, r-u.y:_ w 1-;- -«-.a 'fzflx‘q ‘ . \ .u-Jw . Lavwrs ’ >"~"‘ “‘ ”WIV. .' y I. : ~ ~* 115 '. ‘ NV" “” s ‘. r w. ’ i ‘3‘ .IF} '5 .4 t‘. '3' ‘r'lu‘m’: gym, ._.’ fl"- warm ABSTRACT AN INVESTIGATION OF THE BEHAVIOR OF LARVAL GALLERIA MELLONELLA (L ) IN A LIME ES E‘S‘ITUAT'IUN BY Richard M. Bell Thirty-three Galleria mellonella were tested for 40 trials in a one-way runway to determine the effect of repeated trials, in an aversive situation in which escape is possible, on the time required to escape. The eleven subjects in the experimental group were able to escape. light by crawling into a dark goal box at the end of the runway. Two control groups with eleven subjects in each controlled for factors, other than the opportunity to escape light, which may have had an effect on escape times. Sub- jects in the light control group were not able to escape light by crawling into the goal box. Subjects in the dark control group were never exposed to light. During the first five trials, the experimental and light control groups had significantly shorter escape times than did the dark control group. By the last five trials, the escape times for the dark control group had decreased significantly. The escape times for the light control Richard M. Bell group increased, although not quite significantly. The escape times for the experimental group remained stable and relatively short. The results gave no clear evidence for escape learning by subjects in the experimental group. The light stimulus clearly did have an effect on the subject's behavior during the first five trials, causing the.subjects exposed to light to crawl into the goal box faster than the subjects in darkness did. The decrease in escape times over trials by the dark control group may have been due to sensitization induced by the experimental situation. The increased escape times by the light control group may have been due to the accumulated effect of longer exposures to light than those received by the experimental group. AN INVESTIGATION OF THE BEHAVIOR OF LARVAL GALLERIA MELLONELLA (L.) IN A LIGHT ESCAPE SITUATION By .,:-‘"— Richard MT'Bell A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Psychology 1974 .33? 3‘ fl ACKNOWLEDGMENTS I wish to thank Dr. Stanley Ratner for his sage advice, laboratory space and equipment which made this investigation possible. My appreciation also applies to Dr. Martin Balaban and Mr. Peter Hogan for their invaluable assistance that enabled me to set up my subject colony and for laboratory space and equipment during my pilot experimentation. I thank Dr. James Zacks for his careful reading of my thesis draft and for his very useful comments. ii LIST OF TABLES LIST OF FIGURES INTRODUCTION METHOD Subjects Apparatus Procedure RESULTS DISCUSSION . REFERENCES APPENDICES TABLE OF CONTENTS A. AN ANALYSIS OF FACTORS PERTAINING TO THE SUITABILITY OF GALLERIA MELLONELLA LARVAE AS SUBJECTS FOR BEHAVIORAL RESEARCH . . B. THE MEDIAN ESCAPE TIMES FOR TRIAL BLOCKS 1 THROUGH 8 FOR EACH SUBJECT IN THE EXPERIMENTAL, LIGHT CONTROL, AND DARK CONTROL GROUPS . iii Page iv 26 29 3O 42 Table LIST OF TABLES Page Experimental Paradigm Showing the Stimulus Situations Presented to the Experimental (E), Light Control (LC) and Dark Control (DC) Groups During Each Trial and Interstimulus Interval (ITI). . . . . . . . . . . . 12 iv Figure LIST OF FIGURES Page The Runway . . . . . . . . . . . . 7 Apparatus . . . . . . . . . . . . . 10 Median Escape Times and Interquartile Ranges by the Experimental (E), Light Control (LC) and Dark Control (DC) for Trial Blocks 1, 5, 8 . . 17 INTRODUCTION For the first time, the behavior of the insect species, Galleria mellonella (L.), order Lepidoptera, in its larval stage, has been studied in an experimentally controlled light escape situation. The purpose of this investigation was twofold. First, a behavioral test was designed to determine whether larvae of this species would learn to escape a light stimulus, where escape learning would be exhibited by decreasing escape times in a one-way alley over trials. Although rats have been used in many studies of the one-way alley escape learning situation, there are few reports of the use of this procedure with insects as subjects. The second purpose of this exploratory study was to evaluate the usefulness of Galleria m, as a preparation for behavioral research. This species has proved to be a valuable preparation especially for insect physiologists by virtue of its high rate of growth and metabolism and the relative ease of breeding and colony maintenance. Galleria m, is also economically important as it can infest and damage beehives and stored honeycombs. The escape learning procedure used in the present study was a one-way straight alley situation (Denny and Ratner, 1970). During each trial for the experimental group the subject was released from a start box at the onset of a“ light stimulus. To escape the light, the subject was required to move from the illuminated start box, through an illuminated alley and into a dark goal box. After an 180 sec. rest inside the goal box during the inter-trial interval (ITI), the subject began in the start box again for the next trial. The duration of the light escape response was measured from the time when the subject was able to leave the start box, simultaneously with light onset, to the time when the subject had completely crawled inside the dark goal box. The subject usually did not pause on the threshold of the goal box with only its head in the dark, but crawled all the way inside the goal box, shortly after contact with the threshold. The one-way escape situation was chosen as a test for escape learning because it parallels successful work with insect escape learning (Alloway, 1972), and because it models escape behavior by Galleria in the wax comb described by Paddock (1913) where Galleria larvae escaped from danger by retreating into their tunnels. At this point it is essential to answer the question of whether invertebrates can actually learn and, if so, under what conditions they have been shown to learn. McConnell (1966), in a review article about learning in invertebrates, described experimental findings indicating learning in coelenterates, platyhelminthes, annelids, mollusks, and arthropods. McConnell stated that there was no unequivocal proof that coelenterates could be classically conditioned or were capable of complex associative learning, although they have been shown to be capable of less complex types of learning. Class Insecta contains a tremendous number of species which have evolved a magnificent array of behavioral adapta- tions to their multitudinous ecological niches. Here exists a vast scientific frontier for students of learned as well as unlearned behavior. Already valuable information has been gained from studies of learning by insects. Horridge (1962) found that even a headless cockroach could learn specific leg position with electric shock as the aversive stimulus. Swihart (l970)conditioned butterflies to choose artificial flowers reflecting long wavelength light over flowers reflecting short wavelengths. Schulze (1970) conditioned honeybees to discriminate between various patterns and between colors. Longo (1970) found that running time by cockroaches in a runway with food reward decreased syste- matically over trials. Alloway (1972) conditioned meal worms to turn consistently to one arm of a T-maze to escape light. Using a retraining technique, he found that they retained this learning through.metamorphosis. Although important discoveries about insect learning have already been made there is ample room for further research. Now that many governments have become aware or soon will become aware of the dangers of chemical control of insect pests, the contributions behavioral research can make for the biological control of harmful insects have become increas- ingly important. A number of factors merit consideration in an assess- ment of the suitability of larval Galleria mellonella as a preparation for behavioral research. These include ease of colony maintenance, growth rate of the larvae, activity level, applicable experimental stimuli, the relevance of the experimental situation to natural behavior patterns and their environmental context, and the economic and ecological importance of the species. These factors are discussed in Appendix A. The major emphasis of the present study was to test a representative photo-negative insect species in a light escape learning situation and also to learn about effects of light on behavior other than learning. No one has reported a study in which the design used for the present study was used to test an insect species. The closest approximation was the T-maze light escape design used by Alloway (1972). The present design differs in that escape time was used rather than T-arm choice as the measure of learning. The main purpose for conducting the present study, therefore, was to expand existing knowledge about insect learning and photo-negative responses to light. The independent variable in the present study was the presence or absence of the opportunity to escape from an illuminated start box and alley to a dark goal box. The dependent variable was the time it took for the subject to crawl from the start box, through the alley and into the goal box. The experimental design used in the present study included an experimental group and two control groups. The experimental group was given 40 trials in a runway including an illuminated start box and alley, and a dark goal box to escape to. In addition to the experimental group which could escape light by crawling into the dark box, two control groups were used. The subjects in one control group could not escape the light when they crawled into their goal box. This group controlled for progressive sensitization as a result of light stimulation which might have caused a decrease in response times in the experimental and light control group. The subjects in the other control group were not exposed to light at all. The dark control group con- trolled for response time reducing factors in the experi- mental sitaution independent of the light stimulus. The type of experimental results which would justify a conclusion of possible escape learning would be a combination of a significant decrease in response times over trials for the experimental group and no decrease or an increase in response times for the control groups. METHOD Subjects The subjects used in the present study were 33 larval Galleria mellonella (L.) which are relatively small greyish caterpillars. All subjects were in a late instar and ranged from 12 to 20 mm. in length. Most subjects were raised by this researcher under conditions optimal to growth and development discussed in "Ease of Colony Maintenance," Appendix A. A few subjects were pruchased at a bait shop when laboratory-raised subjects were not available. These subjects were kept in colony medium at 35°C. for at least one day before running. Their general behavior wasrunzobservably different from that of laboratory-raised subjects. Apparatus The subjects were tested in a straight alley runway (see Figure 1). It consisted of an alley (8.125 cm. long x 1.5T cm. inside width x 1.5, cm. inside height) and a removable, interchangeable start box and goal box (2.5 cm. inside length x 1.25 cm. inside width and height). The alley and endboxes were constructed of transparent plexiglas and were equipped with removable transparent plexiglas covers. The endboxes also had transparent doors which could be lowered through slits in the covers to hold a subject “39H rI120m&.I¢ xomozu mooo .>A\>>5434mm m I u. ._ .0 _m illlllllzummellllllli rllzommnli >w44< xomozm r n r £m44< z_>4oazm PrioevmoU inside the endbox. A goal box, when used for the experi- mental group, had an opaque cover and door which kept out light to provide a dark refuge for subjects which had escaped the light. The apparatus was designed so that a subject did not have to be removed from the goal box and be placed into another box in the start position to begin each successive 1 trial. Longo (1970) reported that cockroaches are disturbed ‘ by handling and are reluctant to enter a goal box from which they had been previously removed by hand or by a nylon 1 thread cemented to the thorax. Longo's solution to the problem was the use of interchangeable end boxes. His subjects were removed from the goal position and replaced in the start position without being removed from their compartment. This procedure was used in the present study to minimize escapes as well as to reduce the possible effects of handling on responding. For the experimental group, the light-insulated goal box with a subject inside was changed to a start box in preparation for the next trial by removing the opaque cover and door and replacing it with a transparent cover and door without removing the subject. The clear plastic covers and doors on control group goal boxes were also removed and replaced with new clear plastic covers and doors to keep disturbance of the subject uniform across groups. Removable black construction paper liners were used to cover the bottoms and sides of runways and endboxes and shut out virtually all light from the bottom and sides. They were replaced every two or three trials to prevent a gradual buildup of webbing. The Combination of black paper, opaque covers and doors, and black electrician's tape on the end panel of each end box shut out virtually all light when the goal box door was closed. The present study was conducted in a room with a refrigerator door in which darkness, silence, temperature and humiditv could be maintained with considerable constancy. During experimental sessions the room was illuminated by three 40 watt darkroom red bulbs which were presumed to be invisible to the subjects. The light stimulus was produced by a Bausch and Lombe high intensity microscope lamp which was mounted 52 cm. above the runways (see Figure 2). Its light was passed through a beaker filled with water, 10 cm. deep, which was covered on the sides with black construction paper.‘ The beaker served as a heat filter. No measureable difference in temperature was found using an open bulb thermometer placed in a runway when the light was turned on for 15 min., then turned off for 15 min. At the level of the runway the light made a disk 16 cm. in diameter and the light intensity was measured at 16 fc. using a foot candle meter. 10 HEAT FILTER LAMP DARKROOM REDBULBS I ' r SHEETMETAL RUNWAYIN BRACKETS HYGROMETER LIGHT ~ ATHEMOMETER [HSK A// (D (uscmq INCU BATOR TOP ST OPWATCH FIG-2' APPARATUS 11 For each trial two runways with a start box, alley and goal box were placed side by side with brackets holding the ends in place on top of a 47 cm. square sheet of metal (less than 1 mm. in thickness). Below the sheet metal was a chicken incubator top turned upside down to direct the heat generated by three 60 watt incandescent light bulbs upward to the sheet metal surface. Light leaks were very slight and were not directed at the runways. The incubator thermostat was set so that the temperature in the runways _‘ was maintained at 359C. : 3° which was the same as the colony temperature. A portable rotary "cool steam" humidifier was used in an attempt to maintain a high enough relative humidity to prevent progressive dessication of the subjects which could influence running times. The relative humidity measured with a Luftt Durotherm Hygrometer on the surface of the sheet metal ranged from 43% to 63%. Higher relative humidities were not possible near the surface of the sheet metal at a temperature of 350C. while the laboratory room . was kept at 22°C. The escape times for the two subjects being run simultaneously in the two runways were measured separately using two stopwatches. Procedure The independent variable for the present study, the presence or absence of an opportunity to escape from an 12 illuminated start box and alley to a dark goal box, specifically meant that the experimental group was given an illuminated start box and alley to escape from and a dark goal box to escape to. The light control group was given an illuminated start box and alley, but they could not escape the light by entering the goal box because it was also illuminated. The dark control group was able to crawl inside a dark goal box, but there was no illumination to escape from as the start box and alley were also in darkness. See the experimental paradigm in Table 1. TABLE l.--Experimental Paradigm Showing the Stimulus Situations Presented to the Experimental (E), Light Control (LC) and Dark Control (DC) Groups During Each Trial and Intertrial Interval (ITI). Trial (180 sec.) ITI (180 sec.) Group Start Box and Alley Goal Box Goal Box E Light* Dark Dark LC Light Light Dark DC Dark Dark Dark * For the experimental group, the time in the light could last up to 180 sec. depending upon when the subject entered the goal box. The dependent variable, the time in seconds from the lifting of the start box door until the subject had crawled inside the goal box, was used as a measure of learning for the experimental group. The light control and dark control 13 groups controlled extraneous variables which could have also caused a systematic decrease in running times over trials. The light control group was used as a control for progressive sensitization that could have been caused by repeated light onsets. The light control group was also used as an indicator for adaptation or habituation which would be the most likely explanation if escape times for light control increased along with the times for the experi- mental group. The dark control group was a control for the possibility of wax worms learning to crawl inside the goal box because it provided insulation from outside stimuli. The dark control group also controlled for learning to enter the goal box simply because the result was at least 180 sec. without disturbance during the ensuing inter—trial interval. Both control groups controlled the influence of the experi- mental procedure (handling, dessication, separation from the food medium, etc.) on escape times, as well as the possi- bility that worms crawl from one end of the runway to the other more readily and more directly after having had the opportunity to explore the runway during previous trials. Significant food deprivation was minimized by not allowing an experimental session to last more than three hours. At least ten hours would have been required to complete 40 trials for one subject if there were no breaks. Between experimental sessions the subjeCts were kept inside. end boxes with a chunk of colony medium at 35°C. To lessen 14 the possibility of instar change before 40 trials were com- pleted the subjects were not kept in the storage state for more than a day or two. A reasonably long inter-trial interval of 180 sec. was used in an attempt to prevent significant fatigue, adaptation and habituation. ‘ The length of each subject in milliliters was recorded before the first trial. This measurement was not extremely precise as the larvae are capable of contracting to about 70% of their maximum length. For each trial, the running time was not allowed to exceed 180 sec. Subjects remaining in the start box or alley after the criterion time were gently pushed into the goal box with a soft artist's brush.and the door was closed behind them. To insure that environmental conditions remained stable throughout each experimental session, the incubator and humidifier were activated at least 30 min. before the first trial. The temperature and relative humidity over the sheet metal where the runways were placed were measured before, and were checked for stability during each experi- mental session. During each experimental session a subject from the experimental group was run simultaneously with a subject from the light control group in the two parallel runways. Members of the experimental and light control groups were 15 given the light stimulus simultaneously. During their 180 sec. ITI either a pair of new subjects from the experimental and light control groups or a pair of subjects from the dark control group were run together. In this way, four subjects were run in tandem until completion of their 40 trials. The response time for each subject was recorded after the com- pletion of each trial. The order of running, direction of the runway and placement of the runway in the left or right brackets were varied for the three groups by a predetermined schedule that controlled for possible sequence or position effects on the performance of one group over another. The schedule also determined that roughly half of the subjects in each group would be tested in the right and left brackets on the sheet metal. This controlled for possible localized differences in temperature on the sheet metal and the effect of a possible stimulus on one side of the laboratory. RESULTS The median response times calculated from each of the eight blocks of five trials is presented in Appendix B. The medians for individual subjects were used to calculate group medians for the experimental, light control and dark control groups for trial blocks 1, 5 and 8. The median escape times, along with the interquartile ranges for the groups are displayed in Figure 3. By an inspection of the group medians displayed in Figure 3, it appears that the subjects in the experimental group exhibited neither a systematic increase nor decrease in response times over trials, though the escape times were relatively low in comparison with the times for the control groups. The response times for the light control group showed a fairly large increase from the short times for the lst and 5th trial blocks to longer times for the 8th trial block. The dark control group exhibited the opposite trend with Shorter response times for trial blocks 5 and 8. The interquartile range pictured in Figure 3 are quite large and much overlap occurs between groups and between trial blocks. The exception is the interquartile range for the dark control group for the lst trial block which shows much longer response times than in the experi- mental and control groups and the distributions do not 16 QB mxooam ASE. mo... 69.5528 £55 024 Bi . 305203.10: .Ejfizmémmaxm m5. E 80241 5.543055. ozq mmzfimaqomm 2.38.2 moi m xooam _ .3503 380.; om o... m o.o 8.. m on o... n... P D! .0 mi: 0. $505 w axomN 8 mi... 250m: «on» 6m _ m2; W. A. . . .1 .5323 8. 18 overlap. The extent of overlapping in all of the other cases indicated the need to use statistical tests to determine whether significant within group differences existed between the lst and 8th trial blocks and between groups for the lst and 8th trial blocks. The distributions formed by the response time data did not meet the assumptions necessary for the use of para? metric statistical tests because the 180 sec. cut off made the distributions non-normal. Therefore, non-parametric tests were used. For all tests the significance level, =.05, two-tailed was used. A Kruskal-Wallis one way analysis of variance (Siegel, 1956) was used to test for differences between the median response times for each of the 11 subjects in the three groups for trial block 1 (Appendix C). The Hobs. (6.97) exceeded the critical H (5.99 or more) and the Ho was rejected indicating significant differences among the groups. In order to determine which of the three groups had different response time distributions for trial block 1, a Mann-Whitney U Test (Siegel, 1956) was performed on the subject medians for the experimental group vs. the light control group. The critical U or U' was 30 (or less). Between the experimental group and the light control group, U' = 51.5 and the HO was not rejected and it was concluded that there was no significant difference between the distri- butions for the experimental group and the light control 19 group, U'obs = 26 and between the light control group and the dark control group, U'obs = 27. Ho was rejected in both cases and it was concluded that the distribution of medians from the dark control group was significantly longer than the distributions for the experimental group and the light control group. In conclusion, subjects crawled more rapidly into the goal box when in the light than when in darkness during the first block of five trials. A Kruskal-Wallis Test was conducted in exactly the same way for the experimental group, the light control group and the dark control group for trial block 8 (the last trial block) as it was for the lst trial block. Hobs was 2.01 which was lower than the critical H (5.99 or more) and Ho was not rejected. A.Wann-Whitney U Test was performed on the experi- mental group vs. the light control group, the experimental group vs. the dark control group, and the light control group vs, the dark control group for trial block 8. The U's were, respectively, 41, 43.5, and 62.5 and none of the‘ Ho's were rejected as the critical H was 30 or less. In conclusion, there was no significant difference exhibited in time required for subjects from any of the three groups to crawl inside the goal box during the last block of five trials. The Wilcoxon Test for Two Correlated Samples (Siegel, 1956) was used to determine whether significant 20 increases or decreases in response time had occurred between the lst and 8th trial blocks for any of the three groups: the experimental group, the light control group, and the dark control group. The critical T was 11 (or less). The Tobs for E was 32, and although response time showed a very slight tendency to decrease from the 1st to the 8th trial block, this increase was not significant and Ho was not rejected. The Wilcoxon Test for the light control group resulted in a Tobs of 12 which approached significance and showed a tendency (though not quite significant) for response times to increase from trial block 1 to 8. The HD for the light control group was not rejected. For the dark control group, Tobs equaled 9, enabling Ho to be rejected. In conclusion, the dark‘ control group exhibited a significant decrease in response times from trial block 1 to 8, the light control group exhibited a trend for response times to increase from trial block 1 to 8 that approached significance and the response times for the experimental light-escape group remained virtually the same. No investigation of correlation between subject length and response time was made because subjects of all sizes from 12 to 20 mm. in length were scattered fairly equally among the three groups. Also, size was not highly correlated with maturity as maximum larval growth varies with availability of food during development (Paddock, 1913). DISCUSSION The data obtained from the experimental group's performance do not seem to give evidence for escape learning. Comparing the tendency toward an increase in response time over trials, which approached significance, exhibited by the light control group with the steady per- formance of the experimental group may provide for a weak argument that the light control group exhibited the effects of habituation and (or) adaptation and that the experi- mental group was able to maintain its steady, relatively short response times through learning. The experimental and light control groups were always run simultaneously and were subjected to exactly the same conditions except that the light control subjects always received 180 sec. of light before their 180 sec. ITI in darkness began while subjects from the experimental group received less than 180 sec. of light on the trials during which they entered the goal box before 180 sec. had elapsed. The difference in light received by the two groups per trial was between an average of approximately 80 sec. for the experimental group and 180 sec. for the light control group. The possi- bility that the average of 100 sec. more light per trial for the light control group caused any systematic increase 21 22 in response duration after 40 trials weakens the argument that learning kept the experimental subjects' response times down through the 40 trials as the light exposure difference might account for the different performances by the two groups. If subjects in the experimental group did learn to escape light this learning was not measured very well by duration. Perhaps the performance of a discrete response, such as choosing a specific arm in a T-maze which was successful for Alloway (1970), would provide a better measure. The performance of the dark control group is diffi- cult to interpret. A statistically significant decrease in response times from the lst to the 8th trial block was observed. This seems to argue against the influence of response time increasing fatigue, dessication, or starvation having occurred among the three groups. The systematic decrease in response time may have been due to learning to get around the runway more directly with experience or it may have resulted from progressive sensitization to the experimental situation. In conclusion, if any light escape learning occurred in the experimental group, it was not robust enough to survive the confounding influence of powerful factors working in opposite directions reflected by the data from the two control groups. The performance of the experimental 23 group appears to be at a medium level between the two control groups and may be related to the medium amount of light received. It would not be useful to determine whether the performance of individual subjects in the experimental group was correlated with the amount of light they received. This is because the subjects determine their own durations of illumination by their response times, making a clear inter- pretation along these lines impossible. To be able to make any definite statements from.the results of the present study it would be most prudent to abandon the escape learning concept altogether, and concen- trate on the definite difference between the light and dark control groups. It is clear that the presence or absence of 180 sec. of light during trials had a strong effect on behavior for at least the first five trials. It is less clear how the presentation of light resulted in a tendency (approaching significance) toward an increase in response times for the light control group and how darkness resulted in a significant decrease in response times for the dark control group. As stated above, likely factors causing an increase in response times for the light control group are habituation and adaptation, and factors possibly causing a decrease in response times for the dark control group are sensitization and learning to get around more directly in the runway with practice. 24 In conclusion, when larval Galleria m, were given 40 trials with the entire runway illuminated for 180 sec. for each trial, the increase in response times from trial block 1 to trial block 8 approached statistical significance. With 40 trials in continuous darkness, response times were relatively long during the first five trials and decreased significantly for the last five trials. When the larvae were given the chance to escape inside a dark goal box before the full 180 sec. of light had elapsed, their response times remained stable and relatively short, but probably not near their physiological capacity. Some wax worms responded in 15 sec. in the present experiment com- pared with the 80 sec. average response time for the experimental group. In pilot work two different wax worms crawled approximately 8 cm. in about 1.5 sec. under more intense light than that used for the present study. If, on the other hand, the physiological minimum was actually approached, the stability of the performance by the experimental group could have been caused by a "floor- effect” which would prevent learning from expressing itself through decreasing response times when the response times by the experimental group were short initially. The present study, unfortunately, failed either to support or disprove the occurrence of escape learning but did show some statistically significant differences in performance in the presence and the absence of a light stimulus. 25 The positive and negative factors for larval Galleria m, as a behavioral research preparation are dis- cussed and weighed in Appendix A. Probably the most important thing that can be learned from the results of the present study is that in order to use Galleria m, as a preparation for a behavioral study it must be known whether this species is able to exhibit the process being studied by a clearly observable response. Runway running appears not to be a good response to use when attempting to determine whether wax worms are learning because of the powerful confounding effects of other factors. REFERENCES 26 REFERENCES Alloway, T. N. (1972). Retention of learning through.meta- morphosis in the grain beetle (Tenebrio molitor). American Zoologist, 12: 471-477. Autrum, H. (1968). Color vision in man and animals. Naturwissenschaften, 55 (1): 10—18. Beck, 8. B. (1960). Growth and development of the greater was moth, Galleria mellonella (L.), (Lepidgptera: Galleridae). Wisconsin Academy of Science, Arts and Letters, 49: 137-149. Bullock, T. H. and Horridge, G. A. (1965). Ch. 21, Arthropoda: Details of the groups, p. 1227 in Structure and Function in the Nervous System of Invertebrates. W. H. Freeman and Co., San Francisco and London. Denny, M. R. and Ratner, S. C. (1970). Comparative Psychology: Research in Animal Behavior. The Dorsey Press, Homewde, I11. Horridge, G. A. (1962). Learning of leg position by the ventral nerve cord in headless insects. Proceedings of the ngal Society B, 157: 33-52. Longo, N. (1970). A runway for the cockroach. Behavioral Research Methods and Instrumentation, 2 (3). IIB, I19. McConnell, J. V. (1966). Comparative physiology: Learning in Insects. Annual Review of Physiology, 28: 107-136. Milum, V. G. (1952). Characters and habits of moth larvae infesting honeybee combs. American Bee Journal, 92: 200-201. Paddock, F. B. (1913). The life history and control of the boo-moth or wax worm. Investigations Pertaining to Texas Beekeeping. Texas Agricultural Station, Bulletin 158, June. 27 28 Schulze, S. M. (1970). Discrimination learning of different color and form patterns in honeybees, bumblebees, and ants. Zeitschrift fur Tierpsychologie, 27 (5): 513-552. Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw Hill, New York. Swihart, S. L. (1970). The neural basis of color vision in the butterfly (Pa ilio troilus). Journal of Insect Physiology, 16 : 2 - . APPENDICES 29 APPENDIX A AN ANALYSIS OF FACTORS PERTAINING TO THE SUITABILITY OF GALLERIA.MELLONELLA LARVAE AS SUBJECTS FOR BEHAVIORAL RESEARCH .30 Ease of Colony Maintenance A Galleria colony is not especially difficult to maintain. Late instar larvae can be obtained from bait shops in some parts of the country under the common name "wax worms." This is a great advantage as it eliminates the need for time consuming orders to animal supply houses. For optimum growth and development, the larvae are kept at 35°C in a food medium containing the following ingredients and percentage of content by weight: honey (24.25%), glyceral (21.34%), water (8.73%), pablum (32.95%), brewer's yeast powder (9.7%), and yellow beeswax (3%) (adapted from Beck, 1960). This maintenance procedure requires a large refrigerator-style incubator and containers for the larvae and medium with screen tops to provide adequate ventilation. Hard or tough sided containers should be used as the larvae can chew through soft plastic and thin wood. After the larvae have reached their last instar they find a secure place (usually the screen top) to spin a cocoon and pupate. The newly emerged adult moths will mate in captivity and the females can be induced to lay eggs on pleated wax paper for convenient handling. Strips of egg covered wax paper are placed in containers and the eggs will hatch in about a week. In this way the colony can be 31 maintained indefinitely without the researcher having to buy new animals. Wax worms are especially desirable if troublefree breeding is an important factor as it would be for studies in which all environmental factors must be controlled from the time of hatching. There are some problems with wax worms, however. The colony may die out if there are too many larvae for the medium. If ventillation is inadequate the colony may become diseased. Proper maintenance requires fairly fre- quent checks on the conditions inside the colony containers. Some major esthetic offences by wax worms are the sticky handling consistency and foul smell of the fermenting colony medium. Also, the dust produced by the adult moths' wings causes sneezing and itchy eyes. By comparison, the maintenance procedures for cockroaches (Longo, 1970), meal- worms (Alloway, 1972), and pill bugs (Segal and Gross, 1967) are less trouble than the maintenance for wax worms and the other animals' colony substrates are less offensive. For the above reasons cockroaches, mealworms, and pill bugs might make better preparations for behavioral research that does not specifically ask questions about Galleria mellonella as a species. 33 Growth Rate of the Larvae Galleria m, eggs hatch in 10 to 12 days under the natural conditions described by Paddock (1913). Under Beck's (1960) optimal growth and development procedure eggs probably hatch sooner. Using.Beck's procedure the larvae grow from an average of 0.02 mg. in weight and 3 mm. in length at hatching to 200 mg. and 18 mm. in 15 days. While in the larval stage the wax worms pass through seven instars. Larval weight doubles daily for the first 10 days and then slows down. From the 15th through the 18th day larval weight does not increase. The larvae begin spinning a cocoon on the average on the 18th day. The pupal stage averages two weeks under natural conditions (Paddock, 1913) and probably takes less time using Beck's procedure. Beck describes the growth rate exhibited by wax worms as "fantastically high, even among relatively fast-growing insects." Very high growth rate is both advantageous and dis- advantageous. If a researcher needs larvae at a specific stage of development, he will not have to wait very long after hatching to have appropriate subjects. However, he should test his subjects quickly or they will pass on to another stage of development. Another problem can arise when all larvae have passed into pupal and adult stages. Galleria m, generations tend to synchronize and there are significant periods of time when no larvae are available. 34 Refrigerating larvae will temporarily halt their development and may enable a researcher to save larvae for leaner times, though refrigeration might change their behavior. Activity Level Milum (1952) describes younger larvae as ”extremely active, rapid running." They become somewhat slower for their size as they grow larger. However, Beck (1960) observed that "from the 15th to the 18th day the larvae . were in seemingly constant motion, crawling around the inside of the rearing dishes." This is the period after the larvae have completed their growth during which they seek a place to spin their cocoon and pupate (Paddock, 1913). Beck found the respiratory rates for the larvae to be high compared to rates reported for other insect species. He found the respiratory rate for a feeding, growing larva weighing about 30 mg. to be roughly comparable to those reported for houseflies in full flight. The larvae produce appreciable amounts of heat. Beck cites smith (1941) and Roubaud's (1954) findings that the temperature of a thriving culture can exceed environmental temperature by as much as 25°C. This high activity level is largely a positive factor. for behavioral research. If the experimental situation calls for the experimenter to watch subjects during discrete trials it is helpful if the subject moves faster than at a snail's pace. Time-lapse photography can be used to record 35 a slow~moving subject's behavior in a free response or open field situation but cannot be used in a discrete trials situation. Of course, fast moving subjects present a challenge when the "little rascals" are very adept at getting away from the experimenter. Applicable Experimental Stimuli Light was assumed to be an aversive stimulus in the present study. In order to make this assumption it was necessary to find out what type of visual apparatus wax worms have and whether they do in fact escape and (or) avoid light. The author was not able to find any specific reference to vision in Galleria m, Bullock and Horridge (1965) describe the lateral ocelli in caterpillars (larval Lepidgptera) which correspond to single facets in the com- pound eyes of most adult insects. Lateral ocelli of most caterpillars consist of a few vertically elongated sensory cells surrounding a rhabdom which lies under a thick transparent cuticular lens (Landois, 1965; Pankrath, 1890; and Dethier, 1943). An elementary appreciation of shapes is inferred because some caterpillars crawl toward edges of contrasting areas. The lens can form an image but the receptor would not distinguish it. Therefore, in conjunction with movements of the head, the ocelli are probably able to distinguish only major regions of contrast in the environment and possibly distinguish far and near objects. Primary receptor cells are always figured as having axons in the optic nerve. From Paddock's (1913) account of the behavior of wax worms in beehives it appears that the larvae escape and avoid light. The author of the present article conducted a 36 pilot experiment to learn more about the responses of wax worms to light before the design for the present study was decided upon. Subjects were placed on the illuminated half of the bottom of a crystallizing dish. The other half of the dish was in darkness. The edge between light and dark was quite sharp. A typical response pattern for a subject was to crawl along the side in the dark until its head encountered the edge of the light where the subject would suddenly reverse direction. It would then crawl back along the side in the darkness until its head again hit the edge of the light and it would reverse again. Near the end of the five minute trial the subject would usually make incursions into the illuminated half, apparently habituating to the light. Some subjects did not appear to respond to the light and dark halves. Although no statistical analysis was performed on the data, the fact that sudden reversals occurred virtually nowhere except on contact with the edge of the light establishes sufficiently that light causes escape behavior by Galleria m, and supports the assumption that light is an aversive stimulus. This escape response was not due to a heat differential as both halves measured approximately the same temperature (35°C i.3°C) with the thermometer's bulb touching the bottom of the dish for 15 min. on each side, and any heat differential that did exist would not have a sharp enough gradient to cause such sudden reversals. In addition, light has been used 37 successfully as an aversive stimulus for mealworms by Alloway (1972). Therefore, light is probably a fairly good choice as a stimulus for wax worms. To use light selectively as a stimulus requires that the subject be in darkness when the stimulus is not being presented. This makes the subject difficult to observe if the subject's behavior in darkness is to be compared with behavior in light. Swihart (1970) reports that spicebush swallowtail butterflies (Papilio troilus), which are co- ‘members of the order Lepidoptera with Galleria m,, are insensitive to the infrared end of the light spectrum which is visible to humans. Autrum (1968) reports that honeybees are also insensitive to the infrared end of the spectrum. In the present study no specific behavioral test was made to compare Galleria's responses in dark room red and in darkness. However, the investigator noticed that occasionally the subjects writhed under the light stimulus but never writhed under darkroom red alone. On the assump- tion that wax worms are also insensitive to the infrared end of the spectrum, darkroom red light can enable the experimenter to see the subject, although not very clearly, when it is not being stimulated. Experimental stimuli other than light have been used with insects. Cockroaches have been given electric shock while kept stationary. Their avoidance response was specific leg positioning. Possibly, Galleria larvae could 38- be held stationary by the caudal end and be conditioned to keep their heads in a specific position to avoid shock. Food has been used as a positive reinforCer with insects (Longo, 1970; Schulze, 1970; Swihart, 1970) but wax worms characteristically live in their food medium and do not have to approach it except just after hatching. Vibration should be considered as a possible aversive stimulus. It might be a way wax worms detect the presence of bees which would probably destroy the larvae if they got the opportunity. Relevance of the Experimental Situation There are two possible reasons to test Galleria m, for escape learning in the light-escape runway situation used in this study. The first is to find out whether insects can learn to escape in a similar situation to the one in which white rats have been tested so extensively. For this purpose any insect species that is easy to maintain and has an appropriate locomotory system is as good as another. Therefore, the mealworm would be a more useful preparation than the wax worm since its maintenance is less difficult. If research with Galleria is motivated by an interest in the species itself, the stimuli present in the experi- mental apparatus should be analogous to relevant stimuli in the natural environment in their ability to elicit the behavior under investigation. That is, the SaR relationship studied in the laboratory should yield results which are 39 instructive about an environment-behavior relationship in nature. For example, if the question was whether a photo- negative reaction caused wax worms to begin tunneling inside wax combs farthest from the outside and only tunnel in more brightly lit outer areas when the food is exhausted in the darker innermost areas as was reported by Paddock (1973), a laboratory study designed to elucidate this behavioral phenomenon should provide stimuli and opportunities for response for the subjects and there should be a medium or an apparatus in which the subjects can crawl through tunnels. That wax worms do escape light is quite clear con- sidering the evidence in the literature and the results of the author's pilot study. The experimental apparatus attempted to model the maze of tunnels in a wax worm- infested beehive in its simplest form, the straight alley runway. The light stimulus was analogous to the situation in which a wax worm had found itself in relatively bright light. The light could be a danger signal for the wax worm, perhaps signaling that it had wandered outside of the wax comb or hive where it could perish by starvation, dessica- tion, or attack by enemies. It is possible that Galleria m, evolved in a niche in which the presence of light was paired with destruction. The question asked by the present study, whether wax worms learn to escape light faster when given the 40 opportunity to do so on repeated trials, however, does not model any known behavioral adaptation by wax worms in their niche. It appears that learning to escape light would not be necessary when wax worms already have an innate photo- negative response, though the possibility exists that learning is needed in combination with instinct to perfect the response. The present study, therefore, was not designed primarily to answer a question about behavior peculiar to Galleria m. Economic and Ecological Importance Although the present study is not particularly relevant to species-specific behavioral adaptations of Galleria 9,, any behavioral data on this agricultural pest might aid its control. This species is economically important because it is occasionally a serious pest in apiaries where the larvae feed on the waxy brood combs and pollen stores of honeybee hives (Beck, 1960). It is especially a problem where honeycombs are stored outside the hive and in ailing or queenless hives (Paddock, 1913). In well kept apiaries with vigorous bee colonies the damage is minimal or absent because the bees are able to keep wax worms out (Paddock, 1913). Behavioral data on Galleria m, could help apiarists to keep wax worms out of their beehives and stored honey-combs by the use of stimuli aversive to Galleria m, in the larval or adult form (e.g., install 41 bright lights so that the illumination is too intense for Galleria m,). Galleria m, lives in a parasitic relationship with honeybee colonies. Behavioral studies of species that live in specific ecological niches such as parasitism can help us understand other species which have a similar life- style. The present study investigated a response pattern that seems to keep a parasite species from getting into places where it would be destroyed by the species it parasitizes. APPENDIX B THE MEDIAN ESCAPE TIMES FOR TRIAL BLOCKS 1 THROUGH 8 FOR EACH SUBJECT IN THE EXPERIMENTAL, LIGHT CONTROL, AND DARK CONTROL GROUPS 42 43 The Median Escape Times for Trial Blocks 1 through 8 for each Subject in the Experimental, Light Control, and Dark Control Groups. Subjects Media“ 1 2 3 4 5 6 7 8 9 ' 10 11 EXPERIMENTAL GROUP: Trial Block 1 60 72 180 30 37 20 29 180 52 101 32 Trial Block 2 98 50 180 35 31 23 32 42 46 101 30 Trial Block 3 100 52 180 62 23 127 80 38 47 180 32 Trial Block 4 60 47 180 56 26 40 43 28 93 180 102 Trial Block 5 70 30 67 180 180 180 116 61 52 142 43 Trial Block 6 62 27 100 180 19 180 83 96 83 83 41 Trial Block 7 62 36 93 180 61 180 75 53 84 68 32 Trial Block 8 28 32 73 180 15 180 143 45 57 86 42 LIGHT CONTROL GROUP: Trial Block 1 180 33 49 76 36 65 29 68 43 180 74 Trial Block 2 180 69 180 91 33 37 33 115 49 146 180 Trial Block 3 180 134 180 74 49 62 78 170 39 83 78 Trial Block 4 180 80 180 180 29 103 61 118 54 180 180 Trial Block 5 54 54 180 180 42 39 40 47 122 180 180 Trial Block 6 81 180 180 180 33 43 38 180 143 180 180 Trial Block 7 56 68 163 180 59 56 100 178 180 162 180 Trial Block 8 56 38 180 180 35 57 180 122 63 180 180 DARK CONTROL GROUP: Trial Block 1 180 180 57 79 38 128 180 78 133 180 180 Trial Block 2 180 180 49 180 50 101 56 83 75 180 180 Trial Block 3 180 180 65 180 101 180 180 *180 53 180 180 Trial Block 4 107 75 66 69 144 129 180 83 45 180 163 Trial Block 5 137 69 48 53 67 51 180 50 65 180 173 Trial Block 6 62 56 34 72 91 83 180 83 54 180 180 Trial Block 7 82 90 63 52 101 62 180 137 68 180 180 Trial Block 8 48 63 43 73 43 75 180' 180 43 180 180 .J A. 9v ii- M'cllllfilmflllliililllll WHITMAN 1111111 B 3 1293 03057 6973