure 5). Some of the responses were more common than others however. The most common two responses were increased movement (seen in 27 of the 39 ASRs) and forward movement (seen in 29 of the 39 ASRs) while the least two common responses were ventralflexion (seen in 2 of the 39 ASRs) and movement towards the speaker (seen in 6 of the 39 ASRS) (refer to Table 1 for descriptions of each movement). There was a significant difference between the more commonly seen movements and the least commonly seen movements (Χ2 =59.251, df=8, p<0.001). The individual components of each group of locusts (control 12 kHz and 30 kHz and octopamine injected 12 kHz and 30 kHz) were then looked at individually to see if there was a difference in behavior between the groups (Figure 6). Like in the overall there were certain components that occurred more frequently than others. There was a significant difference in behavior seen between the octopamine injected locusts and the controls in the 30 kHz sound exposed locusts. This was not seen in the 12 kHz sound exposed locusts likely due to there being a lack of overall responses seen particularly in the 12 kHz control.
  25. 25. 24 Figure 5: Different components of the ASR were observed when locusts were presented with either 12 kHz or 30kHz bat-like sounds. Out of 72 videos of locusts exposed to the two sound frequencies, with or without octopamine, 39 of them produced an acoustic startle response (ASR). The most common responses observed were increased movement and forward movement. With the least common being ventralflexion and of the abdomen and movement towards the speaker (which represents the bat echolocation). 2 16 11 17 10 9 27 6 14 29 15 39 0 5 10 15 20 25 30 35 40 45 Number Seen
  26. 26. 25 0 3 2 3 2 1 3 0 2 3 1 6 0 1 2 3 4 5 6 7 Number Seen Control 12khz 0 2 2 3 1 0 6 3 2 6 3 8 0 2 4 6 8 10 Number seen Octopamine 12kHz A B
  27. 27. 26 Figure 6: Each plot shows the components of the ASR for each of the four different groups tested. Certain components occurred more frequently than others. A: Shows the control locusts exposed to a 12kHz sound in which 6 out of 18 locusts produced a response (Χ2 =6.8161, df=8, p=0.557). B: Shows the control locusts exposed to 30kHz in which 11 of the 18 locusts showed an ASR (Χ2 =25.87, df=8, P=0.0015). C: Shows the octopamine injected locusts exposed to a 12kHz sound in which 8 of the 18 locusts produced an ASR (Χ2 =22.026, df=8, p=0.005). D: Shows the octopamine injected locusts exposed to 30kHz in which 14 of the 18 produced a response (Χ2 =19.356, df=8, p=0.013). 1 5 3 4 3 3 8 1 5 10 5 11 0 2 4 6 8 10 12 Number Seen Control 30khz 1 6 4 6 4 5 10 2 5 10 6 14 0 2 4 6 8 10 12 14 16 Number Seen Octopamine 30 khz `C D
  28. 28. 27 VIII. Discussion A. Analysis of the Response Number No significant difference was seen in the number of responses between the 12 kHz sound exposed locusts and the 30 kHz sound exposed ones. A trend can be seen in the results however as there are slightly more responses seen in the 30 kHz exposed locusts than the 12 kHz locusts in both the controls and the octopamine injected locusts, even though it was not significant (Figure 4). The p-value seen in the control data (p=0.1817) is also very close to being considered significant which supports the trend. Previous studies that have looked at locust flight ASR in terms of different sound frequencies have showed results which support that a higher sound frequency of about 30 kHz would elicit higher response rates in flying locusts than lower frequencies and also states that frequencies below 10 kHz would not be likely to an elicit an ASR in a flying locust (Dawson et al, 2010). The lack of major significance seen in the two different sound frequencies could be explained by evolution. The locusts may have an evolved to produce a behavioural response to high ranges of sound frequencies just in case the sound they hear is a predator. It may be that the locusts that have only reacted to a limited range of sounds were more easily preyed upon. The locusts that were treated with the octopamine showed no significant difference in the overall number of responses compared to the controls (Figure 4). This was true for both the 12 kHz and 30 kHz sound frequency exposed locusts. This suggests that octopamine does not have an effect on the overall response number of the locusts, so around the same number of locusts respond to the stimuli regardless of octopamine exposure. While octopamine does not affect the overall number of responses it still may have an effect in other major parts of the locust flight ASR.
  29. 29. 28 B. Analysis of the Components of the Acoustic Startle Response Many varied responses to the bat-like sound stimuli were seen but some more frequent than others (Figure 5). The more common movements such as the forward movements and the increased movements make sense because a locust would be trying to escape the pathway of the bat and an overall increase in movement and forward movement would accomplish that in terms of this experiment due to where the speaker is situated (Robert, 1989). Although some studies have shown that insects producing a flying ASR do not really have much control of what their reaction is, particularly when they are tethered (Dudley, 2002). That being said the fact that these responses were seen at a significantly higher number than the others suggest that at least the locust has some control over the behavioural response. It might also suggest that these may just be typical behavioural responses in tethered locust to an ASR no matter where the source of the stimuli is. The responses that were significantly less common to the others were abdomen ventralflexion and movement towards the speaker. The abdomen ventralflexion is likely uncommon due to it not being a typical movement of a locust in general, as they do not typically move their abdomen upwards. The fact that the locusts are less likely to move towards the speaker, which represents the sound of a possible predator, makes sense, as if they did move towards the speaker it would be counter-intuitive if they are trying to escape predation. This also supports the possibility that maybe locusts may have some control over what their response is, as there was a significantly low amount of locusts moving towards the speaker. However, the amount of locusts moving towards the speaker is not that much lower than the amount moving directly away from it, as well as that some locusts did move towards the sound even though it may have been a predator. The suggestion that a tethered locust does not have much control over their ASR is something that could still be analyzed. A way to examine this could be to look at the
  30. 30. 29 ASR of a locust with sounds coming from a different direction each time, and compare this to see if there is a significant difference in the response. When looking at the components of the acoustic startle response between the octopamine treated locusts and the controls some responses can be viewed more frequently than others (figure 6). This suggests that octopamine may have an effect of the behaviour of the locust during the ASR even though the octopamine did not seem to have an effect on the overall number of locust ASR. This may be due to octopamine being a stress hormone so it may cause more agitated and varied effects due to the excess of it (Verlinden et al, 2010). C. Apparatus The results show that the apparatus made can be used in finding an ASR in a locust. The success of apparatus is quite useful in a couple of ways. In previous experiments ASRs are mostly quantified using high speed cameras (Dawson et al 1997, Dawson et al, 2004). This experiment showed that a camera of only 60 FPS could be used to quantify an ASR in a locust. It also showed that a wind tunnel is not always needed to assess the flight of the locust as the locust was able to fly in an open box using just a fan. D. Possible Sources of Error and Limitations of the Experiment One thing that was seemingly neglected in this study was the analysis of the wing kinematics of the ASR. Originally this analysis was going to be a major factor in this project, as it is a major part of the flight ASR of an insect and is part of many other insect flight studies (Dawson et al, 1997; Dawson et al, 2004; Liu et al, 2008). While the apparatus used was useful in terms of looking at whether the locust produced an ASR or not, the camera was not able to pick up the intricate details of the wings. The camera was limited to 60 FPS, and the wing-beat
  31. 31. 30 frequency of an average locust is about 20 Hz (Camhi et al, 2002). This meant the camera only picked up about a third of the overall wing movement of the locust. This limitation meant that analysis of the wing kinematics proved to be almost impossible, as there would be a lot of guessing of where the wings would be and the results would not have been very reliable. The differences between the controls and octopamine injected locusts were kept as minimal as possible, except for the injection of octopamine; however there was some notable differences. The locust injected with the octopamine had extra fluid in the body this extra fluid may have had some effect on the locust ASR. Saline was originally going to be injected into the controls to counteract this, however this posed the problem that the octopamine injected locusts would then have twice the amount of fluid. A possible solution to this was to inject saline in to different locusts, however this would have taken away from the experiment due to the comparison of different locusts which would have been less helpful in seeing the differences when injecting the octopamine. So in the end the experiment went forward with the controls being simply un-injected locusts, which may have been a source of error but found to be one of the more reliable ways to obtain fair results. While the experiment fairly strictly followed a precise regiment to reduce any possible sources of errors in the results some errors may have occurred. The individual components of the locust ASR were rigorously analyzed individually frame by frame, however there was trouble at times determining whether a certain component had occurred or not. This was due to the locust occasionally moving in a manner that blocked part of its view from the camera. While the mirror was very useful by providing the side view of the locust, there were still a few occasions in which not every part of the body could be seen. A possible solution to this slight limitation would be to add additional cameras or another mirror to see more of the angles of the locust,
  32. 32. 31 however this was not a huge issue; therefore the number of the ASR components that were possibly missed would be very minimal. Another possible error in this experiment is the possibility of exposure of the locust to outside sounds. While efforts were made to minimize this problem such as the experiment occurring at times when the lab was quiet some sounds may still have affected the locust. For example the switch that actually turns on the bat-like sound stimuli, makes a click sound itself, which may have itself caused some sort of response in the locust. This may have given the locust warning that the bat-like stimuli is about to be switched on. An additional issue with the sound is that as a result of the locust being set up in a sort of box the walls of the box may have reflected the sound so it is being heard the from multiple angles, which may have potential effects on the components of the ASR. A possible solution to this would be to have a switch that made no noise, this was originally the plan in this experiment but such a switch was unable to be located for when the apparatus was being set-up. Another possible solution to the issue of outside sound would be to possibly put the locust in a box that limits outside sound and also maybe put it in a room that absorbs sound so that only the sound coming straight from the speakers is heard, not reflections. It is important to consider that there are also diverse sounds in the natural environment of a locust. It may make sense to mimic these sounds as well as it may produce a more natural ASR. While there may have been a few minor sources of error, the majority of the experiment followed precise rules to minimize errors. Most other possible sources of errors were due to limitations of the project itself. E. Future Work This experiment gave some insight of the role of octopamine in the locust ASR in which future studies could possibly expand on. A similar study could be performed which would use a
  33. 33. 32 high speed camera. While the low speed camera was useful in quantifying the ASR the wing kinematics was something that would have been particularly interesting to look at especially since a behavioral difference was seen between the controls and octopamine injected in regards to the ASR. A similar study using a high speed camera would be able to analyze the wing kinematics of the locust. This experiment could also add some of the other aspects that were suggested previously such as adding additional cameras or mirrors so more angles of the locust could be looked at, or adjusting how the locust is receiving the sound and what sound it is receiving, or making the sound stimuli come from different directions to see if it has an effect on the ASR. This experiment could also factor in the two different frequencies (12 kHz & 30 kHz) to see if there is a significant difference between the two in relation to the locust ASR. There are a few other directions that future research on effect of octopamine in the ASR can go in. While the experiment only looked at male gregarious locusts due to time constraints a future experiment could also look at female gregarious locusts as well as both male and female solitary locusts to see if there is a major difference in the acoustic startle response when exposed to octopamine compared to male gregarious locusts. It’d be fairly interesting to look at the solitary locusts as they generally do not fly as well as the gregarious locusts so the octopamine may have a more noticeable effect on them and potentially cause them to fly more (Ayali et al, 1996). Another possibility is that the octopamine could be injected into the locust in a different manner. This may or may not produce a more noticeable effect depending on how the octopamine is injected but it may give insight on how octopamine is used in the body. One way that octopamine could be injected and possibly produce a much more noticeable effect is to somehow inject the octopamine in a way that it bypasses the blood brain barrier. This represents a more difficult challenge however as a technique would have to be developed which would
  34. 34. 33 allow the locust to fly quickly after this was done. Additionally, something similar must be done to the control locust as well that may make it less viable as it is something that would not happen naturally. A similar technique was analyzed at in the lab but it often ended in the locusts dying. Possible solutions for this can be examined for the future. F. Conclusions In this paper the ASR of the locust was analyzed and recorded when exposed to two frequencies and injected with octopamine. This study supports the hypothesis that the octopamine affects the ASR of the locust but it doesn’t show that it is more likely to produce it, nor does it directly support the possibility that an exaggerated ASR would occur. The results show that although the octopamine doesn’t have an effect on the number of the locusts that elicit an ASR it may have an effect on the individual components of the response. Future research should try to focus on these individual components to see the intricate differences while also figuring out new ways to expose locusts to octopamine. The results also didn’t show a significant difference in the ASR when exposed to the two frequencies but there are suggestions that there is a difference here in other studies but there are also evolutionary reasons that this may have occurred as well. Overall this experiment does prove that octopamine has an effect on the ASR and however little it is, and injecting octopamine in the hemocoel does have some effect on the locust.
  35. 35. 34 IX. References Ayali, A., Golenser, E., and Pener, M. (1996). Flight fuel related differences between solitary and gregarious locusts (Locusta migratoria migratorioides). Physio Entomology. 21, 1 6. Buhl, E., Schildberger, K, and Stevenson, P. (2008). A muscarinic cholinergic mechanism underlies activation of the central pattern generator of locust flight. J Exp Biol. 211, 2346-67. Camhi, J.M., Sumbre, G., and Wendler G. (1995). Wing-Beat Coupling Between Flying Locust Pairs: Preferred Phase and Lift Enhancement. J. of Exp Biol. 198, 1051-1063. Cole, S., Camey, G., McClung, C., Willard, S., Taylor, B., and Hirsh, J. (2005). Two Functional but Noncomplementing Drosophila Tyrosine Decarboxylase Genes, Distinct Roles for Neural Tyramine and Octopamine in Female Fertility. Journal Biol Chem. 280, 14948 955. Dawson, J.W, Dawson-Scully, K., Robert, D. and Robertson, R.M. (1997). Forewing Asymmetries During Auditory Avoidance in Flying. J. of Experimental Biol. 200, 2323 2335. Dawson, J. W., Leung, F., and Robertson, R. M. (2004) Acoustic startle/escape reactions in tethered flying locusts: Motor patterns and wing kinematics underlying intentional steering. J. of Comp Physio. 190,581-600 Degen, J., Geweeke, and M., Roeder, T. (2000). Octopamine receptors in the honey bee and locust nervous system:pharmacological similarities between homologous receptors of distantly related species. British J. of Pharma. 130, 587-594 Duch, C., Mentel, and T., Pflüger, H,J. (1999). Distribution and activation of different types of octopaminergic DUM neurons in the locust. Journal of Comparative Neuro. 403, 119 134. Dudley, R. (2002). The Biomechanics of Insect Flight: Form, Function, Evolution. Princeton University Press, Princeton, NJ, W. Evans P,D., Maqueira. B. (2005) Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors. Invertebrate Neuroscience 5, 111–118. Farooqui, T. (2007). Octopamine-Mediated Neuromodulation of Insect Senses. Neurochem Res. 32, 1511–1529 Farooqui, T. (2012). Review of octopamine in insect nervous systems. Insect Physiology. 4, 1 17.
  36. 36. 35 Field, L., Duch, C., and Pfluger, H. (2008). Responses of efferent octopaminergic thoracic unpaired median neurons in the locust to visual and mechanosensory signals. J. of Insect Physio. 54m 240-254. Liu,Y., and Sun, M. (2008) Wing kinematics measurement and aerodynamics of hovering droneflies. J Exp Biol. 211, 2014-25. Orchard, I., Loughton, B.G. and Webb, R.A. (1981). Octopamine and Short-Term Hyperlipaemia in the Locust. General & Comp Endocrinology. 45, 175- 180. Orchard, I., Ramirez J.M. and Lange, A.B. (1993). A Multifunctional Role for Octopamine in Locust Flight. Annu. Rev. Entomol. 38, 227-49. Riede, K. (1992). Prepulse inhibition of the startle reaction in the locust Locusta migratoria (Insecta: Orthoptera: Acridoidea).J Comp Physiol 127, 351-358. Rillich, J., Stevenson, P., and Pflueger, H. (2013). Flight and Walking in Locusts–Cholinergic Co-Activation, Temporal Coupling and Its Modulation by Biogenic Amines. PloS ONE 8(5), e62899. doi:10.1371/journal.pone.0062899. Robert, D. (1989). The Auditory Behavior of Flying Locusts. Experimental Biology. 147, 279 301 Roeder, T. (1999). Octopamine in Invertebrates. Progress in Neurobiology. 59, 533-561 Roeder, T. (2005). Tyramine and octopamine: ruling behavior and metabolism. Annual Review of Entomology 50, 447–477. Schofield P, K., Swales L,S., Treherne J,E. (1984). Quantitative analysis of cellular and paracellular effects involved in the disruption of the blood brain barrier of an insect by hypertonic urea. J Exp Biol 109, 333–340. Verlinden, H., Vleugels, R., Marchal, E., Badiscoa, L., Pfluger, H.J. and Blenauc, W. (2010). The Role of Octopamine in Locusts and Other Arthropods. Journal Insect Phys. 56, 854– 867
  37. 37. 36 X. Appendix Table 1A: A table showing the ASR in each of the locusts and the weight of the locusts used. Yes means that the locust did show a response to the stimuli, no means that the locust did not show a response to the stimuli. Locust Weight Control 12 Control 30 Octopamine 12 Octopamine 30 1 1.17 No Yes Yes Yes 2 1.15 No Yes No Yes 3 1.3 No No No Yes 4 1.26 Yes Yes Yes Yes 5 1.27 No No No No 6 1.43 No No No No 7 1.62 Yes Yes Yes Yes 8 1.35 No Yes No Yes 9 1.55 Yes Yes Yes Yes 10 1.32 No Yes Yes Yes 11 1.58 No Yes Yes Yes 12 1.56 No Yes No Yes 13 1.72 No No No No 14 1.49 No No No No 15 1.56 No Yes No Yes 16 1.33 Yes Yes Yes Yes 17 1.2 No No Yes Yes 18 1.24 No No No Yes

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