External morphology of the immature stages of Neotropical heliconians: X. Heliconius sara apseudes (Lepidoptera, Nymphalidae, Heliconiinae)

The biology and morphology of the immature stages of Heliconius sara apseudes (Hubner, [1813]) are still little known. External features of the egg, larvae and pupa of H. sara apseudes are described and illustrated, based upon light and scanning electron microscopy. Eggs with smooth carina, first instar larva with scaly setae, and body of second to fifth instars covered with scattered pinnacles distinguish H. sara apseudes from other heliconiine species.

With a broad distribution in Central and South America, most subspecies of H. sara occur in the Amazonian region (Rosser et al., 2012). Heliconius sara apseudes (Hübner, [1813]) is the only subspecies found throughout the South American Atlantic Forest (Emsley, 1963;Holzinger & Holzinger, 1994;Rosser et al., 2012). Recently, Cardoso (2005) and Iserhard et al. (2010) recorded new localities of H. sara apseudes, extending its northern and southern distributions to states of Bahia and Rio Grande do Sul, respectively.
Heliconius sara lays egg clusters at the branch apex of passion-vines (Passifloraceae) and larvae are semigregarious. Five species of host plants are recorded for H. sara apseudes in southeastern Brazil (Beccaloni et al., 2008): Passiflora edulis Sims, P. pentagona Masters, P. rhamnifolia Masters [=P. sidaefolia (M. Roemer)], and P. ovalis Vell. ex M. Roem. Additional field observations made by us indicate that this subspecies also uses P. truncata Regel and P. haematostigma Masters in Santa Catarina and Paraná states, respectively.
Even though resources exist to enable species identification at early stages and to elucidate taxonomic and phylogenetic relationships (e.g. Penz, 1999), the natural history and external tegumentary ultrastructure of immature stages of species of the sara/sapho clade are still poorly documented. Descriptions of immature stages of H. sara are available only at the gross-morphology level for the subspecies occurring in Trinidad and Tobago (Beebe et al., 1960;Fleming, 1960), and few species diagnostic characteristics have been supplied by Brown (1981).
As part of a series of articles focusing on the external morphology of the immature stages of Neotropical heliconiines (Antunes et al., 2002;Kaminski et al., 2002Kaminski et al., , 2008Tavares et al., 2002;Paim et al., 2004;Silva et al., 2006Silva et al., , 2008Barão & Moreira, 2010;Vargas et al., 2014), here we describe and illustrate in detail the early stages of H. sara apseudes based on light and scanning electron microscopy.

MATERIAL AND METHODS
Eggs and larvae were collected from leaves of P. haematostigma in Quatro Barras municipality, Paraná, Brazil. Eggs were placed on moist filter paper in Petri dishes for incubation. After hatching, larvae were grown on branches of P. haematostigma, kept in plastic bottles equipped with a wire support. The bottles were filled with tap water, covered with a fine mesh, and kept under laboratory conditions (25± 1ºC, 14 h light/day). Head capsules were collected after each ecdysis, fixed in Dietrich's fluid, and preserved in 70% ethanol. Additional eggs, larvae and pupae were also similarly fixed and preserved. The examined material was deposited in the collection of the Laboratório de Morfologia e Comportamento de Insetos (LMCI), of the Departamento de Zoologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brazil, under accession numbers 132 and 136.
Aspects of general morphology of immature stages were analyzed from material preserved in ethanol 70%. Head capsules were rehydrated, cleared in a 10% potassium hydroxide solution (KOH), and slide-mounted in glycerin jelly. Drawings were made from preserved specimens, using a reticulated ocular attached to a Leica ® M125. The tegumentary ultrastructure was studied at the Centro de Microscopia Eletrônica (CME) of UFRGS. Specimens were dehydrated in a Bal-Tec ® CPD 030 critical-point dryer, mounted with double-sided tape on metal stubs, coated with gold in a Bal-Tec ® SCD 050 sputter coater, and analyzed on a JEOL ® JSM-6060 scanning electron microscope.
To distinguish larval instars, the greatest width of the head capsule in frontal view was measured with a micrometer scale mounted in the ocular of the stereomicroscope. By using the least-squares method, the resulting data were adjusted to the power function y = ae^bx (Snedecor & Cochran, 1980). The nomenclature of Dell' Erba et al. (2005) is used for eggs, Peterson (1962) for larval body regions, Stehr (1987) for primary chaetotaxy and crochet of prolegs, Kitching (1984) for the setae of the paraproct and of the anal prolegs, Beebe et al. (1960) for the scoli, and Mosher (1916) for the pupa.
Chorion ornamented with vertical (Vr) and horizontal (Hr) smooth carinae (Fig. 1) (Dell'Erba et al., 2005). Vertical carinae twice as thick as horizontal ones (Fig. 5). Carina number varying from 13 to 14 Vr and from 8 to 10 Hr. Beebe et al. (1960) and Brown (1981) found slightly lower values for Hr (6-8 and 7, respectively). Intersections of vertical and horizontal carinae delimit cells; surface of cells rugose close to carinae and smooth medially (Fig. 3). Lower cells (Lc; Fig. 5) tetragonal; upper cells (Uc; Fig. 3) varying from tetragonal to hexagonal. Apical Hr border the micropylar region ( Fig. 2), but do not project over it as found in H. erato phyllis . Micropylar region ornamented with polygonal, rugose cells peripherally, forming the annulus, and smooth cells centrally, forming the rosette, in the center of which are the micropyles (Mp;    Erba et al. (2005) by having the surface of cells rugose close to the carinae and smooth medially. From Heliconius species also studied by these authors, H. sara apseudes is distinguishable by its smooth carinae.
Head with pair of short, stout cephalic scoli (Sc), measuring approximately half height of head; apex of cephalic scoli rounded, with more than one apical seta (Fig.  24). Mouthparts and stemmata (Fig. 21) as in first instar.
As in the first instar, the gross morphology and chaetotaxy of the subsequent instars of H. sara apseudes fit the general pattern described for Heliconiini (Beebe et al., 1960;Antunes et al., 2002;Kaminski et al., 2002Kaminski et al., , 2008Tavares et al., 2002;Paim et al., 2004;Silva et al., 2006Silva et al., , 2008Barão & Moreira, 2010;Vargas et al., 2014). From the second to fifth instars, the coloration of H. sara apseudes differs from those of H. besckei, H. erato phyllis and H. ethilla narcaea, which have the head in varying shades of yellow and the body white with black spots. The multiple setae on apex of Sc and So on T2 projected anteriorly and larger than any other scoli are also unique features of H. sara apseudes larvae (Beebe et al., 1960;Tavares et al., 2002;Kaminski et al., 2002Kaminski et al., , 2008Paim et al., 2004;Silva et al., 2006Silva et al., , 2008Barão & Moreira, 2010;Vargas et al., 2014).
Instar identification. Besides the striking differences in chaetotaxy and coloration between the first and remaining instars, there are insufficient morphological or coloration changes to precisely identify instars of H. sara apseudes. However, they can be accurately distinguished by the size of their head capsule, because head widths of successive instars do not overlap (Tab. I). The following exponential growth equation was adjusted for the five instars: y = 0.0289e^0.533x; n = 59; r = 0.98; p = 0.0001. The mean growth ratio among instars was 1.67, similar to ratios previously reported for other Neotropical heliconiines (Antunes et al., 2002;Kaminski et al., 2002Kaminski et al., , 2008Tavares et al., 2002;Paim et al., 2004;Silva et al., 2006Silva et al., , 2008Barão & Moreira, 2010;Vargas et al., 2014).
Pupa . Yellowish with scattered black markings (Fig. 36); dorsal pairs of golden spots on T1, T3, AB1, and AB2. Beebe et al. (1960) noted the absence of dorsal golden spots on T2 for populations of H. sara in Trinidad.
The gross morphology of the pupa of H. sara apseudes is similar to other Heliconiini, following the pattern proposed by Mosher (1916) for Nymphalidae. The presence of spike-like tubercles on the antennae, thorax and abdomen, well-developed latero-dorsal flanges at least on AB3, and three to five pairs of dorsal golden spots fits the morphological pattern of "Group C" proposed by Beebe et al. (1960). Other species of Heliconius (e.g. H. erato, H. wallacei and H. melpomene) also fit this pattern (Beebe et al., 1960).