The osmeterium of B. polydamas archidamas was composed of: i) tubular arms made up by epidermal cells, with an apparent structural function; and ii) columnar ellipsoid glands, located at the base of the dorsal side of each arm, which possess a secretory function. Our results agree with previous general descriptions of the osmeterium for other Papilionidae species, which also describe a glandular patch on the posterior side of each osmeterium arm [13, 14, 17, 19]. Nevertheless, it is highly different from the description given for the osmeterium in Zerynthia polyxena (Papilionidae), in which the ellipsoid glands are opened almost at the apex of each arm and possess a reservoir [34]. The mechanisms of eversion and retraction of the osmeterium in B. polydamas archidamas is dependent on the hemolymph and the longitudinal muscles connecting the larva abdomen with the apex of the osmeterium. Upon disturbance, larvae curl up the body, increasing the hydrostatic pressure inside of it. Thereby, the abdominal musculature is contracted and the hemolymph pushes out the osmeterium against the prothorax cavity, which leads to its eversion. When returning to its original position, the same muscles are contracted, leading to the retraction of the osmeterium.
Epidermal cells in the tubular arms lacked specific organelles or structures that may allow the secretion of compounds. Thus, vesicles and ribosomes present there might be part of the lysosome-cytolysome vacuolar apparatus, related to the proper maintenance of cells, such as the conformation of the external surface and osmeterium cuticle, and substitution of old cells for new ones [13, 19, 35, 36]. Furthermore, Martinez et al. [14] suggested that these vesicles likely relate to the coloration of the tubular arms in the osmeterium of Papilio thoas. On the other hand, cells in the ellipsoid glands showed organelles and structures related to secretory activity, which coincides with previous studies in other Papilionidae species, including Graphium agamemnon agamemnon and Papilio thoas, which described the role of the osmeterium in the synthesis of substances [13, 14]. Tracheas and tracheoles were observed running through the space between the cytoplasm and the basement membrane of the ellipsoid gland cells. Similarly, these tracheas have been observed in the osmeterium of Papilio demoleus [36]. Tracheas would enable an efficient exchange of gasses, nutrients and residues. The latter would provide the surrounding cells a large amount of energy supply for the mitochondria, allowing a robust secretory activity [36]. Cuticles were also distinct between both non-secretory and secretory regions. The well composed and flexible cuticle of the tubular arms is likely a morphological adaptation, which allows the right and fast eversion of the osmeterium as well as the exposure of the glandular pore [5, 13]. In contrast, the highly modified cuticle of the ellipsoid glands exhibits several microchannels, which suggests that secreted compounds are released throughout the cuticle.
The osmeterial secretion of B. polydamas archidamas is composed of monoterpenes and sesquiterpenes. The monoterpenes sabinene and ß-pinene and the sesquiterpene (E)-β-caryophyllene were detected in both the osmeterium and the hemolymph, suggesting that the origin of these compounds is not the osmeterium itself and that they are likely a by-product of the hemolymph. These VOCs are toxic [11, 37, 38], and have been previously reported in the osmeterium of more than 20 species from the tribu Papilionini [11, 14, 26] and in two species from the tribu Zerynthiniini [11]. In addition, sabinene and ß-pinene constitute an important part of the volatiles present in Aristolochia chilensis [39], the host plant of B. polydamas archidamas. In contrast to the monoterpenes, most sesquiterpenes (with the exception of (E)-β-caryophyllene) were detected exclusively in the osmeterium, and not in the hemolymph. The latter suggests that they are secreted by the specialized glands located in the osmeterium. Germacrene A, the main compound in the secretion of B. polydamas archidamas, is relatively widespread in nature, probably because it is an important intermediate in the biosynthesis of many sesquiterpenes of the eudesmane- and guaiane-type and structures derived thereof [40]. Furthermore, it is synthesized by defensive glands in numerous insects [3, 41–43] and it has been found in the osmeterial secretions of various Papilio species [11, 20–22]. Germacrene A is an unstable compound that easily isomerizes to more stable compounds. The odd gas chromatographic behavior has been described before [24] and is due to a thermally induced Cope rearrangement resulting in the formation of β-elemene, which takes place partly in the injector and partly in the column [24]. Acidic conditions induce cyclization reactions leading to the formation of α-selinene, β-selinene, and selina-4,11-diene as main products. In the presence of water, the intermediate carbocation formed from germacrene A can also react to give sesquiterpene alcohols such as selin-11-en-4α-ol or neointermedeol [44]. Furthermore, Eisner et al. [45] reported the isolation of β-selinene and selin-11-en-4α-ol from the osmeterial secretion collected from a population of Battus polydamas. It remains unclear if these two compounds are artefacts from the transformation of germacrene A, or if there are differences between the geographically distant populations.
Behavioral assays showed a repellent effect of the osmeterial secretion of B. polydamas archidamas against the predator, L. humile ants. Previous studies have reported a toxic function for the osmeterium either when predators where exposed to the larvae fully displaying the osmeterium or when predators were fed with artificial diets containing single VOCs [23, 26, 46, 47]. Here, we showed additional evidence that the osmeterium-associated chemistry has a direct defensive efficacy against the natural predator. Considering that the hemolymph showed also a repellent effect against ants, our study suggests that B. polydamas archidamas combines different chemical defensive systems, including repellent volatiles in the osmeterium with volatile and non-volatile toxic compounds (i.e., aristolochic acids, [39]) present in the hemolymph. The combination of two defense lines (including toxic hemolymph) has been previously described for Coccinelidae larvae [48, 49], which may synergistically help insects to defend against a wide array of predators (invertebrates and vertebrates).
This study used a multidisciplinary approach, including morphology, structure, chemistry and ecology to describe and understand the mode of action and function of the eversible defensive organ in Papillionidae. Our results lead us to conclude that the osmeterium, besides serving as an aposematic warning for potential enemies, is an efficient chemical defense against natural predators, with its own synthesis of toxic volatiles.