Acta Biológica Colombiana

Tropane alkaloids (TAs) constitute the main secondary metabolites in six tribes of Solanaceae: Datureae, Solandreae, Solaneae, Mandragoreae, Hyoscyaneae, and Physaleae. Scopolamine and atropine are the most distinctive TAs, and they are implicated in the resistance against various herbivore insects (Griffin and Lin 2000; Wink 2003).

Brugmansia suaveolens (Humb. & Bonpl. Ex Willd.) Bercht. & Presl. (Solanaceae: Datureae) is a Neotropical shrub widely distributed around the world as an ornamental plant. The leaves and flowers of B. suaveolens contain several secondary metabolites (Petricevich et al. 2020), with TAs being the most abundant (Freitas et al. 1996). In Southeastern Brazil, the leaves of B. suaveolens are primarily consumed by the larvae of the TA specialist Placidina euryanassa (Felder & Felder, 1860) (Lepidoptera: Nymphalidae). While TAs are effective against generalist herbivores, P. euryanassa incorporates ingested TAs into their cuticles (Freitas et al. 1996). Nonetheless, there is limited research on the interaction between TAs and other insects.

At the Atlantic Forest Reserve of the Serra do Japi, located in the Jundiaí Municipality, São Paulo State, Brazil (23°13’S, 46°58’W), leaves of B. suaveolens serve as a food source for various insects. These include, P. euryanassa, the grasshopper Chromacris speciosa (Thunberg, 1824) (Orthoptera: Rhomaleidae) as well as both the larvae and adults of the leaf-beetle Lema daturaphila Kogan & Goeden (Coleoptera: Chrysomelidae). Insects were collected and kept in plastic boxes and supplied with fresh B. suaveolens leaves under controlled conditions (25º C; 60 % relative humidity). Before chemical analysis, insects were kept starved for 24 h in order to eliminate TAs contained in the food and feces. Rooted cuttings of 10 B. suaveolens genotypes were grown in individual 5 L pots filled with soil and kept in a greenhouse under natural temperature and photoperiod conditions.

TAs from B. suaveolens and insect samples were extracted in EtOH. The solution was acidified with 1N H₂SO₄ and extracted three times with CH₂Cl. The aqueous solution was reduced with Zn dust for 3 h, alkalinized with NH₄OH and eluted ten times with 5 ml CH₂Cl. The residue represented the total alkaloids as free bases. TAs were identified by gas chromatography coupled with mass spectrometry (GC-MS). Before both chemical analyses, samples were derivatized with N-Methyl-N-(trimethylsilyl) trifluoroacetamide (Acros Organics™) to prevent the alkaloid pyrolysis. TAs were quantified by gas chromatography coupled with flame ionization detection (GC-FID) using sparteine (Acros Organics™) as standard.

In this research, we report two new records of insects sequestering TAs from B. suaveolens: the grasshopper C. speciosa and the larvae of the leaf-beetle L. daturaphila. Both insect species are known to feed on the leaves of B. suaveolens and other solanaceous plants (Kogan and Goeden 1970; Turk and Barrera 1977; Schmitt 1988) (Fig. 1a). B. suaveolens contain five TAs being scopolamine the most abundant (Fig. 1c) and (Table 1). We identified four TAs in the extracts of C. speciosa and larvae of L. daturaphila, indicating these insects sequester these alkaloids (Table 1). Total concentration of TAs was 16.34 ± 13.20 mg/g dry weight for B. suaveolens, 11.54 ± 18.21 mg/g dry for L. daturaphila larvae and 16.42 ± 12.06 mg/g dry for C. speciosa. We did not detect TAs in L. daturaphila adults.

Figure 1.: a) Detail of an adult of Chromacris speciosa. b) Larvae and an adult of Lema daturaphila. c) GC chromatogram of the TAs profile of Brugmansia suaveolens leaves.

Table 1.: Tropane alkaloids (TAs) concentration (mg/g dry weight) in the bodies of Lema daturaphila (Coleoptera), Chromacris speciosa (Orthoptera) and leaves of Brugmansia suaveolens (Solanaceae)

Characteristic ions of mass spectra with parent peak in bold. TAs profile of B. suaveolens corresponds to one genotype. Only larvae of L. daturaphila contained TAs.

This is the first report of L. daturaphila and C. speciosa sequestering TAs from B. suaveolens. While TAs are potentially neurotoxic to insects, specialist herbivores have developed tolerance to these compounds. Consequently, the selective advantage of TAs on plant fitness appears to be more significant when plants are attacked by generalist herbivores (Arab and Trigo 2011; Griffin and Lin 2000). In many insects, a substantial portion of accumulated toxic plant secondary metabolites is excreted. However, specialist insects have developed adaptations to sequester these compounds from their host plants and employ them as a defense against their own natural enemies (Ode 2006). Notably, the adults of L. daturaphila did not sequester TAs from the host plant. This discrepancy might be since immature insect stages are more vulnerable to predators than adults. Therefore, the higher accumulation of TAs in their bodies could provide an advantage during this developmental stage.

Other alkaloids sequestered by insects have been found to be effective against natural enemies (Bernays et al. 1977; Hartmann 1999). For instance, TAs sequestered by P. euryanassa have been observed to protect the larvae of this butterfly from avian predators (Arab, A, personal observation). Both the larvae of L. daturaphila and the adults of C. speciosa exhibit moderately aposematic coloration and behavior (Fig. 1a-1b), which could be linked to their unpalatability (Pasteels et al. 1988; Turk and Barrera 1977). Nevertheless, further research is needed to elucidate the specific defensive role of TAs in these insects.