Defense mechanisms of Nyssodesmus python (Polydesmidae) Sheiphali Gandhi Pott College of Science and Engineering, University of Southern Indiana ABSTRACT The purpose of this study was to examine the effects different characteristics of Nyssodesmus python had on the defense mechanisms of the organism. The defense mechanisms of N. python include: curling into a spiral and chemical defense. It was predicted that sex would not affect defense mechanisms, whereas size and level of calcification would. Populations were found in two locations: La Estación Biológica Monteverde and San Gerardo on different sides of the continental divide. One hundred and twenty millipedes were collected, 42 in Monteverde and 78 in San Gerardo. They were artificially threatened, and the time spent in a protective position and the release of toxin were recorded. The size of the individuals and level of calcification were also recorded. T-tests and chi-squared tests showed that there were significant differences in the defense mechanisms of males and females. Regression analysis showed a significant trend between size of an individual and the time it remained in the protected position, but the relationship may not be due to causation. The millipedes of different populations had some distinctive morphological and behavioral characteristics; additionally, there was a significant difference in their length of time spent in a curled position. RESUMEN El objetivo de este estudio fue examinar los efectos de las diferentes característcas de Nyssodesmus python sobre los mecanismos de defensa de estos organismos. Los mecanismos de defensa de N. python incluyen: doblarse sobre sí mismos y la defensa química. Se predijo que el sexo no afectaría a los mecanismos de defensa, mientras el tamaño y el nivel de calcificación si lo harían. Los individuos se encontraron en dos lugares: La Estación Biológica Monteverde y San Gerardo, en lados diferentes de la división continental. Ciento veinte milpiés se estudiaron en total, 42 en Monteverde y 78 en San Gerardo. Se amenazaron artificialmente, y se registraron el tiempo que pasaron en una posición protectiva y la presencia de la secreción de toxina. También se anotaron el tamaño de los individuos y el nivel de calcificación. Los experimentos indicaron una relación significativa entre el sexo de un milpiés y sus mecanismos de defensa. El análisis de regresión mostró una relación significativa entre el tamaño del individuo y el tiempo que pasó en una posición protectiva. Los milpiés de poblaciones diferentes mostraron diferencias morfológicas y etológicas características; hubo una diferencia significativa en el tiempo en que permanecieron en una posición protectiva. INTRODUCTION Nyssodesmus python, commonly known as the large forest-floor millipede, is usually found on the Caribbean slope of Costa Rica in patches of uncut understory. They feed primarily on rotting wood, like most other Polydesmidae, and are important decomposers in the rainforest ecosystem (Heisler 1983). Adult N. python have twenty body segments, nineteen of which display a pair of horizontal keels that are large and flat, resembling a large isopod. Individuals are dull light yellow with two dark brown longitudinal stripes running down their backs. Sex can be easily determined because on the ventral side of the seventh body segment of male individuals, the first pair of legs is modified into a set of small curved “gonopods.” These structures are used in the transfer of sperm to the female millipede. Additionally, female N. python at 70-100 mm in length are larger than males, who measure on average 65-90 mm (Heisler 1983). Nyssodesmus python molts throughout its entire life cycle even after it has stopped growing; after each molt, an adult N. python is soft and unpigmented. Approximately a month passes until the process of calcification and pigmentation is complete (Heisler 1983). The calcified exoskeleton plays an essential role in resisting the large pressures developed while burrowing. It is thought that calcium salts increase resistance without making the keel unwieldy. In general, the exoskeleton of males is stronger and more resistant than females. Also, as the body mass of females increases, the strength of their exoskeleton increases as well; while this trend does exist in male exoskeletons, it is much less apparent (Borrell 2004). N. python has two forms of protection: an extremely rigid exoskeleton and a toxic chemical excretion. When threatened, it curls into a spiral, protecting its vulnerable underside, and secretes a toxin from its hindgut (Heisler 1983). The predominant components of their cyanogenetic defense secretions are hydrogen cyanide and benzaldehyde, together with other compounds such as phenol, benzoic acid, benzoyl cyanide, and mandelonitrile (Kuwahara et. al. 2002). Their characteristic secretion glands are known as oxadenes. They open laterally on the individual diplosegments to exude the repugnant liquid (Wright 1999). The composition of each toxin secretion is species-specific (Kuwahara et. al. 2002), and the secretion of N. python is commonly known for its pleasing cherry-almond scent. This liquid can be expelled violently up to a distance of 30 cm (Heisler 1983). Research has been done on the reproductive behavior of this species by Hyatt (1993) and Arnold (1998), but little else is known about the natural history of N. python (Heisler 1983). The present study explored significant trends in defense mechanisms dependent on sex, size, and population. It was predicted that sex would not affect defense mechanisms, whereas size and level of calcification would. The hypothesis was based on the fact that little distinction exists between the morphology of males and females, whereas a clear disparity can be seen in size and calcification. MATERIALS AND METHODS Study site Nyssodesmus python were collected in two locations: the forest close to La Estación Biológica Monteverde and the forest in San Gerardo. The forest close to La Estación Biológica is on the Pacific slope in the lower montane wet forest life zone with an elevation of 1400-1800 m (Haber et al. 2000). San Gerardo sits at approximately 1300 meters in elevation on the Atlantic slope in the premontane rain forest life zone. Collection occurred close to trails due to accessibility (Fig. 1). Data collection The millipedes from the forest above La Estación Biológica Monteverde were collected in plastic bags and brought to the station by CIEE students. The millipedes collected in San Gerardo by Alan Masters were placed in a plastic container containing leaf litter and brought back to La Estación Biológica Monteverde to be tested. All subjects were collected between July 21, 2005 and August 1, 2005. They were kept at the biological station in terraria containing leaf litter collected on station property and covered with plastic wrap to maintain a moist environment. The millipedes from the two life zones were kept in separate terraria. The millipedes were left alone for one day to allow for acclimation. Multiple terraria were maintained so that simultaneous acclimation periods could be used for millipedes collected on different days. No more than ten millipedes were kept in a terrarium at the same time. After the acclimation period, the millipedes were taken out of the terraria individually. The subject was then placed on a table and was artificially threatened by roughly turning it over onto its dorsal side. The amount of time the millipede remained curled in a spiral was recorded. The presence of toxin was determined by the presence of a sweet/cyanide smell and recorded. The length and width of the millipede were measured in millimeters by holding the millipede on it ventral side against a ruler on the table. These measures were multiplied to produce a size index for the comparison of different individuals. The new layer of exoskeleton, after molting, is unpigmented; therefore, the amount of calcification of the exoskeleton can be determined by its pigmentation. This was rated on a scale from one to ten with ten being the darkest and one the lightest. Calcification was recorded for only the Monteverde population because the pigmentation trend is only known for this population of millipedes (Heisler 1983). Statistical analysis Parametric tests were used to compare values. The log of the time of protection was taken to generate a normal data set. Unpaired t-tests were used to compare the protection time and size index of millipedes found in different locations. A chi-squared test was used to determine if the secretion of toxin differed between populations at the two sites. Unpaired t-tests were used to compare the curling time of males and females. Because there is a significant size difference between the two sexes (Heisler 1983) (Fig. 2), separate tests compared the size index and the protection time of each sex using simple regression analysis. Similarly, separate unpaired t-tests were run comparing the size index and presence of toxin of each sex. A chi-squared test was run to compare the presence of toxin for both sexes. Lastly, simple regression analysis was used to see if the protection time was a function of calcification. RESULTS A total of 120 Nyssodesmus python were collected. Close to La Estación Biológica Monteverde, 42 millipedes were collected, whereas 78 were collected in the San Gerardo forest. There were 46 females collected (16 in Monteverde and 30 in San Gerardo), while 74 males were collected (26 in Monteverde and 48 in San Gerardo). The ratio of females to males for each population was approximately 5 to 8. Effect of sex The protection time for the different sexes was found to be significantly different (p=0.0034). The mean protection time for females was 266.32248.530 seconds, the mean protection time for males was 141.59722.641 seconds. There was also a significant relationship between the sex of a millipede and the secretion of toxin (X2=10.781, DF=1, p=0.0010). Females were more likely to secrete toxin than males.