Immature Stages of the Neotropical Butterfly, Dynamine agacles agacles

The external morphology of the immature stages of Dynamine agacles agacles (Dalman, 1823) (Lepidoptera: Nymphalidae: Biblidinae) is described, including photos, drawings, and scanning electron micrographs. Data on the adult and larval behavior are given based on observations in the host plant Dalechampia triphylla Lam. (Malpighiales : Euphorbiaceae). The results are compared and discussed with other described species of Biblidinae, allowing to make further observations on the natural history and evolution of Dynamine.


Introduction
Biblidinae is a well defined clade of Nymphalidae, and their members are distributed primarily in the Neotropical region, with some species also present in the Paleotropics (Munroe 1949;Van Son 1979;Ackery 1988). Besides being conspicuous and well represented in most Neotropical biomes, many Biblidinae genera are still poorly known, with early stages described for just a small fraction of taxa in the larger genera (Freitas et al. 1997; Barbosa et al. 2010).
Considering the importance of early stages as sources of characters for establishing phylogenetic hypothesis and for taxonomic studies in higher Lepidoptera taxa (Freitas et al. 1997;Greeney and Gerardo 2001;Hasenfuss and Kristensen 2003;Casagrande and Mielke 2005;Souza et al. 2006;Freitas andBrown Jr. 2004, 2008;Dias et al. 2010aDias et al. , 2010b, the present study has the objective of presenting detailed morphological and behavioral descriptions of the immature stages of Dynamine agacles agacles, aiming to contribute to the knowledge of Neotropical Biblidinae.

Materials and Methods
Adults and immatures were studied in three localities in Brazil: (1)  Immatures for morphological analysis (at least three of each stage) were killed in boiling water, fixed in Kahle-Dietrich solution, and after three days transferred to 70% ethanol. Shed head capsules and exuviae were collected and preserved dry for measurements. Measurements, drawings, and general aspects of morphology were done in a Leica® MZ16 stereomicroscope equipped with a micrometric scale. Head capsule width of larvae was the distance between the most external stemmata; maximum total length for both larvae and pupae corresponded to the distance from the head to the posterior margin of the tenth abdominal segment in dorsal view (as in Freitas 2007).
Scanning electron microscopy (SEM) was conducted using a Jeol® JSM-6360LV microscope (www.jeol.com). Samples were prepared in accordance with the following protocol: critical point dried in a Bal-tec® CPD030 Critical Point Dryer (www.precisionballs.com) and attached with double stick tape to aluminum stubs; gold/palladium coated with a Bal-tec® SCD030 Sputter Coater.

Results
Egg 28,29). Diameter 0.6 mm, height 0.5 mm (n = 14). Coloration uniformly greenish yellow; truncate, flatten in the base, with maximum diameter in the basal third, progressively narrowing toward the convex micropilar region in the apex; decorated with 14 vertical ribs forming prominent keels, and ca. 16-18 horizontal ribs visible only when crossing the vertical keels. Duration 7-8 days (n = 7).

Fifth (last) instar
Pupa (25)(26)(27)(44)(45)(46)(47)(48)(49). 10 mm long. Duration 7 days (n = 6). Adecticous obtect, suspended by the cremaster. Initially dark green with light ventral region, changing to dark green or brown after one day, with a narrow cream dorso-longitudinal line from T1 to T3 and a narrow dark green ventral stripe from A4 to A8; profile elongated, with two conspicuous dorsal indentations, in T2 and A2, the abdominal indentation slightly forked. Head with two small projections on vertex; frons smooth; clypeus quadrangular; tentorial pit conspicuous dorso-lateral to the clypeus; labrum pentagonal and small; galea elongated from the labrum to the margin of the forewing cap; antennae with several transverse stripes from dorso-posterior head to distal galea; eye caps oval and lateral to anterior third of antenna, covering most of anterior head. Pronotum subrectangular and small; mesonotum large, with dorsal longitudinal ridge projecting as a point in the inferior portion; thoracic spiracle evident, lateral to the antenna in the T1-T2 intersegmental region; metanotum with antero-median margin convex. Prothoracic legs two-thirds the length of mesothoracic legs, arising adjacent to the anterior portion of eye cap and ending about half the galea length; mesothoracic legs lateral to the antennae, from the anterior third of the last until the posterior third of galea. Abdomen with antero-dorsal projections spined from A3 to A7; ellipsoidal spiracles from A2 to A8, the last two smaller than the others. Cremaster dark green, with simple dark brown distal hooks.

Adult and larval behavior
In the three study sites, the only recorded host plant was Dalechampia triphylla Lam. (Malpighiales: Euphorbiaceae), a scandent vine common in clearings and forest edges. Females were observed flying over only on plants bearing developed inflorescences. Females typically lay single eggs, in general on the involucral bract (either inside or outside the inflorescence), but eggs were also observed in other floral parts. Larvae eat part of the eggshell after hatching, and then feed preferentially on floral parts inside the inflorescences, including ovary, styles, pollen, the viscose resin, and even on the involucral bracts. Eggs were observed especially on flower buds, and the development of the larvae is closely linked with flower maturation, with no serious damage caused until the larva is fully-grown. At this time, the seeds are being formed, and the larva feeds on these until pupation.
In the first three instars, larvae remain inconspicuous nearby the floral parts, both due to their cryptic coloration and also due to pollen adherence in the setae and scolli tips. The greenish coloration after the fourth instar improves camouflaging with nearby fruits and seeds, where larvae usually rest in a semicircular posture.
In the laboratory, pupation was observed in secondary branches of D. triphylla, and pupae were observed suspended by the cremaster in a silk base made by the larva. Prepupa lasts about 24 hours, with larvae remaining suspended by the anal prolegs in a semicircular posture with head almost touching segment A6. Pupa remains motionless, but the abdominal segments are mobile, and can wriggle if the pupa is disturbed. Adults emerge through an opening in the dorsal region near the head, expelling a reddish brown meconium.

Morphology of immature stages
The eggs of D. agacles are similar in morphology with those of most Biblidinae species, being truncate and with well-marked vertical ribs (Freitas and Oliveira 1992;Freitas et al. 1997;Greeney and Gerardo 2001). Most eggs of Biblidinae share this general pattern, with notable exceptions observed in the tribe Biblidini and Eurytelini (sensu Lamas 2004), which present unique "pilose" eggs (Johnston and Johnston 1980;Larsen 1991;Van Son 1979;Freitas and Brown Jr. 2008).
The first instar is characteristic within the Biblidinae in having relatively long body setae (ratio between setal length/segment height about 1.0), a feature also shared with all known Biblidini and Eurytelini, and with the genera Cybdelis Boisduval, 1836 and Sea Hayward, 1950, though the setae in the last two genera are somewhat shorter (ratio about 0.7) (Freitas et al. 1997). First instar of D. agacles has less than eight crochets on the prolegs; this character is shared with D. postverta (Cramer, 1779) (Freitas and Brown Jr. 2004). The only other Nymphalid genus observed with less than eight crochets on the prolegs of first instar is Hypanartia Hübner, (1821) (Nymphalinae) (Freitas and Brown Jr. 2004).
As described here, body scoli of D. agacles bear small viscose vesicles on the tip of the spines of the apical rosette. This unique structure of body scoli was first noted by Müller (1886) in D. postverta, and is also a possible synapomorphy of the genus Dynamine. However, the nature and function of this viscose substance is unknown and deserves further investigation.
Feeding on inflorescences probably resulted in various modifications of the larvae, deviating from the general Biblidinae pattern, such as the lack of head scoli, reduction in body scoli length, and sluglike appearance. All these traits are probably adaptations to facilitate movement and feeding inside buds and flower parts. Additionally, the small size of most species of Dynamine (compared with remaining Biblidinae) could be related to use of a limiting larval resource (in this case inflorescences). As proposed by Thompson (1983), species feeding on small plant parts could be potentially smaller than their relatives, particularly those that feed internally on plant tissues being, therefore, unable to move easily between individual host plants; this hypothesis warrants investigation. By placing natural history information and morphological traits (including size) in a phylogenetic framework, we could attempt to understand the evolution of such life history traits in Dynamine butterflies in particular, and the association between body size and specialized feeding habits in general.