Chemical defence of the warningly coloured caterpillars of Methona themisto (Lepidoptera: Nymphalidae: Ithomiinae)

The caterpillars of the butterfly Methona themisto (Nymphalidae: Ithomiinae) are conspicuously coloured and feed exclusively on Brunfelsia uniflora (Solanaceae), a plant that is rich in secondary plant substances, which suggests the caterpillars are chemically protected against predators. Results of experiments indicate that predators determine the survival of Methona themisto caterpillars in the field and laboratory bioassays that this organism is eaten by ants and spiders but not chicks. Both the conspicuous orange and black striped colouration and chemical compounds of Methona themisto caterpillars seem to be related to protection against predation by visually hunting predators. Chicks ate proportionally more of the cryptically coloured 1st instar caterpillars than of the conspicuously coloured later instar caterpillars. That Methona themisto caterpillars are chemically defended is supported by the activity of the dichloromethanic extract of 5th instars in preventing predation by chicks. Caterpillars of Methona themisto are aposematic as they are both (1) unpalatable, and (2) their warning signal is easily recognized by potential predators. Chicks learned to avoid the aposematic 3rd or 5th instar caterpillars after one encounter. Mealworms painted to look like caterpillars were also rejected by chicks that had previously encountered Methona caterpillars. Naïve chicks did not avoid eating the painted mealworms, which indicates they do not innately avoid this specific colour pattern. 253 * Corresponding author; e-mail: [email protected] have a conspicuously black and orange striped colouration in the later intars (3rd–5th) and feed mainly on the abaxial surface of leaves (Fig. 1). Caterpillars take 20 days to complete their development and then, pupate away from the host plant. The life cycle of Methona themisto is described in more detail in Lamas (1973) and Brown & Freitas (1994). The host plant Brunfelsia uniflora is a Neotropical shrub used as an ornamental (Plowman, 1979) and medicinal plant (Schultes, 1976). This genus of plants is known to contain brunfelsamidine (Lloyd et al., 1985), scopoletine (Mors & Ribeiro, 1957) and calistegines (Bekkouche et al., 2001). Caterpillar survival in the field In order to verify the role of predators in the survival of Methona themisto caterpillars in nature we compared the survival of caterpillars that were either protected or unprotected against predators on plants in a fragment of forest near Campinas, São Paulo state, Brazil [Mata Ribeirão-Cachoeira (22°50 ́S, 46°55 ́W)] and in an urban environment [Campus of Universidade Estadual de Campinas (UNICAMP) (22°49’S, 47°05’W)]. Eggs were obtained from adults kept in a greenhouse in the Departamento de Zoologia, IB, UNICAMP. We used 20 potted Brunfelsia uniflora grown in a greenhouse in order to minimize the influence of cues due to herbivory, which might attract natural enemies, and allowed the standardization host plant size (1 m in height). Newly hatched first instar caterpillars (n = 20 individuals/plant) were placed on the abaxial surface of Brunfelsia uniflora leaves (one caterpillar per leaf) and their survival monitored daily until the 5th instar. We randomly choose the leaf which each caterpillar was placed. At each site the plants were either unprotected or protected against predators (n = 5 for each treatment). The plants were protected against predators using the method described by Nogueira-de-Sá & Trigo (2002). The plants were placed in exclusion cages made of PVC (1 × 1 × 1 m) and covered with a fine mesh, which excluded predators such as birds, wasps and bugs. TanglefootTM resin placed around the base of each plant prevented ants reaching the plants. Pairs of treatments were placed close to each other (1 m apart) and each pair randomly assigned to positions 10 m apart. The percentage survival of the last instar (5th) was arcsin transformed and compared for the different sites and treatments using repeated measures Anova (Zar, 1999), assuming that as the treatments within each pair were positioned close together they should be similarly attacked by predators. Bioassays using live caterpillars Since ants will attack and kill caterpillars that are not usually ant attended (Smiley, 1985; Montllor & Beranys, 1993; Freitas & Oliveira, 1996) and are deterred by distasteful caterpillars (Dyer & Floyd, 1993; Dyer, 1995a, b; Dyer & Bowers, 1996; Dyer et al., 2003) ants were used as the predator in laboratory bioassays. The neotropical carpenter ant, Camponotus crassus Mayr (Formicinae), was used as the predator in the bioassays. This species nests in live trees and in dead and decaying logs (Kusnezov, 1951), and is frequently seen patrolling plants in a range of habitats. Ten colonies of Camponotus crassus were collected and maintained in the laboratory following Portugal & Trigo (2005). The colonies were fed late instar mealworms Tenebrio molitor L. (Coleoptera: Tenebrionidae) 48 h before the bioassays. In a bioassay one 5th instar Methona themisto caterpillar was offered on a mature leaf of its host plant, Brunfelsia uniflora. The leaves were collected immediately before the bioassays from plants growing in the field at UNICAMP and their petioles temporarily placed in a vial water to prevent the leaves from dessicating. Caterpillar mortality was recorded after 24 h. In addition to C. crassus, two other ant species maintained as above, Camponotus rufipes Fabricius (two colonies) and Camponotus rengeri Emery (three colonies), were used as predators in the bioassays. The wolf spider, Lycosa erythrognatha Lucas (Arachnida: Araneae: Lycosidae), was also used as a predator (n = 30). Like ants, they are voracious invertebrate predators but avoid eating chemically protected organisms (Eisner & Eisner, 1991; Gonzáles et al., 1999; Framenau, 2000; Carrell, 2001). As prior to pupation Methona themisto caterpillars leave their host plant, generally in the 5th instar, they may encounter this predator, as well as ants (see below) in nature. The bioassays using wolf spiders were carried using the method described by Gonzáles et al. (1999). The spiders were collected on the campus of UNICAMP and kept individually in transparent plastic containers (15 × 12 cm, d × h) with a ball of water saturated cotton wool and fed weekly with one mealworm. Two weeks prior to their use in the bioassays they received one mealworm. In each bioassay, a single 5th instar caterpillar of Methona themisto, held in a pair of forceps, was offered to the wolf spider by placing it in front of the spider’s chelicerae. The response of the spider was recorded as successful if the caterpillar was captured and killed, or not successful, if the caterpillar survived the spider’s attack. The spider’s response was validated if after the capture of a Methona themisto caterpillar it accepted a mealworm. Young individuals of Gallus gallus domesticus (L.) (Galliformes: Phasianidae) are often used as models of visually hunting predators (Begossi & Benson, 1988; Nogueira-de-Sá & Trigo, 2005). The bioassays were conducted following Nogueira-de-Sá & Trigo (2005) by first offering a control mealworm. If the mealworm was eaten a caterpillar of Methona themisto was offered and the chick’s responses recorded as pecked and eaten, pecked and not eaten, or not pecked (response categories modified from Brower 1958). After offering the caterpillar the chick was offered another mealworm and if the chick ignored or rejected the mealworm the result of that trial was not included in the analysis. Each individual chick was used in only one trial (n = 30 for each trial). In order to determine whether they could complete their development after their encounter with the predator the pecked and not eaten, and not pecked caterpillars were reared individually. The frequencies of each chick’s response were compared using a G-test for contingency tables, followed by a subdivided contingency table as a post hoc test when needed (Zar, 1999, p. 502). Bioassays with caterpillar and host plant extracts To verify if caterpillars of Methona themisto were chemically protected against predators, bioassays using dichloromethanic and aqueous extracts of caterpillars and of its host plant were carried out under laboratory and field conditions. Caterpillars of Methona themisto or fresh leaves of Brunfelsia uniflora were homogenized three times in MeOH : H2O 4 : 1 (10× volume/weight). The homogenates were filtered, combined and evaporated at low pressure (40°C) until only the aqueous phase remained. The aqueous phase was extracted three times with dichloromethane, yielding two phases: dichloromethanic and aqueous. The dichloromethanic phase was washed three times with water, which was then discarded. The same procedure was followed with the aqueous phase, which was washed three times with dichloromethane, which was then discarded. This procedure reduces the presence of polar compounds in the dichloromethanic phase, and neutral and apolar compounds in the aqueous phase (J.R. Trigo, pers. obs.). Then the dichloromethanic phase was treated with anhydrous Na2SO4 and the solvent evaporated as above, yielding a dichloromethanic extract, and the aqueous phase was freeze-dried, yielding the aqueous extract.