Emanations Volume 4

ABSTRACT

Caterpillars are likely to possess defensive adaptations because of their sedentary and vulnerable lifestyle. When studying an insect’s chemical ecology, identifying its food source is important in order to determine if it produces its own chemical defenses or sequesters them from plant sources. Three species of Lepidoptera, Pseudosphinx tetrio, Automeris sp., and an unknown species in the family Limacodidae, were examined due to their warning coloration and/or defensive spines. Their host plants, Himatanthus sucuuba, Croton schomburkii, and Rheedia madruno, respectively, were also collected and studied. Each organism’s chemistry was analyzed using thin-layer chromatography (TLC) and anti-oxidant tests. In addition, biological assays were conducted to test for anti-bacterial properties against Bacillus cereus (gram-positive) and Pseudomonas aeruginosa (gram-negative), anti-fungal properties against Candida albicans, and cytotoxicity using brine shrimp. Results show that Limacodidae appears to acquire the anti-bacterial properties of its host plant, while Automeris sp. does not demonstrate the anti-bacterial activity observed in its host. In contrast, Pseudosphinx tetrio appears to synthesize anti-bacterial chemicals that are lacking in its host. All of the host plants showed anti-fungal activity, but these properties were not present in the larvae. All three Lepidoptera species were found to exhibit cytotoxic properties, although no comparative conclusions can be made because host plant cytotoxicity data is unavailable. This preliminary study defines the interactions and bioactive properties of these insects and host plants so that further analyses and comparisons can be performed that investigate the biochemical cascade.

The relationship between organisms and their food source is a central aspect of chemical ecology. Tracing biochemical cascades can reveal information about chemical function and alteration. Many Lepidoptera species have been found to sequester bioactive compounds from their host plants. While the host plant occasionally can be substituted with a related species, the relationship is often specific. In fact, a larva may spend its entire immature life on a single host individual. As a result, the relationship between larvae and host is easily identifiable. Additionally, because Lepidoptera larvae have limited mobility and are vulnerable to predation, they have developed defensive characteristics to maximize their chances of survival. Defensive spikes and warning coloration serve to identify the larvae to potential predators.

Because of specific physical warning characteristics, three Lepidoptera species were selected in the Esbaran Preserve: Pseudosphinx tetrio, Automeris sp., and an unknown species within the family Limacodidae. The P. tetrio has a black body with yellow bands located equidistantly along the body with a bright red-orange head. The caterpillar makes itself even more conspicuous through its unusual behavior: it flicks its body violently and waves a posterior projection when approached. Automeris sp. has a bright green body with multiple-tipped thorns covering its dorsal side. The Limacodidae species has bright green coloration and smaller thorns at both its posterior and anterior ends.

The host plants of the larvae were also collected. P. tetrio was found to be associated with Himatanthus sucuuba; Automeris sp. with Croton schomburgkii, and the Limacodidae species with Rheedia madruno. Known human applications of the host plants indicate bioactivity. Bark extracts of H. sucuuba are commonly used as an anti-inflammatory and to treat numerous ailments including gastritis, anemia, arthritis, and cancer (de Miranda et. al. 2000). Although a human application of C. Schomburgkii could not be found, Croton lechleri, a closely related plant, has been used traditionally in the Amazon to treat gastric ulcers, gastritis, diarrhea, skin lesions, and insect stings. (Miller, M. J.S. et. al 2000).

In this study, the anti-fungal, anti-bacterial, anti-oxidant, and cytotoxic properties of the insects and their host plants were analyzed and compared to define their chemical relationships.

MATERIALS AND METHODS

Specimens were collected with their natural host plant, with the exception of Automeris sp., which was found on a small tree that had been cut down and trampled. A related species was located and utilized to feed this caterpillar.

In preparing extracts, the caterpillars were dissected into several parts, depending on the species. The P. tetrio caterpillars were dissected to separate the skin, gut, and fat tissue. The Limacodidae caterpillars, because of their small size, relatively few spines, and lack of intestinal content, were only dissected to the extent of removing the skin for extraction. The Automeris’ numerous long spines were trimmed from the body, and extracted separately from the skin. The Automeris’ gut was not extracted, as there was very little intestinal content. Host plants were dried and crushed in preparation for extraction. All extracts were made in ethanol, using macerated plant or insect material, which was allowed to extract for about 24 hours. The extracts were concentrated under a hair dryer before all analytical tests.

The cytotoxic activity of the extracts was determined using the brine shrimp assay described in Meyer et al.1982. One replicate of each extract was tested, each consisting of approximately 30 individual brine shrimp.

The anti-bacterial and anti-fungal assays were performed as described in Aregullin and Rodriguez 2000. Bacteria or yeast were streaked onto nutrient agar media. Sterile disks were impregnated with 20uL of extract, allowed to dry, and placed on the inoculated plates. Plates were incubated for 24 hours at 35 C. The zones of inhibition of Bacillus cereus (gram-positive) (ATCC#11778), Pseudomonas aeruginosa (gram-negative) (ATCC#27853) and Candida albicans (ATCC#90028) were measured in each assay and reported in Figure 1. B. cereus and P. aeruginosa were plated on nutrient agar (Difco 0001), while C. albicans was plated on Sabouraud Dextros Broth (Difco 0382).

Chemical analysis was performed on Lepidoptera extracts using thin-layer chromatography (TLC) on aluminum-backed plates coated with silica gel. All solvent systems consisted of a 3:1 mixture of methanol to ethyl acetate. A 2,2-diphenyl-1-picrylhydrazil spray reagent was applied to a developed TLC plate to test for antioxidants. Upon contact with an antioxidant compound, this purple reagent turned yellow.

RESULTS

The P. tetrio skin extract displayed anti-bacterial activity against both gram-positive and gram-negative strains. However, no anti-fungal activity was observed. Both the skin and the fat extracts displayed 100% mortality in the cytotoxicity experiment. Its host plant, Himatanthus sucuuba, showed anti-fungal activity but no anti-bacterial behavior. Host plant cytotoxicity was not determined. The TLC analysis of the skin extract revealed chemicals at Rf values of 0.3-0.49 (red) and between 0-0.6. The skin also had anti-oxidant compounds at Rf ranges between 0-0.6, 0.27-0.36, and 0.43-0.52 on the TLC plate. The fat extract revealed chemical spots at Rf values of 0.68, 0.72, 0.76 and 0.79. Antioxidant compounds was found between 0.71-0.79.

The Automeris sp. and its host, Croton schomburkii, also showed biochemical activity. Neither the larva’s spines nor the skin revealed any anti-bacterial or anti-fungal properties, but the spines displayed 94% mortality in the cytotoxicity study. Its host plant showed anti-fungal and anti-bacterial activity. Its cytotoxicity was not determined. The TLC analysis of the spine extract showed a well-defined spot at an Rf value of 0.46, and the skin showed spots at 0.16 and 0.4. Antioxidant chemicals appeared between 0.37 and 0.55 in the skin and at 0.52 in the spines.

The Limacodidae (species unknown) revealed no anti-fungal activity, but did exhibit anti-bacterial activity for both gram-postive and gram-negative bacteria as well as strong cytoxicity (100% mortality). The host plant, Rheedia madruno, had anti-fungal and anti-bacterial activity, and demonstrated 91% mortality in the cytotoxicity assay. The TLC analysis revealed one compound at an Rf value of 0.41 and anti-oxidant activity was found at a range between 0.38-0.42.