Diapause Aggregations in Stenotarsus sp. Endomychidae Alison Bernhardt Department of Biology, University of Minnesota Â€ Twin Cities Abstract Stenotarsus sp . Endomychidae, Coleoptera are handsome fungus beetles, which feed on fungus and rotten wood . The genus Stenotarsus is well known for its formation of large aggregations often composed of several layers, during diapause. Stenotarsus sp . has been shown to form these aggregations for mating purposes, predator avoidance and water conservation. The refore, given these reasons for aggregating this study predicts that larger individuals will have an advantage at acquiring positions low in the stacks and towards the center where they are less likely to be picked off by predators and will have an advanta ge in conserving water. This study also predicts that males will be found in the upper layers or on top of the females in order to copulate. A sample of Stenotarsus sp . was collected from the diameter of an aggregation and analyzed to determine if there we re any relationships between sex, size, stacking and distance from the center. A biased ratio of females was found which could suggest that many of the males had already dispersed. Males were found to be significantly larger than females for two differen t size measurements showing that the beetles are sexually dimorphic. The results did not support the predictions that larger individuals would be found in the best positions and that male s would be found above females. Resumen Stenotarsus sp . Endomych idae, Coleoptera son escarabajos que comen hongos y madera podridos. El gÃ©nero Stenotarsus es bien conocido por su formaciÃ³n de grupos grandes de capas mÃºltiples durante la diapausa. Se ha demostrado que estos grupos se constituyen para copular, evitar depredado res y para conservar agua. Por l o tanto, este estudio predice que los individuos mÃ¡s grandes van a tener una ventaja al obtener las posiciones mÃ¡s bajas en las agrupaciones y mÃ¡s cercanas al centro del grupo, donde es menos probable que un depreda dor los atrape y donde es mÃ¡s fÃ¡cil conservar agua. TambiÃ©n se predice que los machos se encontrarÃ¡n en las capas altas encima de las hembras para copular. Se colectÃ³ una muestra de Stenotarsus sp. de un grupo y se analizÃ³ para determinar si habÃa alguna re laciÃ³n entre el sexo de los individuales, su tamaÃ±o, su ubicaciÃ³n en las capas y dÃ³nde se encontraban en relaciÃ³n al centro del grupo. Se descubriÃ³ una desproporciÃ³n a favor de las hembras que puede indicar que los machos ya se habÃan dispersado. TambiÃ©n se encontrÃ³ que los machos son significativamente mÃ¡s grandes que las hembras en dos aspectos corporales diferentes y esto demostrÃ³ que los escarabajos presentan dimorfismo sexual. Los resultados no apoyan las predicciones de que los individuos mÃ¡s grande s se ubican en las mejores posiciones y de que los machos se localizan encima de las hembras. Introduction Stenotarsus sp . Endomychidae, Coleoptera, also known as handsome fungus beetles, are tiny reddish brown beetles, which feed on fungi, rotten w ood, or decayed fruit White 1983. The genus Stenotarsus has over 250 species with nearly 100 described from the New World Roubik and Skelley 2001. Three site specific aggregations of Stenotarsus
sp . have been found in Monteverde, Puntarenas, Costa Ri ca. The natural history of the species in Monteverde is largely unknown; however it has been shown in a previous study to reflect behavioral characteristics of a population of Stenotarsus subtilis found on Barro Colorado Island, Panama Cagle 1999. Sten otarsus species have been the subjects of many studies since they are well known for forming diapausing aggregations in the dry season. Diapause in insects is an adaptive response to intermittent occurrence of unfavorable conditions such as winter, rain, o r desiccating heat Huffaker and Gutierrez 1999. During diapause most insects become relatively inactive and sometimes development and reproduction are even arrested Daly et al. 1998. Typically diapause occurs in a particular development stage and usua lly only once in an insect s lifetime Tanaka and Zhu 2003. Diapuase is common among temperate insects since the seasons are much more pronounced in temperate regions. However, diapause is not a rare phenomenon in the tropics Tanaka and Zhu 2003. Ins ects still react to seasons in the tropics even though the seasonal changes in temperature are minimal. The main cues driving diapause in the tropics are humidity and rainfall Wolda1988. A study on seven Cassidines sp. Chrysomelidae in Santa Rosa and Guanacaste National Parks showed that these beetles were aggregating in the moistest habitats available in order to pass the dry season Flowers 1991. This is very similar to the Stenotarsus sp . found in Monteverde, which has been found aggregating and going through diapause on cool surfaces near streams. Stenotarsus subtilis , the endomychid beetle found on BCI, has been shown to diapause at the same site for a specific duration of time each year since 1980 Wolda and Denlinger, 1984. Beginning in Jun e S. subtilis migrates to a specific palm tree and forms large aggregations. These beetles stay there in diapause for up to ten months until the beginning of the wet season when they disperse after the first heavy rain occurs Wolda and Denlinger, 1984. The Stenotarsus species in Monteverde also forms large aggregations often composed of multiple layers at specific sites for a certain period of time and then disperses at the onset of the first heavy rains. There are several reasons or benefits to formin g aggregations. The first reason that Stenotarsus sp . form aggregations is for mating purposes. A previous study conducted on Stenotarsus observed the beetles mating a week prior to dispersal Cagle 1999. The second reason aggregates is for predator avo idance. Individuals in the aggregation benefit from predator avoidance because they are so numerous and tightly packed, therefore only the individuals in the outer most layer are more likely to be picked off by predators. The beetles resemble a selfish herd in which each individual is responsible for their own survival. Furthermore, Stenotarsus sp . has been shown to be unpalatable and not acceptable to Sceloporus malachiticus Montalvo 1999 and therefore the aggregations are less of a target for hungry predators. Stenotarsus sp. also aggregates to prevent water loss since they are found on humid, cool rocks only during the dry season Cagle 1999. Other Stenotarsus sp . do this as well such as Stenotarsus subtilis from BCI, which has been shown to co nserve water by aggregating Yoder et al. 1992. Given these benefits of aggregating, it is logical to predict that larger individuals will have an advantage at acquiring the best positions in the aggregation, i.e. low in the stacks and towards the center where they are less likely to be picked off by predators and will have an advantage in preventing water loss. It is also logical to predict that males will be found above
females in order to copulate with them. The purpose of this study is to test thes e predictions based upon evidence that the reasons for forming aggregations are mating, predator avoidance and water conservation. Materials and Methods The study site utilized is located next to the Quebrada MÃ¡quina between the Hotel Belmar and the Bi ological Station in Monteverde, Puntarenas, Costa Rica. Stenotarsus sp. had formed numerous aggregations on the downward surface of a 4m tall rock located on the edge of a path approximately 25m north of where the stream and the clay road to Cerro Amigos meet. Collection of specimens The beetles on the rock were arranged in large aggregations composed of multiple layers. The diameter of one of the aggregations was measured using a tape measure and then the beetles were collected from every half centimet er along the diameter. When the beetles were stacked every beetle in the stacking was collected and their location within the stack was recorded. The beetles were collected using tweezers and each beetle was placed in its own test tube. The diameter of the aggregation collected from was 21 cm. A sample of 96 individuals was collected from the diameter on the morning of Tuesday, April 18, 2006. Sex Ratio and Size The sample of 96 individuals was then examined by using a dissecting microscope to determine the sex of each individual. Males were confirmed by having a penis beneath the skin on their lower abdomen and tooth like structures on their hind tibias. Measurements of pronotum length and width, and femur length were taken on the 96 individuals by usin g an ocular micrometer on an Olympus SZ40 telescope. Data Analysis The sex ratio of the sample was tested for deviation from random 1:1 by using a chi square test. To see if males and females differed in size, an unpaired t test was run for eac h of the three size measurements. To determine how male and females were distributed in relation to the levels, or stacks, of individuals a contingency table was used and a chi squared test performed. An unpaired t test was then used to reveal whether fem ales and males differed in their distance from the center of the aggregation. Next, to determine whether females of different stacking levels differed in size, a Kruskal Wallis test was run for all three size measurements. The same was repeated for males. To look at whether size influences distance from the center a simple regression was performed for each size measurement for both sexes. Finally, to establish whether size of an individual influenced how far it was found from the center in each of the thre e layers, three Spearman Ranks were done for each of the three size measurements for each level of stacking for both sexes.
Results Sex ratio The sex ratio of the 96 individuals was 39 males to 57 females approximately 2:3; Ã°l 2 = 3.38; df = 1; P > 0.05 . A previous study conducted here in Monteverde also found a ratio of 2:3 Montalvo 1999. Difference in size between males and females The mean pronotum length and femur length were significantly different between sexes, which reveals that these male and female beetles are dimorphic in size Unpaired t test, t = 5.16; p < 0.0001; t = 8.95; p < 0.0001. The mean sizes show that, on average, males have a greater pronotum length and femur length while females have a greater pronotum width Figure 1. 0 10 20 30 40 50 60 70 80 90 Female Pronotum Length Male pronotum Length Female pronotum width Male Pronotum Width Female Femur Length Male Femur Length Size Measurements Length Figure 1: Differences in mean pronotum length, mean pronotum width, and mean femur length between male and females demonstrating that females and males are morphologically distinct. Average male pronotum length x = 42.95 Â± 3.07 was greater than that of the females x = 39.63 Â± 3.12. Average femur length for males x = 56.85 Â± 2.37 was also larger than that for females x = 51.81 Â± 2.92. Average female pronotum
width x = 87.47 Â± 3.97 was greater than the average pronot um length for males x = 86.64 Â± 4.09 Relationship between Sex and Stacking The beetles were observed to be stacked up to four layers deep on the rock; however the fourth layer was excluded from the data analysis since there were only a few individual s. Furthermore, there were a few individuals on the perimeter of the aggregation who were not stacked at all and therefore they were excluded. There was no relationship found between sex and stacking Ã°l 2 = 3.81; df = 2; p = 0.1487. Males and females we re randomly distributed throughout each layer Table 1. Table 1: Locations of males and females in the aggregation with relation to stacking. Distance from Center vs. Sex Ther e was no difference found in the average distances of males and females from the center of the aggregation Unpaired t test; t = 0.85; p = 0.3971. Stacking vs. Size Females did not differ in size in different levels of stacking, nor did males Table 2. For male pronotum length, however, a nearly significant value was obtained which showed that males with larger pronotums have a tendency to be found on the second stacking level Kruskal Wallis; p = 0.0579. Table 2. Correlation of size and stacking level for three size measurements for both males and females. Pronotum Length P value Pronotum Width P value Femur Length P value Females 0.9392 0.5452 0.1696 Males 0.0579 0.1206 0.6219 Distance from center vs. size There was no association found b etween the distance from the center of the aggregation and size for any of the three size measurements Figures 2 7. Influence of Size on distance from center in three stacking types Bottom layer Second layer Third layer Males 12 19 6 Females 22 15 12
There was no influence of size on the distance of individuals from the center in the three different layers. A Spearman Rank Test was used to analyze each size measurement for each of the three layers for both sexes Table 4. Table 4. Influence of three different size measurements on the distance of males an d females from the center of the aggregation in each stacking layer. No significant relationship was found between distance and size in any of the three layers for males or females. __Bottom Layer_ __ __Second Layer__ __Third Layer___ p value r ho n p value rho n p value rho n Pronotum Length Male 0.32 0.30 12 0.17 0.32 19 0.75 0.14 6 Female 0.95 0.13 22 0.54 0.16 15 0.89 0.04 12 Pronotum Width Male 0.63 0.14 12 0.60 0.13 19 0.42 0.36 6 Female 0.80 0.55 22 0.36 0 .25 15 0.81 0.07 12 Femur length Male 0.41 0.25 12 0.94 0.02 19 0.29 0.47 6 Female 0.85 0.42 22 0.91 0.03 15 0.39 0.26 12 Discussion Sex ratio and size The ratio of 2:3 is very similar to the previous results obtained by Montalvo 19 99 and Cagle 1999. In addition, the results from a study conducted on BCI were also substantially female biased where females often outnumbered males by a factor of two or three Roubik and Skelley 2001. One explanation for this bias is that males ma y face a higher mortality during diapause. Another explanation may be that males do not join the aggregation until the end of the diapause period when the females are ready to mate. A final explanation may be that males disperse earlier than females. Ea rlier dispersal would explain the results obtained in Monteverde since all the studies were conducted right before Stenotarsus sp . dispersed. Males tended to be larger than females for two of the size measurements: pronotum length and femur length. This shows that there is sexual dimorphism in Stenotarsus sp. found in Monteverde. Reasons for Aggregation Formation There are various reasons that animals form aggregations, such as for maintenance of water content, heat conservation and regulation, protecti on from adverse conditions, predator avoidance, increased vigilance and for mating purposes Allee 1931. Stenotarsus sp . has been shown to form aggregations for mating purposes, predator avoidance and water conservation. Based on these reasons it is pr edicted that the larger individuals will occupy the center and lower layers of the aggregation since they have a
size advantage and males will be above the females in order to mate with them. The purpose of this study was to test those predictions. One reason why Stenotarsus forms aggregations is for mating purposes. However, the data implies that the beetles in the aggregation were not mating since males were distributed randomly throughout the layers. If Stenotarsus sp. were mating the majority of th e males would have been found on the upper layers since they need to be on top of the female in order to mate with her. A study conducted on Stenotarsus subtilis , the Endomychid beetle from BCI, found that sexually mature male beetles abstained from matin g during the dry season Tanaka et al. 1987. The researchers believe that the reason for not mating is to ensure survival until the end of the dry season by minimizing expenditure of fat and water reserves by suppressing locomotion Tanaka et al. 1987. Cagle 1999 observed that the Stenotarsus species in Monteverde also did not begin mating until a few days before they dispersed. One reason why Stenotarsus sp. was not found mating during this study is that maybe the males and females had already mated and thus males were not attempting further copulation. Another reason may be because mating might occur later after the aggregation has dispersed and disappeared. The second reason that Stenotarsus sp. form large aggregations is to avoid predation. In a s tudy conducted on the aphid Aphis Varians Apidae it was found that as aggregation size increased the predators also showed a strong aggregating response, however the predators could not keep up with the aphids and as a result the risk of predation to ind ividual prey was diluted by an increase in prey numbers Turchin and Kareiva 1989. Aggregation reduces predation risks to individuals since when the beetles are tightly packed together only the individuals in the outermost layer are likely to be eaten. Each individual in the aggregation is responsible for his or her own survival and therefore it is to his or her advantage to be in the center or near the bottom. Therefore it was predicted that the larger individuals would have an advantage and be able t o push themselves to the center and to the bottom layer of the stack more easily than the smaller individuals. However, the results from this study did not support this prediction. This may be because having a greater pronotum length or femur length does not mean that that individual has an increased ability to move to the best sites. The third reason that Stenotarsus sp . aggregate is to conserve water. The dry season in the tropics can be harsh, hot and desiccating and consequently the beetles want to tr y and avoid these conditions. Monteverde is much cooler and moister than the lowlands, and therefore it is a good place to avoid the harshness of the dry season. A study carried out on Stenotarsus subtilis in BCI showed that aggregation promoted water co nservation Yoder et al. 1992. A similar study conducted on the American house dust mite, Dermatophagoides farinae established that the water loss of an individual in isolation was greater than water loss by individuals in an aggregation; in fact, the gr oup of mites was nearly twice more effective in conserving water than the solitary individual Glass et al. 1998. Since Stenotarsus sp. aggregates to conserve water it was predicted that the larger individuals would be in the center and towards the bottom s ince they have a size advantage. However, as mentioned earlier none of the data supports this prediction. This may be because a few centimeters difference does not affect water conservation because the rock surface and ambient conditions are uniform and therefore the larger individuals have no interest in being in the center. Another reason, which was previously
mentioned, is that having larger pronotum or larger legs does not mean that that individual has an increased ability to move to the best site. No significant data was found to support either of the predictions. Larger individuals did not occupy the lowest and most centered positions in the aggregation and males were not found to be above females. This could be because this study was conducted only three days before Stenotarsus sp. dispersed and therefore the beetles were more concerned about getting ready to disperse than trying to compete for the best site or trying to mate. Future studies should repeat the study for larger aggregations and at a different period during their diapause given that this study was only conducted a few days before dispersal. Acknowledgements I would especially like to thank Karen Masters for all her positive advice and guidance through everything. I would also li ke to thank Karen for her endless ideas every time I had to alter my project. I would also like to thank Ollie Hyman and Maria Jost for all the help with supplies and statistics that they gave me during my project and Javier MÃ©ndez for helping me with my resumen . I would also like to thank my host family for showing me the aggregation of beetles and sparking my curiosity in them. Lastly, I would like to thank my parents for allowing me to have this wonderful experience and for supporting me all the way th rough. Literature Cited Allee, W.C. 1927. Animal Aggregations. Quarterly Review of Biology 23: 367 398. Cagle, Amber. 1999. Dry Season Aggregations of a Tropical Beetle in the Monteverde Cloud Forest. CIEE Spring 1999. Daly, H.V., J.T. Doyen a nd A.H. Purcell III. 1998. Introduction to Insect Biology and Diversity. Oxford University Press, New York, pp. 81 82. Flowers, R.W. 1991. Aggregations of Cassidinae Chrysomelidae in Santa Rosa and Guanacaste National Parks, Costa Rica. Biotropica 23: 308 310. Glass, E.V., J. A. Yoder and G. R. Needham. 1998. Clustering Reduces Water Loss by Adult American House Dust Mites Dermatophagoides farinae Acarri: Pyroglyphidae Experimental and Applied Acarology 221: 31 37. Ed. Huffaker, C. B. and Ed. A. P. Gutierrez. 1999. Ecological Entomology: Second Edition . John Wiley and Sons Inc., New York:162 168. Montalvo, Alex J. 1999. Population Behavior of an Aggregating Stenotarsus sp . Endomychidae in the Monteverde Cloud Forest during the Wet Seaso m. CIEE Summer 1999. Roubik, David W. and P. E. Skelley. 2001. Stenotarsus subtilis Arrow, the Aggregating Fungus Beetle of Barro Colorado Island Nature Monument, Panama. The Coleopterists Bulletin 553: 249 263. Tanaka, Seiji. and Dau Hong Zh u. 2003. Presence of Three Diapauses in a Subtropical Cockroach: Control Mechanisms and adaptive significance. Physiological
Entomology 28: 323 330. Tanaka, S., H. Wolda, and D. L. Denlinger. 1987. Abstinence from Mating by Sexually Mature Males of the fungus Beetle, Stenotarsus rotundus , During a Tropical Dry Season. Biotropica 193: 252 254. Turchin, P. and P. Kareiva. 1989. Aggregations in Aphis Varians: An Effective Strategy For Reducing Predation Risk. Ecology 704: 1008 1016. White, Ric hard E. 1983. Beetles. Houghton Mifflin Company, New York, pp. 237 238. Wolda, H. 1988. Insect Seasonality: Why? Annual Review of Ecology and Systematics 19: 1 18. Wolda, H., and D. L. Denlinger.1984. Diapause in a large aggregation of a tropical bee tle. Ecological Entomology 9: 217 230. Yoder, J.A., D. L. Denlinger and H. Wolda. 1992. Aggregation Promotes Water Conservation during Diapause in the Tropical Fungus Beetle, Stenotarsus Rotundus. Entomologia Experimentalis et Applicata 632: 203. 30 35 40 45 50 0 2 4 6 8 10 Distance from center Pronotum length Figure 2: There was no significant relationship found between distance from center and the pronotum length of females Simple Regression; p = 0.6037; R 2 = 0.006; N = 49.
70 75 80 85 90 95 100 0 2 4 6 8 10 Distance from center cm Pronotum width Figure 3: There was no signi ficant relationship found between distance from center and the pronotum width of females Simple Regression; p = 0.4583; R 2 = 0.012; N = 49. 40 45 50 55 60 0 2 4 6 8 10 distance from center cm femur length Figure 4: There was no significant relationship found between distance from center a nd the femur length of females Simple Regression; p = 0.3275; R 2 = 0.020; N = 49.
30 35 40 45 50 0 2 4 6 8 10 Distance from center cm Pronotum Length Figure 5: There was no significant relationship found between distance from center and the pronotum length of males Simple Regression; p = 0.99 23; R 2 = 2.712E 6; N = 37. 70 75 80 85 90 95 100 0 2 4 6 8 10 Distance from center cm Pronotum Width Figure 6: There was no significant relationship found between distance from center and the pronotum width of males Simple Regression; p = 0.2440; R 2 = 0.039; N = 37.
50 55 60 65 70 0 2 4 6 8 10 Distance from Center cm Femur Length Fi gure 7: There was no significant relationship found between distance from center and the femur length of males Simple Regression; p = 0.8784; R 2 = 0.001; N = 37.