This study reports on the embryogenesis of Heliconius erato phyllis between blastoderm formation and the prehatching larval stage. Syncytial blastoderm formation occurred approximately 2 h after egg laying (AEL) and at about 4 h, the cellular blastoderm was formed. The germ band arose from the entire length of the blastoderm, and rapidly became compacted occupying approximately two-thirds of the egg length. At about 7 h AEL, protocephalon and protocorm differentiation occurred. Continued proliferation of the germ band was followed by penetration into the yolk mass, forming a C-shaped embryo at about 10 h. Approximately 12 h AEL, the gnathal, thoracic and abdominal segments became visible. The primordium of the mouthparts and thoracic legs formed as paired evaginations, while the prolegs formed as paired lobes. At about 30 h, the embryo reversed dorsoventrally. Approximately 32 h AEL, the protocephalon and gnathal segments fused, shifting the relative position of the rudimentary appendages in this region. At about 52 h, the embryo was U-shaped in lateral view and at approximately 56 h, the bristles began evagination from the larval cuticle. Larvae hatched at about 72 h. We found that H. erato phyllis followed an embryonic pattern consistent with long-germ embryogenesis. Thus, we believe that H. erato phyllis should be classified as a long-germ lepidopteran. The study of H. erato phyllis embryogenesis provided a structural glimpse into the morphogenetic events that occur in the Heliconius egg period. This study could help future molecular approaches to understanding the evolution of Heliconius development.
Studying the development of different model animals is important for understanding how differences in developmental programs create diversity, either in whole-body plans or in structural details. In insects, segmentation mechanisms have been heavily studied (Sander 1984, 1988; Patel 1994; Davis & Patel 2002; Liu & Kaufman 2005; Nakao 2012). However, earlier key stages, such as blastoderm formation, are still poorly studied and have only recently begun to be surveyed (Nagy et al. 1994; Ho et al. 1997; Masci & Monteiro 2005). Data from embryological studies on insects are relevant for comparative embryogenesis and for understanding insect body plan evolution.
Insect embryos have been classified by segmentation as short-germ, intermediate-germ or long-germ embryos. In the short-germ and intermediate-germ embryos, posterior segments are generated sequentially from an uncommitted growth zone. In long-germ embryos, all segments are specified simultaneously from cells covering the entire blastoderm. The difference between the short-germ, intermediate-germ and long-germ modes is considered to be a case of developmental heterochrony, since timing shifts in the process of segmentation and patterning are involved in the generation of different phenotypes (Davis & Patel 2002). In general, ancestral insects (e.g., grasshoppers) show short-germ embryogenesis and more derived insects (e.g., Drosophila) show long-germ embryogenesis. Historically, lepidopteran embryos are classified as intermediate-germ (Anderson 1972); however, closer inspection of different species shows that multiple germ types exist in this order (Kraft & Jäckle 1994; Masci & Monteiro 2005; Nakao 2012). Manduca sexta and Bombyx mori have the most studied lepidopteran embryonic development. M. sexta represents intermediate-germ embryos and B. mori represents long-germ embryos (Kraft & Jäckle 1994; Nakao 2010).
The neotropical genus Heliconius has been used for research in ecology, evolution and behavior (Benson 1972; Mallet & Barton 1989; Jiggins et al. 2001). Diversification of color patterns in the genus is known for intraspecific divergence and interspecific convergence and has been the focus of evo-devo studies (Joron et al. 2006; Reed et al. 2011; Hines et al. 2012; Aymone et al. 2013; Hill et al. 2013).
In addition to wing color pattern, Heliconius species show variation in egg morphology (Dell'Erba et al. 2005) and in single or cluster oviposition modes (Brown 1981). The Heliconius egg is subcylindrical and flattened at the base. The micropylar axis (an imaginary line between the anterior and posterior poles) is perpendicular to the substrate (Beebe et al. 1960). After oviposition, the anterior pole has a superior position and the posterior pole has an inferior position (Hinton 1981). The egg chorion has longitudinal and transversal ribs that delimit rectangular cells (Dell'Erba et al. 2005). Although the external morphology of Heliconius eggs is well known, aspects of embryo morphogenesis are unknown for this group of butterflies.
This study examined the eggs of a Heliconius species. Our objective was to describe the embryogenesis of Heliconius erato phyllis from blastoderm formation to prehatching larvae. We also described features of the yolk and serosa cells, which are functionally integrated into the egg structure. The analysis of H. erato phyllis embryogenesis is important not only for comparative studies within lepidopterans, but also for future molecular approaches to understanding the evolution of Heliconius development.
Materials and methods
Egg-laying butterflies were captured in natural populations around the city of Porto Alegre (30°01′59″S, 51°13′48″W), State of Rio Grande do Sul, Southern Brazil, and transferred to open air insectaries at the Genetics Department of the Federal University of Rio Grande do Sul. Each insectary unit was 2 × 3 × 3 m with seminatural conditions and host plants, mainly Passiflora misera. Butterflies were fed daily with a mixture of honey, water and pollen that was refreshed each morning. After oviposition, eggs were collected, brought to the laboratory, and transferred to Petri dishes with humid filter paper kept at 25 ± 1°C and 14 h/light per day photoperiod.
Blastoderm formation was observed using a protocol developed by Masci & Monteiro (2005) for visualizing Bicyclus anynana embryos. At time intervals of 1 h including the moment of oviposition, until 10 h, eggs were fixed in Carnoy (n = 10/collection point). The egg chorion was removed and dechorionated eggs were transferred to 75% ethanol/25% phosphate-buffered saline (PBS), rehydrated in an ethanol series with PBS, stained with Hoechst 33258 (Molecular Probes) and observed under a spectral confocal microscope (Olympus FV1000) with dye excitability in the spectral range of 405 nm.
Light microscopy and scanning electron microscopy (SEM) were used to observe embryogenesis. For light microscopy, eggs were fixed in Bouin's solution at time intervals of 1 h until prehatching stage (n = 20/collection point). Because the H. erato phyllis chorion was opaque and impermeable, it was removed to allow observation of tissues within the egg. Previous attempts to permeabilize or make the chorion translucent using chemicals were unsuccessful (data not shown). Egg preparation was followed by dehydrating with an ethanol series, diaphanizing, paraffin embedding, microtomy to 3 μm, and staining with hematoxylin and eosin or toluidine blue.
For SEM, eggs (n = 20/collection point) were fixed in glutaraldehyde. Embryos were isolated from the yolk mass, washed in PBS buffer, dehydrated in a ketonic series, dried in a critical point dryer (BALTEC CPD-030), mounted on metal stubs with double-sided carbon tape, metalized with approximately 15 nm of gold (BAL-TEC SCD-050) and observed by SEM (JEOL JSM-6060), at 10 kV.
Serosa cells and vitellophages were observed under a transmission electron microscope (JEM 1200EXII). Eggs were previously fixed in Karnowisky, postfixed in osmium tetroxide 2%, washed in PBS buffer, dehydrated in a ketonic series, embedded and included in resin (EMBED Epon 812). Ultramicrotomy was performed in LEICA ULTRACUT UC7 ultramicrotome, with 100 nm sections, which were contrasted with uranyl acetate and lead citrate. Figure representations were by technique rather than chronological order.
The general external morphology of H. erato phyllis eggs is in Figure 1A. The egg period was around 72 h under controlled conditions. Below, we describe the main events throughout embryonic development.
The blastoderm was visualized by confocal microscopy of the egg periphery approximately 2 h after egg laying (AEL). Staining with Hoechst showed intense labeling of cleavage nuclei (Fig. 1B). H. erato phyllis followed the same meroistic pattern found in most holometabolous insects with a syncytial blastoderm initially formed. The syncytial blastoderm lacked membranes and all cleavage nuclei were contained in a common cytoplasm. Approximately 4 h AEL, membranes started to develop around each nucleus, creating individual cells that formed the cellular blastoderm (Fig. 1C).
Yolk and vitellophages
At the moment of blastoderm formation, cleavage nuclei that did not migrate to the periphery began yolk cellularization, forming vitellophages (Fig. 2A–C), at about 4 h AEL. Vitellophages had one or two nuclei, which were indented (Fig. 2C, E). Some nuclei were densely packed in a mass of cytoplasm (Fig. 2D) with a large amount of Golgi (Fig. 2F), secretory vesicles (Fig. 2G), glycogen granules (Fig. 2H), and mitochondria (Fig. 2I). The cytoplasmic mass was surrounded by carbohydrates and lipid droplets.
Formation of the germ band
As division of the blastoderm continued, the ventral region thickened and became the embryonic primordium (Figs 1D, 3A–B), at about 5 h AEL. Cells fated to form the germ band arose from the entire length of the blastoderm (Figs 1D, 4A). Approximately 7 h AEL, the germ band became compacted occupying approximately two-thirds of the egg length. Differentiation of the protocephalon and protocorm occurred. The protocephalon was the most prominent region at this stage and became large and bilobed (Fig. 4B). Continued proliferation of the germ band (Fig. 4C) was followed by separation from the extra-embryonic ectoderm and penetration into the interior of the yolk mass at about 9 h AEL. The anterior and posterior ends of the germ band grew dorsally into the yolk with the anterior end forming the stomodeum and the posterior end forming the proctodeum. The germ band elongated antero-posteriorly, assuming a C-shape (Fig. 3C) at about 10 h AEL.
Separation of the germ band and extra-embryonic ectoderm constituted the serosa (Figs 3D, 5A–C). Serosa cells of H. erato phyllis were mononucleated in most cases (Fig. 5A) with the cytoplasm full of mithochondria and prominent Golgi complexes that were concentrically structured, indicating intense secretory activity (Fig. 5B, C). At serosa formation, the amnion formed, enclosing the embryo in a fluid-filled amniotic cavity (Fig. 3D). Formation of the amnion separated the germ band from the extraembryonic ectoderm and allowed blastokinesis.
At approximately 12 h AEL, segmental grooves became visible along the entire embryo, constituting the gnathal, thoracic, and abdominal segments (Fig. 3C, E, F). The gnathal segments corresponded to the labral, mandibular, maxillary and labial appendages (Fig. 6A, B). The abdominal region divided into 10 segments (Fig. 3E). The most posterior region of the embryo corresponded to the telson, a nonsegmental terminal region (Figs 3E, 6D, H). At about 16 h AEL, the primordium of the mouthparts and thoracic legs formed as paired evaginations (Fig. 6A, B), while the prolegs formed in the abdominal segments as paired lobes (Fig. 6A). At segmentation, the central nervous system (brain, suboesophageal, thoracic and abdominal ganglions) originated from the ectodermal cells of the embryonic ventral region (Fig. 7A–D).
Embryo revolution and dorsal closure
At approximately 30 h AEL, the embryo became wide and broad and reversed dorsoventrally, changing the relative position of the yolk (Figs 3G–I, 4D). Earlier, the embryo was positioned within the yolk, but after revolution at 42 h AEL, the yolk was contained within the embryo. At approximately 32 h AEL, the abdominal end turned ventrally, reaching the level of the ventral abdomen (Fig. 6D). The embryo assumed a J-shape in lateral view. At about 48 h AEL, the abdominal end reached the level of the thorax and the cephalognathal region turned ventrally (Fig. 6E). At the abdominal end, the tenth abdominal segment and the telson fused (Fig. 6H). At about 52 h AEL, the embryo assumed an approximate U-shape in lateral view. The yolk mass gradually decreased with consumption by the embryo (Fig. 3G–I).
After embryo revolution, the lateral walls of the embryo grew dorsally to replace the amnion, which was provisionally covering the embryo dorsal surface. At about 54 h, dorsal closure occurred. At approximately 56 h AEL, bristles began evaginating from the larval cuticle (Fig. 6I, J).
Gut development occurred throughout the embryo revolution (Fig. 3G–J). Cells at the anterior and posterior ends of the C-shaped embryo migrated inward to form the foregut, hindgut, and midgut at about 32 h AEL. The foregut arose from ectodermal cells of the stomodeum and the hindgut from the proctodeum. Endodermal cells derived from the stomodeum and proctodeum enclosed the yolk in a tube creating the midgut (Fig. 3G–J). Vitellophages enclosed by the midgut integrated into the endoderm and formed the midgut epithelium (Fig. 3H–J). The elongating hindgut subdivided into the small intestine, large intestine, and rectum, each containing cells of different sizes (Fig. 3I). Shortly before hatching, at about 70 h AEL, the blind ends of the foregut and hindgut opened and the continuity of the digestive tract was established (not shown).
Formation of the head, thoracic and abdominal appendages
After embryos widen and broadened (Fig. 3E, 6A) at 30 h AEL, the protocephalon and gnathal segments began to fuse and morphogenetic movement of these regions began so the relative position of the rudimentary appendages in the region shifted to form the larval head. At approximately 32 h AEL, labral rudiments fused and the proximal part of each labial rudiment moved toward the median line and fused to a single structure (Fig. 6C). The fused labial rudiment moved anteriorly between the maxillary rudiments (Fig. 6F) at about 48 h. At this stage, the thoracic legs became cylindrical and grew antero-posteriorly, but were not yet segmented (Fig. 6G) as were the prolegs on the third to sixth abdominal segments (Fig. 6H).
At about 56 h AEL, the labrum and mandibles became thin and the incisor region of the mandibles formed, while the labium and maxillas became sharp and segmented (Fig. 6J). The antennae became segmented and projected multiple sensillae. Six stemmata arose beside each antenna (Fig. 6J). At this stage, the thoracic legs and prolegs were fully segmented and the tarsal claw on the leg tips achieved its final shape (Fig. 6K) and crochets on the proleg plantae appeared (Fig. 6L).
Before hatching at about 70 h AEL, the definite form of the head capsule and mouthparts were established (Fig. 6M). At first, the mandibles were sclerotized and began abduction-adduction movements. At approximately 72 h AEL, the embryo ingested the fragmented embryonic membranes. The anterior pole of the chorion was ruptured by gnawing with mandibles and the larvae hatched.
Microscopy study of H. erato phyllis embryogenesis provided structural insights into the morphogenetic changes that occur during its development. Starting with blastoderm formation, H. erato phyllis showed the same pattern as B. mori (Nagy et al. 1994) and Bicyclus anynana (Masci & Monteiro 2005), which first form a syncytial blastoderm followed by cellularization of the cleavage nuclei. In Lepidoptera, blastoderm formation starts at the center of the egg cytoplasm where the zygote nucleus undergoes meroblastic cleavages. After a series of mitosis events, nuclei migrate to the egg periphery in cytoplasm islands and continue to divide (Kobayashi et al. 2003). Cleavage nuclei that do not migrate to the periphery form vitellophages with one or two nuclei. Some nuclei are densely packed in a dense mass of cytoplasm, possibly because of a high quantity of lipid droplets and carbohydrates from the yolk.
We believe that H. erato phyllis should be classified as a long-germ lepidopteran. The current insect embryology literature indicates three main types of embryos with long-germ, intermediate-germ, or short-germ bands. The characteristics of long-germ embryos include a large germ band within the egg, relatively short embryogenesis, and inability to regulate embryogenesis in response to environmental perturbation (Davis & Patel 2002). Derived lepidopteran species such as B. anynana and M. sexta follow this developmental pattern (Kraft & Jäckle 1994; Masci & Monteiro 2005). More ancestral lepidopteran species, however, have shorter germ bands, and a longer embryogenesis with an ability to respond to environmental changes during embryogenesis. The fate of each cell in the short-germ embryos is believed to be determined later in development, allowing for adjustment in cell fates (Nagy 1995; Davis & Patel 2002). Another feature that distinguishes short-germ and long-germ embryos is segmentation pattern. In many long-germ insects (e.g., Drosophila) all segments are specified almost simultaneously within the blastoderm. In short-germ insects, only head segments are specified in the blastoderm with remaining segments of the thorax and abdomen forming progressively from a posterior growth zone (Davis & Patel 2002). Although complete segmentation of H. erato phyllis was not visible in the blastoderm immediately, at about 12 h AEL, segmental grooves were present along the antero-posterior axis, suggesting long-germ embryogenesis.
The size variation of the H. erato phyllis germ band during the first 10 h of development was different from variation in B. mori. In B. mori, germ band size is initially large, occupying most of the diameter and almost the entire length of the egg. However, the germ band soon contracts to only half the egg length for a short-germ appearance and subsequently undergoes an elongation phase (Nagy et al. 1994). During this phase, segmental grooves are formed, first in the gnathal and thoracic regions and then sequentially in the abdomen. Based on these observations and molecular data, B. mori is considered an evolutionarily intermediate state in the transition from short-germ to long-germ type (Nakao 2010). In H. erato phyllis, the early germ band did not have a large diameter as in B. mori; the band was slender but occupied the entire egg length, indicating that it arose from the entire length of the blastoderm. Following compaction, the H. erato phyllis germ band occupied approximately two-thirds of the egg length; thus, it did not reduce as much as the band in B. mori.
Bombyx mori embryogenesis resembles M. sexta embryogenesis in some aspects. In M. sexta, segmental grooves first appear in the gnathal-thoracic region followed by the sequential appearance of abdominal segments (Broadie et al. 1991). Despite this intermediate-germ feature, molecular data for M. sexta show a genetic prepattern simultaneously existing for all segments. Therefore, M. sexta is considered a long-germ lepidopteran (Kraft & Jäckle 1994). Both M. sexta and H. erato phyllis undergo a phase in which only the head lobes and a nonsegmented terminus are apparent; at this stage, no signs of morphological segmentation were apparent.
In Lepidoptera, three modes of embryonic membrane formation (serosa and amnion) are known: invaginate, amnioserosal fold, and fault types (Kobayashi et al. 2003). The invaginate type is found in Neomicropteryx (Zeugloptera) and Endoclyta (Exoporia), and is thought to be the most ancestral type. In these genera, a relatively short germ band invaginates into the yolk forming a vesicular germ band; extraembryonic ectoderm closes above it, forming the serosa. One layer of the vesicular germ band differentiates into the germ band and the other layer into the amnion. The vesicular lumen represents the amniotic cavity (Ando & Tanaka 1980; Kobayashi & Ando 1982). The amnioserosal fold type is found only in Eriocrania (Dacnonypha), whose embryonic membranes are formed by invagination of the anterior and posterior regions of the germ band, which form folds that extend to the ventral midline and merge to form the amnion and serosa (Kobayashi & Ando 1987). The fault type is known only in ditrysians and the monotrysian Stigmella. In this type, the germ band is cut off from the extraembryonic ectoderm and the serosa forms by cell proliferation; the amnion forms independently by extension of the germ band edges. This mode of ontogeny of embryonic membranes is considered an apomorphic characteristic of Heteroneura (Kobayashi 1996). In H. erato phyllis, the separation between germ band and extraembryonic ectoderm and the consequent formation of the serosa and amnion followed the fault-type pattern.
The serosa is crucial for the secretion of the egg cuticle and the amnion is critical for dorsal closure of the embryo. The serosa cuticle, which is deposited beneath the chorion, is generally assumed to be a protection for the embryo. This is plausible when the chorion is thin or can rupture during embryogenesis. In Lepidoptera, however, the chorion is sturdy and might not need the support of the relatively thin serosal cuticle (Larink 1972). Nonetheless, Lamer & Dorn (2001) used electron microscopy to study the structure and function of the serosa throughout M. sexta embryonic development, revealing that the serosa cells in this species show intense cuticle secretion and might adopt several sequential functions according to changing embryonic needs. Features of the serosa cell cytoplasm in M. sexta include a high quantity of concentrically structured Golgi complexes; these were also found in H. erato phyllis serosa cells. Therefore, we propose that the serosa of H. erato phyllis is involved in cuticle secretion, despite the egg chorion stiffness.
Serosal cells of long-germ eggs tend to be mononucleated, in contrast to the multinucleated serosal cells of short-germ species (Wall 1973; Kobayashi & Ando 1984). The H. erato phyllis serosa cells were mostly mononucleated, suggesting the phylogenetic trend of long-germ embryos.
During embryonic development, Insecta have 11 abdominal segments, excluding the telson. The 11th segment tends to be greatly reduced or lacking, especially in holometabolous insects. The absence of the 11th abdominal segment is considered an apomorphic character. In Lepidoptera larvae, this segment is absent. However, the embryos of Neomicropteryx and Endoclyta temporarily have an 11th segment that later disappears (Tanaka 1993). In H. erato phyllis embryos, the 11th segment did not appear, which was phylogenetically consistent with Heteroneura, which lacks this segment (Kobayashi & Ando 1987).
Heliconius erato phyllis followed the same pattern of morphogenetic movements involved in the formation of the head, thoracic and abdominal appendages observed in M. sexta appendages (Dow et al. 1988). H. erato phyllis developed relatively quickly, completing embryogenesis at about 72 h AEL. Developmental times are about 96–120 h for B. anynana (Masci & Monteiro 2005) and 100 h for M. sexta (Kraft & Jäckle 1994). Time of egg stages is another feature that distinguishes short-germ and long-germ insects (Davis & Patel 2002).
Short-germ and intermediate-germ embryos are widely found in various insect orders, while long-germ embryos are mostly restricted to orders using nurse cells during oogenesis (e.g., Lepidoptera and Diptera). Nurse cells allow an increased and spatially polarized maternal contribution to the developing oocyte; this evolutionary innovation might have been an important precondition for rapid development or a product of selection (Davis & Patel 2002). Therefore, the provisioning of maternal information by nurse cells might have been a precondition to the evolution of long-germ embryogenesis.
Closer inspection of the phylogenetic distribution of germ types reveals, however, that the long-germ type is more closely correlated to holometaboly, since the long-germ type is found in multiple clades in Holometabola (Davis & Patel 2002). This suggests either that long-germ embryogenesis was secondarily lost in orders with representatives of multiple germ types, such as Lepidoptera, or it evolved more than once.
This paper showed that H. erato phyllis, a derived species whose embryogenesis has not been studied, appears to be a long-germ band lepidopteran. The analysis of H. erato phyllis embryonic development from blastoderm formation to prehatching larvae, provided morphological information about Heliconius embryogenesis. Furthermore, this study could help future molecular approaches to understanding the evolution of Heliconius development.
We thank our colleagues at the Ecological Genetics Laboratory for their help with collecting butterflies and daily feeding of caterpillars. Electron and confocal microscopy were performed at the Electronic Microscopy Center from the Federal University of Rio Grande do Sul, Porto Alegre, Brazil. Thanks also to the Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, for financial support and for a doctoral scholarship to the first author.