The embryonic development of the central American wandering spider Cupiennius salei

Embryonic stages

The numbered stages into which we divide development in C. salei are intended to replace the existing hAEL staging system by Seitz [26]. Besides a number, each stage is also given a colloquial name for practical use. Figure 1 gives a general overview of our new system, and additional file 1 provides a detailed comparison between the old and new systems. The number of stages allocated to early development (up to stage 6) is higher in the Seitz system, where the early stages are separated into numerous time (hour) intervals after egg laying (hAEL). The resolution we chose for the early stages reflects the degree of detail we were able to observe using our methods, and follows the staging nomenclature of A. tepidariorum[25] until stage 9 (prosomal limb buds). From stage 6 to 10, the staging resolution of the two systems is similar, and after stage 10 our new system is more detailed (additional file 1).

Stage 1, Early cleavages

During the process of egg-laying, the female spider produces a liquid secretion that guides the soft and ovoid-shaped eggs from the genital opening into a silk pouch that is then formed by the female into a round cocoon [7]. The liquid secretion is absorbed by the eggs, which as a result increase in size, become more solid and take on a spherical shape of roughly 1.2 mm in diameter. During these first hours of development the eggs are sticky and fragile. Because of these conditions, we waited 12 to 24 hours before it was possible to remove intact single eggs for further investigation. Eggs at stage 1 largely consist of yolk, which is distributed as fine homogeneous granules. In these early stages, the nuclei are surrounded by a mass of yolk and not yet enclosed within cell membranes. The first cycles of nuclear division are superficial (nuclear mitosis without cytokinesis which results in a polynuclear cell). The divisions take place intralecithally in the centre of the egg (Figures 2a, b). Based upon earlier observations [26] we assume that the first cleavage cycles are synchronous. During these cleavages the nuclei start to migrate towards the egg surface. This migration results in an egg with an even distribution of nuclei (Figure 2c).

Stage 4, Primary thickening

In the egg hemisphere that contains the advanced blastopore, scattered divisions result in an uneven distribution of the blastodermic cells. As a result, this half of the egg becomes more patchy (white arrows; Figure 3a). The blastodermic cells in the opposite egg hemisphere are evenly distributed though they appear to be fewer in number (Figure 3b). The blastopore region displays high nuclear density with the nuclei arranged in several layers. The pore-like composition of the blastopore disappears (Figure 3a). This indicates the end of gastrulation, i.e., the inward migration of cells at the blastopore. As development proceeds, the cellular tissue of the region where the blastopore formed becomes thicker and appears to bulge outwards (visible around movie frame 700 of Additional file 2). This conspicuous structure is known as the primary thickening, or cumulus anterior [25, 31, 32].

Stage 5, Cumulus migration

From the primary thickening (formerly the blastopore) clusters of internalised cells migrate in radial directions. Two different types of cellular movement are observed: The most prominent movement follows the asymmetrical fission of the primary thickening into two cell groups (visible in Additional file 2 at about movie frame 750 and in Additional file 3 at movie frame 570). A smaller group remains at the centre of the embryonic portion of the egg and continues to be identified as primary thickening. The larger cell group, the cumulus or cumulus posterior, is visible as a little bulge on the egg surface (Figures 3c-e; Additional files 2 and 3). The cumulus migrates about 90 degrees underneath the egg surface. The second type of cellular movement is a radial migration of single cells or groups of a few cells starting from the primary thickening. These cells (primordial mesendoderm) migrate directly underneath the ectodermal layer up to 90 degrees along the outer egg curvature (Additional files 2 and 3). At the end of the process, the migrating cells appear to be evenly distributed but are restricted to the egg hemisphere that has the primary thickening in its centre (Figure 3e).

The embryo now has two dissimilar egg hemispheres. The embryonic hemisphere appears to be more opaque because of the new layer of cells (primordial mesendoderm) underneath the ectoderm. The more translucent hemisphere is made up of extra-embryonic tissue, mainly filled with yolk in this and subsequent figures (e.g., Figures 3, 4, 5 and 6). The transition between the subsurface cell layer and the more translucent hemisphere has been called the 'equator' [26], and is clearly visible in living eggs (Figure 3e; Additional file 2). The cumulus is embedded at the edge of the equator and marks the anterior and dorsal region of the embryo body, while the primary thickening (in the centre of the embryonic hemisphere) is at the ventral and caudal pole of the embryo body (Figure 3e).

Stage 11, Opisthosomal limb buds

At this stage, the precheliceral region is partitioned medially into bilateral precheliceral lobes (Figures 7a', b). Each lobe has about 30 point-like depressions (invagination sites, sensu[14]) that are presumably neural precursor tissue (black arrow heads, Figure 7b). The anlage of the stomodeum becomes visible. It is formed by a somewhat depressed antero-medial precheliceral region, which bears a small longitudinal furrow with two lateral adjacent invaginating neural precursor groups (Figure 7b).

In older embryos of this stage, the stomodeal anlage has migrated posteriorly, leaving behind a shallow cleft between the precheliceral lobes (Figure 7b). The cheliceral limb buds have become more flattened and are approximately twice as long as they are wide. They have twisted slightly ventro-posteriorly, and their distal parts are cone-like (Figures 7a', b). The pedipalps have a proximo-medial swelling formed by the anlage of an endite (en, Figure 7b). The ventral sulcus has extended posteriorly, reaching the sixth opisthosomal segment in older embryos of this stage (Figures 7a'', d). Small spots (black arrow heads; Figures 7a', a'') in a segmentally iterated pattern are barely visible laterally adjacent to the ventral sulcus. This area is the ventral neuroectoderm and the point-like depressions correspond to neural precursor tissue [27]. In addition, medially and between the developing limb buds, larger spots of neural precursor tissue are observable on the ventral surface of each prosomal segment (black arrows, Figure 7c). Together with the point-like depressions of the precheliceral region (black arrow heads; Figure 7b) they are the first external indications of neurogenesis.

The pedipalps and walking legs continue elongation and start to bend ventrally. Two annulations develop, dividing the limb buds into three regions (black dotted lines; Figure 7c). For a short time, a small structure is visible on the first opisthosomal segment. This small structure can be interpreted as a vestige or remnant of an appendage (white arrow; Figure 7c). With a developmental gradient from anterior (more developed) to posterior (less developed), primordial limb buds have appeared as small bulges on opisthosomal segments two to five (Figures 7a, a'', d). The initial shape of these buds is not as broad as the prosomal limb buds when they first appeared (compare with stage 9; Figures 5a', b). Depressions at the medio-posterior insertion of opisthosomal limb buds two and three can be seen (white arrows; Figure 7e). These invaginations are precursor tissue for the book lung system. At least seven separated segments are visible anterior to the growth zone (Figures 7a'', d).

Stage 12, Lateral furrow

The outer edges of the precheliceral lobes have become very distinct, and the lobes stand out clearly from the surrounding tissue (Figures 8a', b). Alongside the point-like depressions, a slight relief begins to form on the precheliceral lobes. Lateral to the stomodeum, invaginating kidney shaped folds of neural tissue are visible (lateral furrow LF; Figures 8a', b). Medially adjacent to these folds a minimal elevation can be seen in some specimens.

The stomodeum has now subsided and moved further posteriorly so that the cleft separating the precheliceral lobes is more evident (Figure 8b). Anterior to the stomodeum, the small bi-lobed anlage of the labrum is visible (Lb; Figure 8b). The prosomal limbs (pedipalp and walking legs) have elongated, and the tips of the walking legs from each body halve approach each other. The pedipalpal endite on the most proximal segment (coxa) is clearly visible (en; Figure 8b). The pedipalps and all the walking limbs display signs of annulations. It is not clear how these annulations relate to later leg segments.

The ventral sulcus extends posteriorly to the seventh opisthosomal segment, and has slightly widened (Figures 8a'', c). All opisthosomal limb buds have a globular shape (Figures 8a'', c). Small depressions of the primordial respiratory system are evident at the posterior insertion of the limb bud on opisthosomal segment two (black arrows; Figure 8c). Medially and between the opisthosomal limb buds, large point-like depressions of neural precursor tissue can be seen. These depressions are similar to the large spots on the prosoma in stage 12 (compare Figure 8c with Figure 7c). Up to eight separate opisthosomal segments are visible anterior to the growth zone (Figures 8a'', c).

Stage 13, Labrum

The distance between the posterior end of the opisthosoma and the anterior border of the precheliceral lobes is at its smallest at this stage (white line; Figure 9a). In the forming brain, the lateral furrows have deepened (Figure 9b). Two distinct fields of neural precursor tissue are evident within the crescent-shaped precheliceral lobes: the medial subdivision and the lateral subdivision (ms and ls, sensu[37]). These are positioned between the lateral furrow and the anlage of the labrum (Figure 9b). The two lobes of the labrum are clearly evident at this stage (Figure 9b) but are still separate structures (compare with later stages; e.g. Figure 12 b).

The cheliceres have a proximal base and the anlagen of the distal fangs are visible (f; Figure 9b). The pedipalps and walking legs show clearer annulations and a subdivision into podomeres is evident. The pedipalps are divided into four segments, and the walking legs have five segments (Arabic numbers in Figures 9b, c). The most proximal leg segments (coxa and trochanter/femur) are broader than the more distal leg segments (Figure 9c). Large invagination sites are positioned at the distal tips of the pedipalps and walking legs (black arrows; Figure 9c).

All opisthosomal limb buds retain their globular shape. A slit-like invagination is evident at the posterior base of the limb bud on opisthosomal segment two (black arrow, Figure 9d) and will be the first opening of the primordial respiratory tissue (book lung system). The fifth opisthosomal limb buds are still smaller than the more anterior buds. The opisthosoma has up to nine separated segments and the ventral sulcus, which has again slightly widened, extends posteriorly to the eighth opisthosomal segment (Figures 9a, d).

Stage 14, Inversion I

The gradual widening of the ventral sulcus, which from stage 11 to 13 is a relatively slow process, significantly accelerates during stage 14 (Figures 10a'', e). This marks the start of inversion, a complex sequence of tissue movement and growth that results in a rearrangement of the body and incorporation of the yolk mass into the embryo. Apart from the precheliceral region and the posterior-most opisthosomal segments, the two halves of the germ band move separately over the yolk mass until they connect again on the dorsal side (Figure 11d gives a schematic overview). As a result of this movement, the distance between the precheliceral region and the posterior opisthosomal region increases. Simultaneously, the germ band continues to extend with the addition of the final opisthosomal segments. The precheliceral region, which until inversion was an extension of the rest of the germ band, gradually folds posteriorly. In order to precisely map the various developmental events that occur during inversion, we distinguish four separate stages.

At Inversion I, the dorsal edges of the body halves have not yet reached the upper hemisphere of the egg. The precheliceral lobes are characterized by a high density of point-like depressions and even more pronounced anterior rims (Figures 10a', b). In addition, the medial and lateral subdivisions are more evident. Anterior to the medial subdivision, a crescent shaped anterior furrow has formed (AF, Figure 10b). The anterior furrow has also been termed the semi-lunar or cerebral groove in other arachnids [e.g. [33, 45]]. The lateral subdivision migrates in the direction of the lateral furrow, partly covering it (black arrows; Figure 10b). The cheliceres are now two-segmented. The proximal segment (basal segment) widens distally and the tapering distal segment (fang) sits slightly off-centre on the basal segment (Figure 10c). The proximal segments (coxa and trochanter/femur) of the pedipalps and walking legs are wider than the more distal segments (white stars; Figure 10c). This widening is probably related to anterior and posterior invagination sites on each of these leg segments. The neuroectoderm medial to the prosomal limbs displays a grid-like formation of point-like depressions (white arrows; Figure 10d).

The buds on opisthosomal segment two have become dorso-ventrally elongated. On the posterior ends of these buds, the opening of the pulmonary sac and one or two pulmonary furrows are evident (Figures 10e, f). The buds on opisthosomal segments three to five are undifferentiated and still more or less globular in shape. Dorsal to the opisthosomal limb buds, the anlagen of the tergite plates are evident (black stars; Figure 10e). At the posterior end of the embryo, nine opisthosomal segments have separated from the growth zone (Figures 10a, e). The growth zone now protrudes slightly from the yolk, marking the start of the tail-like formation of the 'post-opisthosoma' (name derived from 'Postabdomen'[46]).

Stage 16, Inversion III

By the third stage of inversion, the tergite plates of the opisthosoma are completely enclosed within the dorsal hemisphere of the egg: from a caudal perspective the opisthosomal limb buds of both halves lie more or less in one line (Figures 12a'', 11d). The distance between the precheliceral lobes and post-opisthosoma has increased and is about a quarter of the total circumference of the embryo (Figure 12a). The lateral furrows are completely covered by tissue from the kidney-shaped lateral subdivisions (Figure 12b). The medial subdivisions are growing anteriorly, partially covering the anterior furrows (black arrows; Figure 12b). The anterior furrows are partially closed by anterior expansions of medial subdivisions (Figure 12b). The tip of the labrum is stretched medially and points in a ventral direction (Figures 12a', b). Posterior to the stomodeum, the unpaired anlage of the labium is formed (Figure 12b). The mouth area (labrum, stomodeum and labium) has migrated further and lies posterior to the lateral furrow/lateral subdivision, on a level with the insertion of the cheliceres (white dotted line; Figure 12a'). The bases of the cheliceres have further widened, and at the ventral base of the pedipalp a prominent endite is evident (Figure 12b). The coxae of the pedipalps and walking legs still have a 'bi-lobed' appearance, and these appendages have elongated (Figures 12a, c). Anlagen of the segmental sternites become visible medial to the pedipalps and walking legs (white dotted line: Figure 12b). The prosomal tergites start to extend dorsally (white arrows; Figure 12a).

The posterior base of the limb bud on opisthosomal segment two bears three pulmonary furrows (black arrows) and the lateral opening of the pulmonary sac (PuS, Figure 12d). At the latero-posterior insertion of the limb bud on opisthosomal segment three, the invagination of the tubular trachea is visible (white arrow; Figure 12d). The globular limb bud on opisthosomal segment four will eventually differentiate into the anterior spinneret (ASp), while the dorso-ventrally elongated limb bud on opisthosomal segment five will differentiate into the posterior (PSp) and medial (MSp) spinnerets (Figure 12d).

On the dorsal surface, the opisthosomal tergite plates have further expanded, and their dorsal edges start to approach each other (Figures 12a'', e). Eleven opisthosomal segments have formed anterior to the growth zone. In between the eleventh opisthosomal segment and the growth zone (GZ), small bilateral lobes probably represent the twelfth opisthosomal segment (arrowheads; Figure 12e).

Stage 17, Dorsal closure

Dorsal closure completes inversion. The tergites of both body halves meet along the dorsal midline, covering all of the dorsal yolk with embryonic tissue. This gradual event starts posteriorly with the tergites of the caudal region and progresses anteriorly. The laterally expanding tissue of the prosomal tergites meets the tissue posterior to the precheliceral lobes (white arrows; Figure 13b). During the process, the dorsal prosomal surface has a crumpled appearance (Figure 13b). Underneath the area where the tergite plates touch each other, tissue that eventually forms the heart becomes evident (Figure 13a'''). Cuticle covers the sternites in the prosoma (white arrows; Figures 13c) and the brain region is also overgrown by epidermal and cuticular formations (white arrow; Figure 13a'). The embryonic tissue anterior to the pedipalp has bent posteriorly, marking the start of the process in which the supraoesophageal area folds onto the suboesophageal area. As a result of the positioning of the cheliceres and stomodeum, the labrum is positioned between the bases of the cheliceres (Figures 13a'. c). The pedipalps and walking legs have further extended and meet each other medially in a zipper-like manner. The posterior sides of the limb buds on opisthosomal segment two have become concave (Figure 13d). Evident on each bud are four pulmonary furrows (Figure 13d; black arrows) and the opening of the pulmonary sack (Figure 13d; white arrow). The segments posterior to opisthosomal segment eight have become compressed, giving them a swollen appearance (Figure 13e).

Stage 18, Prosomal shield

The rim of the precheliceral lobes grows in the direction of the mouth opening and covers the brain, which has thickened substantially (Figures 14a, a'', b). The lateral and medial subdivisions are the last parts of the brain to be overgrown. Dorso-posteriorly to the precheliceral region, cuticle continues to expand marking the start of the formation of the prosomal shield (white arrows; Figure 14b). The labrum is now posterior to the cheliceres, which in frontal view partially cover the labrum with their bases (Figures 14a', b, e). Tiny egg teeth appear laterally on the most proximal segment of the pedipalps (ET; Figure 14d). The walking legs are more slender than before and show their final segmentation into seven podomeres (black lines; Figure 14c).

The dorsal yolky mass is divided into at least three distinct yolk sacs (YS; Figures 14a, a''). In parallel, yolk moves from the prosomal segments into the posterior part of the embryo, while simultaneously the petiolus starts to constrict, causing the embryo to lose its spherical shape. Together, these events mark the start of the division into what will later become the tagmata (prosoma and opisthosoma). The opisthosoma has grown in relation to the rest of the embryo: at this stage the width of the petiolus is about 60-70% of the width of the opisthosoma (trapezoid line; Figure 14a'').

Opisthosomal segments two, three and four broaden substantially, especially at their dorsal ends, giving the embryo a crooked appearance (Figures 14a, c). The third opisthosomal segment widens ventrally, with the result that the distance between the book lung primordia and the tracheal tubercles increases (Figure 14g). Ventral closure initiates: The anlagen of the prosomal sternites start to close from anterior to posterior in a process that will eventually result in a single sternum plate (white arrows; Figure 14e). The sternites posterior to opisthosomal segment five have also moved closer together (white arrows; Figure 14g). The most posterior opisthosomal segments, probably segments nine to twelve, have swollen up even more ventrally and form a square-shaped protrusion (Figures 14a, c, g).

Stage 19, Heart

The protocerebral part of the brain no longer sits on the yolk but has sunk into the prosoma (Figure 15a). The prosomal shield has almost completely covered the brain, save for a wedge-shaped opening directly dorsal to the cheliceres (white dotted line; Figure 15a'). In frontal view, the labrum is now fully covered by the cheliceres (Figures 15a', c). The labium has started to protrude, and together with the labrum forms a beak-like structure. Posterior to the mouth, the prosomal sternites have fully closed (white arrows; Figure 15d). The prosomal tergites are dorso-ventrally reduced, and the inserts of the pedipalps and walking legs have moved dorsally (Figures 15a, a'', b). From a lateral perspective, the brain and the inserts of the walking legs no longer form a continuous arch but lie at an acute angle to each other. All in all, the prosoma has become more compact, a process probably also accompanied by further movement of yolk from the prosoma into the opisthosoma.

The petiolus has constricted further, and at this stage is about 50-60% of the width of the opisthosoma (trapezoid line; Figure 15a''). Dorsally on the opisthosoma, nuclear staining shows a tubular heart (Figures 15a'', e). Ventrally, the opisthosomal sternites have not yet fully closed. Due to a broadening of the sternite of opisthosomal segment three, the book lung primordia have migrated anteriorly and are now almost completely lateral to the petiolus (Figure 15a''). The posterior-most segments of the opisthosoma have further compressed and together form the anal tubercle (Figure 15b).

Stage 20, Ventral closure

Nuclear staining of the developing brain shows distinct regions that correspond to brain parts such as the optic ganglia (Figure 16a). The prosomal shield covers the whole prosoma, including the brain region directly dorsal to the labrum (Figures 16a, a', b). The cuticular border of the prosomal shield is now visible between the prosoma and the opisthosoma (Figure 16b). In lateral view, the insertion sites of the walking appendages lie in a straight line (Figure 16a). The width of the petiolus is about 40-50% of the width of the opisthosoma (trapezoid line; Figure 16a''). The embryo has become even more crooked, such that the first pair of walking legs almost touches the anal tubercle (Figures 16a, a'). Ventral closure of the opisthosoma is complete, and the book lungs have moved antero-medially in the direction of the petiolus (Figure 16d). The spinnerets of both body halves lie close together and form the spinning field. The spinning field has moved close to the anal tubercle (AT; Figures 16a', d).

Stage 21, Petiolus

In the course of this stage, a complete cuticle develops underneath the embryonic cuticle, making it impossible to obtain information about the inner morphology using nuclear staining of whole mounts. Externally, the fangs of the cheliceres become pointed and are directed towards each other (Figures 17a'', c). The final restriction of the petiolus takes place; the embryo starts unfolding and loses its crooked appearance (Figures 17a-d). Seitz [26] observed in advanced embryos (probably corresponding to stage 21) that lateral parts of the prosoma had not yet been fully covered by the dorsal shield. We cannot confirm this however. Towards the end of this stage, air appears between the prosomal appendages, indicating that the embryo is taking up the exuvial liquid which will allow it to exert pressure on the egg membranes (white arrow; Figure 18a).

Postembryonic stages

First instar

The first instar emerges from the postembryo after about 3 days (at 25 C). Contrary to earlier observations [48] we never witnessed a first instar hatching directly from the egg. The walking legs of the first instar extend laterally (Figures 19b, c-e). The cheliceres have two so-called retromarginal teeth on their bases (black arrows; Figure 19a'). Both teeth are positioned opposite the folded fangs. Distal-laterally, the fangs bear an opening to the poison gland (white arrow head; Figure 19a').

Although the first instar cannot walk, it bears sensory hairs on the cuticle and reacts to tactile stimuli by wiggling its appendages (Figures 19a, b). The opisthosoma is spherical in shape, still contains yolk reserves, and is about twice as big as the prosoma (Figures 19b-e). The first instar probably obtains nutrients mainly from this yolk reserve, but animals were frequently observed feeding on unfertilized eggs as well.

The cuticle of the first instar is transparent and not pigmented. After about 10 days an increase in cuticle pigmentation can clearly be seen (Figures 19c-e). In addition, the pigmentation of the eyes becomes more intense. The first silk is produced from the spinnerets, as evidenced by the fine threads that hold the first instars together when the cocoon is opened at this stage.

The germ disc and the equator

As noted by previous authors [5, 26]C. salei is significantly different from many other spider species because no dense germ disc is formed during its development. A germ disc (a circular aggregation of blastodermic cells) represents the first visible formation of embryonic tissue and is found in many arthropod groups (e.g. onychophorans [49], myriapods [50], crustaceans [51] and insects [52]). Germ discs are usually made up of cells that are almost free of yolk compartments and arranged in a more compact way than the remaining cells. A consequence of germ disc formation is that there are relatively few blastodermic cells in the remainder of the egg; the extra-embryonic region. In many spiders, including A. tepidariorum, the germ disc is very dense in comparison with the extra-embryonic part of the egg. It takes up roughly half of the egg, and a pronounced border with the extra-embryonic region circumscribes the egg more or less half-way between the poles [e.g. [5]]. In C. salei however, no aggregated cells are noticeable at the point when gastrulation starts with the formation of the blastopore (stage 3, Figures 2h-j: for comparison see stage 3 in [25]). Instead, the blastodermic cells appear to remain evenly distributed over the egg surface and a relatively large amount of yolk surrounds the periplasm. We cannot conclude from our data that no germ disc forms, but if an aggregation of cells does occur around the blastopore, it is very subtle and no clear border is visible during gastrulation (stages 3, 4 and the first half of stage 5). We therefore describe C. salei as not having a dense germ disc.

After gastrulation (end of stage 5) in C. salei a visible border appears that circumscribes the egg (Figure 3a). Seitz refers to this border between the embryonic and extra-embryonic portions of the egg as the 'equator', and accurately interprets it as the anterior border of the embryo [26]. However, the way in which this structure develops was unclear at the time of publication, as well as how it relates to the germ disc in other spider species. One hypothesis that has been presented is that the equator becomes visible because of an increase in cell density in the embryonic half of the C. salei egg, due to ongoing cell division during stage 5 [5].

Our live-embryo movies provide further insight into the nature of the equator, because the lack of a non-transparent germ disc in C. salei facilitates the visualisation of cell movements underneath the surface (ectoderm). Shortly before and during the instant when the cumulus separates from the primary thickening, blastodermic cells move towards the blastopore. Simultaneously with this radial movement of the cumulus, additional cells (also from the primary thickening) disperse radially underneath the ectoderm (e.g. following movie frame 560 of Additional file 3). When the cumulus finishes its migration of about 90 degrees over the yolk mass (end of stage 5) these radially migrating cells are distributed over the embryonic hemisphere. This multi-layered hemisphere appears to be more dense than the extra-embryonic hemisphere (Figure 3e; end of movie of Additional file 2). We interpret these uniformly distributed cells as mesendodermal (mesoderm and endoderm) and it is likely that most of them were internalised during formation of the primary thickening. This data thus shows that cell migration from the primary thickening (end of stage 5) rather than cell division in the blastoderm is responsible for the formation of the equator.

The question then arises how the mode of gastrulation of C. salei relates to that observed in spider species that do form a dense germ disc [e.g. [32, 33, 36, 53]]. Sections and time-lapse movies of an example of the latter category, Zygiella x-notata, showed similar cell movements: 1) The cumulus migrates away from the blastopore; 2) additional cells underneath the ectoderm spread over the embryonic primordium from the blastopore and 3) ectodermal cells move towards the blastopore [32]. Since our data for C. salei is similar to the data for Z. x-notata, we propose that both species with and without a dense germ disc follow what has been called the 'canonical mode of gastrulation' [32] in that all mesendodermal cells have their origin in the region of the blastopore/primary thickening. Furthermore, we propose that in species with a dense germ disc, a border is also formed that for some as yet undiscovered reason the migrating mesendoderm does not pass. In those species, such a structural equivalent of the equator of C. salei is masked by the edge of the germ disc and therefore not readily visible.

These proposals are in discord with one interpretation of gastrulation that has been proposed for A. tepidariorum[54, 55]. In these publications, the 'rim of the germ disc' is given a prominent organising role. To achieve this, expression of a twist homolog (At-twist) is used as a molecular marker of spider mesoderm. Expression of At-twist is shown in embryos fixed towards the end of stage 5, in cells underneath the blastoderm at the border of the germ disc. Next, in embryos fixed at stage 6, At-twist expressing cells are shown distributed over the early germ band. Combining these patterns, the authors conclude that At-twist expressing cells internalise at the rim of the germ disc and subsequently spread over the embryo. This data is taken as evidence that the border of the germ disc is an additional site of gastrulation [54] giving rise to so-called 'peripheral mesoderm'. This would imply that A. tepidariorum differs from the canonical mode of spider gastrulation.

We argue against this hypothesis. First, similarly to what we show for C. salei (Additional files 2 and 3), also in A. tepidariorum we have observed centrifugal movements of individual subsurface cells coming from the centre of the germ disc in a time-lapse movie of live embryos (unpublished data, CW). In C. salei, cells originating from the blastopore have reached the equator towards the end of stage 5, and it is plausible that this is also true for late stage 5 embryos of A. tepidariorum. Second, it cannot be concluded from gene expression in fixed embryos alone that the putative 'peripheral' mesoderm in A. tepidariorum internalises at the rim of the germ disc [54] and that it does not derive from the centre of the germ disc. Third, the putative spreading of At-twist expressing cells at stage 6 [54, 55] is clearly a later event than the centrifugal movement that we observe during stage 5. This spreading therefore does not necessarily depend on internalisation of cells at the rim of the germ disc and does not contradict the canonical view of gastrulation.

Concluding, our data provide an explanation for the appearance of an equator in the absence of a dense germ disc in C. salei. Furthermore, we propose that in C. salei all mesendodermal cells have their origin in the region of the blastopore/primary thickening, and that this also holds true for species with a dense germ disc such as Z. x-notata and A. tepidariorum. Consequently we propose that the equivalent of an equator also forms in species with a dense germ disc, and that the nature of this border deserves further attention. For example, which factors prevent mesendoderm from migrating past the equator in C. salei? How do these factors relate to those that define the edge of the germ disc in A. tepidariorum? These questions show that a combined study of more than one species is necessary for a better understanding of the early development of spiders. Also, from a broader perspective, the variation in density of the germ disc in spider embryos provides evidence for the idea that early developmental stages can be modified during evolution without affecting later stages, and that homologous developmental stages are not necessarily based on homologous earlier stages [56].

Brain development

The first signs of neurogenesis can be observed in stage 11 of C. salei once gastrulation has taken place, the a/p axis has formed and the germ band has started to elongate. Evenly dispersed point-like depressions appear on the precheliceral lobes and ventral neuroectoderm (Figures 7a', a'', b). These depressions are caused by the immigration of cell groups and are also called 'invagination sites' [e.g. [57]]). In addition, depressions are visible on the stomodeal anlage (see below). The internalised cells detach from the embryonic ectoderm and develop directly into neural tissue [57].

Differentiation of the brain starts with the formation of the lateral furrows in stage 12 (Figures 8a', b). On each precheliceral lobe, a lateral field of neural primordia cells folds inwards and differentiates into the lateral eyes (anterior lateral and posterior lateral eyes, Figures 19g, g'). These bilateral structures have been termed lateral vesicles or invaginations in other arachnids and have been identified as the primordia of the lateral eyes [e.g. [27, 39]]. As development progresses and the cells of the lateral furrow continue to immigrate (stages 14-16) a medially adjacent cluster of cells - the lateral subdivision - begins to cover the lateral furrow completely (ls; Figures 14b, 16b). This neural primordial tissue differentiates as development progresses into the median eyes (anterior median and posterior median eyes; Figures 19g, g' and [27]).

Simultaneously, a large field of cells at the anterior rim of each precheliceral lobe folds inward to form a characteristically crescent-like shape - the anterior furrow (AF; stage 14, Figure 10b). This is the most prominent structure seen during early chelicerate brain development and is often described as the semi-lunar or cerebral groove [e.g. [33, 45]]. The invaginating cluster of cells that results in the anterior furrow is the neural primordium of the arcuate body. In a procedure similar to that described for the migration of the lateral subdivision (median eyes), a medial subdivision of neuroectodermal cells starts to overgrow the anterior furrow (stages 15-17). The medial subdivision fills the space occupied by the anterior furrow and represents the neural primordium of the mushroom bodies (Figure 12b, figure 3 in [27]). The now separate brain compartments continue to differentiate and are eventually overgrown by a hood (Figures 14b, 15c, stages 18-19, see [27]). The tissue for the hood comes from the outer rim of the precheliceral lobes and encloses not only the entire brain complex but also the dorsal prosomal region to form what is known as the prosomal shield. At the same time, an extreme backwards bending of the precheliceral region during the process of inversion brings the brain compartments into their final position.

For the insights they provide about the sequence of early brain differentiation, our data complement earlier findings [27] regarding the three dimensional arrangement of brain parts such as the arcuate body, the mushroom body and the optic ganglia. Our data provide information about the area from which these primordial tissues originate. We are able to map the precursor cells of single brain regions (arcuate body, mushroom body and optic ganglia) onto the two dimensional tissue layer of the precheliceral neuroectoderm. As development progresses, the neural tissue undergoes complete spatial reorganization and differentiates into the mature brain via complex morphogenetic processes

Mouth region

The mouth develops in close association with the developing brain. The first depression of the stomodeal anlage appears in the course of stage 11 between the precheliceral lobes (Figure 7b). By stage 12 the labrum (upper lip) appears as a bi-lobed structure directly anterior to the stomodeal anlage, which by now has subsided somewhat (Figure 8b). As development progresses, the mouth opening gradually migrates posteriorly, while in the course of stage 14 the labium (lower lip) appears and the lobes of the labrum fuse (Figure 10b). The result of the posterior migration of the mouth opening is an increasingly pronounced cleft between the two precheliceral lobes (visible for example in Figure 12b). After the first true moult into the first instar, the endites of the pedipalps (also known as the maxillae) form the lateral border of the mouth opening (Figure 19a').

The early development of the mouth region of chelicerates has been the subject of recent publications [37, 40]. Three small point-like invaginations were observed in the centre of the stomodeal anlage of the spider L. geometricus[37]. Since the proboscis of pycnogonids (sea spiders) has a tri-radial construction and this tri-radial symmetry is established during the formation of the stomodeum [40, 58, 59], the occurrence of a triangular stomodeal invagination has been used as an argument for a sister group relationship between pycnogonids and euchelicerates (vs. the Cormogonida hypothesis, according to which the sister group relationship is between pycnogonids and extant euarthropods) [59]. In C. salei at stage 11 (Figure 6b) and in A. tepidariorum (unpublished data, CW) we have observed three invaginations in the stomodeal anlage that we also interpret as the result of internalised neural precursor cells. Due to ongoing mouth formation, these invaginations are no longer externally visible by stage 14 (Figure 10b). Previously [27] a crescent-shaped array of neural primordial cells has been described in the dorsal part of the stomodeum at 220 hAEL of C. salei development (comparable to stage 15; see Figure 1). The authors assume that these invaginating neural precursors give rise to the stomatogastric nervous system, which controls the motions of the gut. To test whether the triangular pattern is a valuable character for making phylogenetic inferences, we need to know more about the neural structures that result from these particular neural precursor cells. A Y-shaped stomodeal invagination was also found in the isopod crustacean P. scaber[24] for example, indicating that this structure may actually represent an arthropod plesiomorphy (ancestral character) rather than a synapomorphy of any particular arthropod subgroup. With the techniques available for C. salei, the species would be an appropriate starting point for such investigations, especially in light of the fact that its central and peripheral nervous system has been the focus of previous studies [27, 48].

The nature and evolutionary origin of the labrum is another open question relating to the mouth region in arthropod evo-devo studies [e.g. [60]]. It is well documented that the spider labrum develops from a bi-lobed structure (also known as the 'rostral appendages') that fuses into a single structure (or 'rostrum') [33, 36, 37, 61]. We observed the same sequence in C. salei; two bulges first appear at stage 12 (Figures 8a', 8b), become more pronounced during stages 13 and 14 (Figures 9a', 9b, 10a', 10b) and fuse at stage 15 (Figures 11a', 12a', 12b) to form the labrum. Additionally, gene expression studies show that several leg patterning genes are expressed in the labrum in an appendage-like way [62]. These data support the argument that the labrum consists of appendages from the protocerebral region (ocular segment) that fuse in the course of embryonic development [60].

The bi-lobed nature of the labrum is also described in a recent study of L. geometricus[37]. Furthermore, it has been observed that before the first appearance of the labrum, a distinct and slightly depressed field appears between the two precheliceral lobes which the authors refer to as the 'mouth region' [37]. The authors hypothesise that it is within this mouth region that the stomodeal opening forms and the two labral bulges (which the authors call 'hypostome') develop [37]. We do not observe a distinct field of this nature between the precheliceral lobes in C. salei (Figures 6 and 7). Moreover, the raw SEM data presented for L. geometricus (Figure 3b in [37]) is not entirely clean, and the impression of a depression might derive from shrinkage during fixation.

Inversion and the post-opisthosoma

As described in the Results section, inversion in C. salei involves a drastic rearrangement of different parts of the body due to simultaneous migration and expansion of tissue over the yolk mass (Figures 10, 11, 12 and 13). Yolk is incorporated into embryonic tissue, and parts of the brain fold backwards over the ventral nerve cord (Figure 1 in [27]). It is generally agreed that inversion starts with a pronounced increase of the widening of the ventral sulcus [e.g. [30, 63]] which corresponds to stage 14 of C. salei (Figure 10a''). The precise end, however, is less well defined. A study of the species Latrodectus mactans described the end as when "the shift of tissues toward the mid-ventral line is completed" [[36], p.49], which would imply that inversion finishes at ventral closure, i.e. at stage 20 in C. salei. Other interpretations suggest that inversion ends when all the yolk is covered by ectoderm [35] or that it finishes with the dorsal closure of the prosoma [30]. Dorsal closure is more practical to observe than ventral closure because for observation of the latter the legs must be removed. Furthermore, during inversion the ventral sulcus consists of a (thin) layer of tissue and thus covers the yolk. We therefore use dorsal closure to define the end of inversion in C. salei (stage 17). We then divide the inversion movements preceding dorsal closure into three separate stages (stages 14-16; Figure 11d) based on the progress of the movement of the germ band halves over the yolk mass.

Inversion itself has undergone some significant changes over the course of spider evolution. In some species like the basally branching Heptathela kimurai, the ventral sulcus does not widen to incorporate the yolk mass. Instead, most opisthosomal segments develop as a protuberance that flexes ventro-anteriorly, extending from the yolk mass [33]. This so-called post-opisthosoma initially contains very little yolk, but gradually fills up with more yolk from the more anterior segments [33]. It has been proposed that this way of internalising the yolk represents the ancestral state [63]. Other species show an intermediate morphology. In Segestria bavarica for example, the ventral sulcus of opisthosomal segments 1 to 3 widens ventrally and the two halves of the germ band move dorsally over the yolk mass, similar to most opisthosomal segments of C. salei. The rest of the opisthosomal segments, however, form a forwardly flexed post-opisthosoma [46], similar to in H. kimurai. Entelegyne spiders, such as C. salei or A. tepidariorum, are thought to display the most derived character state [46, 63] with a strongly reduced post-opisthosoma. In C. salei, the post-opisthosoma consists of the tissue posterior to opisthosomal segment nine only. The tissue lies flat on the tergites of opisthosomal segments seven to nine (Figure 12e). Additional file 4 compares the post-opisthosomas of S. bavarica and C. salei.

It is unclear why inversion evolved in the first place, and why the post-opisthosoma seems to have become reduced over time. One proposed scenario is that this happened as "an adaptation to a secondarily increased amount of nutritional yolk in the egg" [[64], p.213]. Another possibility is that the evolution of inversion relates to a change in speed of development. In order to try to answer these questions, one could compare the developmental modes of different spider species taking into account factors such as relative yolk content and duration of development. Furthermore, since the molecular basis of spider segmentation and segment specification has been studied intensively in C. salei and A. tepidariorum [e.g. [65] and references therein] one might investigate factors involved in the opening of the ventral sulcus and the specification of the post-opisthosoma in these species.

Respiratory system

During inversion, the breathing organs (the book lungs and tracheae) start to differentiate. It is widely accepted that arachnid book lungs are derived from the book gills of an aquatic xiphosuran-like ancestor [30, 66, 67] and that the tracheal systems found among arachnids are a derived character [41, 68]. However, there is no consensus on the homology of the book lungs and the tracheal system. The traditional view [35, 36, 42] holds the spider tracheal system to be derived from a book lung system (making them homologous structures) while more recent studies question whether tracheal systems really do originate in the book lungs [e.g. [66]]. Purcell [42] proposes that the tracheal system in spiders originates either from a book lung system or from an ectodermal tendon [42] and argues that it thus evolved independently.

Our data contributes to this discussion by showing the progression of development of the book lungs in opisthosomal segment two and the tracheal system in opisthosomal segment three. Development in opisthosomal segment two starts with a conspicuous depression of the cells posterior to the limb buds which later differentiate into the transverse slit-like openings of the book lung system (Figures 7e, 9d). As development progresses, the opening of the pulmonary sac is formed laterally, followed by the addition one by one, medially, of the book lung openings. In opisthosomal segment three, initial depressions form in the course of stage 11 that look similar to those of the book lung primordia (white arrows; Figure 7e). However, by stage 12, these depressions are no longer visible, and the surface of the embryonic tissue is smooth again (Figure 8c). At stage 16 the third book lung opening is formed in opisthosomal segment two. Only at this stage, a new opening of the tracheal system becomes visible in opisthosomal segment three (TrO; Figure 12d). The position of this new opening is posterior to the limb buds of segment three, but slightly more lateral than the initial opening of the book lung system at opisthosomal segment two (compare with stage 11; Figure 7e). We interpret these early depressions on opisthosomal segment three as remnants of an ancient book lung system in this segment. The opening that emerges later, more medially at the posterior border to the limb bud, is a different structure. The observation that the tracheal opening appears much later than the opening of the book lung system in opisthosomal segment two has also been made for L. mactans[36]. This supports our hypothesis that the book lungs and the tracheal system have different developmental origins in C. salei and are therefore not homologous structures.

Our data also has implications for the interpretation of a more recent study that uses molecular markers to investigate the evolutionary relationship between arthropod respiratory structures. It is shown that in C. salei, the gene pdm/nubbin is expressed in all four developing opisthosomal limb buds in a similar expression pattern, and from this it is concluded that structures such as book lungs, tracheal systems and spinnerets might represent evolutionary modifications of an ancient gill structure that was a limb branch (the so-called epipod) [10]. However, in C. salei it is difficult to understand which parts of the opisthosomal limbs give rise to which part of the respiratory system. Our data show that both respiratory structures (book lung system and tracheal system) have their origin not in a clear limb structure, but obviously posterior to the limb buds. The possible contribution of a limb structure and its surrounding tissue therefore should be re-investigated in more detail at both a morphological and a molecular level before any conclusions can be drawn as to the homology of spider respiratory structures with breathing structures in other arthropod lineages.

Finally, our data also give some indication of the developmental mechanisms that contribute to the generation of the respiratory structures. Our SEM samples often showed cellular debris in the form of small globules in the area of the forming book lung opening (Figure 6e). This could indicate that the book lungs are not only the result of an ectodermal invagination, as observed by some authors [e.g. [67]] but that cell death could also play a role in their formation. The relative contribution of both of these mechanisms must be investigated further if we are to improve our understanding of the development of this complex system and its evolutionary origin.

Prosomal and opisthosomal sternites

After dorsal closure, the general morphology of the embryo gradually changes from a round shape to a typical spider body plan with two separate tagmata (prosoma and opisthosoma, Figures 14, 15, 16 and 17). This process involves the constriction of the petiolus (see the trapezoids in Figures 14a'', 15a'', 16a'') and movement of yolk from the prosoma into the opisthosoma. In parallel, both the prosoma and opisthosoma close ventrally.

In the prosoma, ventral closure marks the beginning of the formation of a sclerotised plate, the 'sternum', in between the coxae of the walking legs. Few studies deal with the formation of the sternum, and no definitive information is available on the origin of the sternal tissue [33, 36, 37, 69]. For example, it is unclear whether the single cuticular sternum in adults has a segmental origin and if so, what segments are involved [69]. However, it is known that in this region the ventral neuroectoderm is overgrown by an epidermal layer of cells from lateral regions [14, 33]. In C. salei this takes place when neurogenesis is over, shortly before ventral closure [14]. We have been able to confirm the overgrowth process using phalloidin staining (data not shown). The overgrowth becomes visible for the first time in the course of stage 18. Unfortunately, this epidermal layer lacks morphological features, such as borders of sclerites or muscle attachments. Therefore, this observation does not provide any conclusions regarding the segmental nature of the sternum.

Nevertheless, the ventral neuroectoderm does exhibit pronounced segmental organization when the prosoma starts to close medio-ventrally (stage 18; Figure 13e). We interpret these segmentally iterated structures as anlagen of sternites. During ventral closure (stages 18-20) the sternal units become more substantial and finally merge into the sternum. In the course of this process, newly formed cuticle covers the ventral prosomal tissue, thus masking the segmental appearance of the sternites (in SEM samples, cells underneath a cuticle are typically no longer detectable).

In the opisthosoma, the process of ventral closure is more complex and our data do not show exactly what happens here with regard to the segmental (sternal) compartments. Significant morphogenetic transformations and extensive tissue reduction in the medio-ventral region of this tagma are probably necessary for the successful differentiation of organ systems such as the respiratory system, the genitals, and the spinning apparatus. The second opisthosomal segment widens, while the more posterior segments are reduced. More information about the tissue covering the opisthosomal neuroectoderm would be extremely helpful in enabling us to draw conclusions about the segmental organisation of the opisthosomal region within arachnids [e.g. [70]].

The post-embryonic stages

Embryonic development finishes with eclosion (Figures 18a, b). The outer appearance of the final embryonic stage (stage 21; Figure 17) is very similar to the first post-embryonic stage. Nonetheless, sections of C. salei post-embryos (unpublished data, MH) and late embryonic stages of L. mactans[36] show that many internal changes are taking place, including the development of the reproductive system and the silk glands. After eclosion, the peripheral nervous system also continues to develop [48].

Disagreement about nomenclature and lack of consistent usage of nomenclature complicate comparative studies of the late embryonic and post-embryonic stages of spiders [47]. Part of this problem is likely to stem from the fact that there is great variation in the number and nature of stages in different spider species [46]. Instead of taking this variation into account, research papers on single spider species often stage this period of development according to secondary literature that is over-simplified and outdated regarding this subject [41]. In accordance with a comprehensive review of the post-embryonic stages and a proposed standardization of the terminology [47] we have revised the naming of the stages of C. salei (Figure 1).

One of the most confusing and yet persistent terms is 'larva' [41, 71]. Its use is unfortunate because, like 'nymph' and 'imago' [71] the term larva derives from the terminology of insect holometabolic development, which implies metamorphosis. We therefore reject this term for the staging of C. salei. Instead, we name the stage directly following eclosion and simultaneous shedding of the embryonic cuticle 'postembryo' (after [47], see Figure 18). This postembryo is completely immobile and lacks sensory hairs. The stage was previously referred to as: 'second pre-larva' [7] or 'stage 1 larva'[48].

After the next moult, the 'first instar' emerges (Figures 19a-e). This stage was previously identified as 'larva' and 'third incomplete stage' [7] or 'stage 2 larva' [48]. The first instar is followed by the 'second instar' (Figure 19f-h). The second instar was previously called 'first complete stage' [7] or 'stage 3 larva' [48]). Subsequent instars are numbered further until the sexually mature adult emerges from the penultimate instar. Typically, the adult stage is the twelfth instar. When underfed, sexual maturity can be reached as early as the tenth or eleventh instar [5, 7]. Such developmental plasticity is not uncommon for spiders [72].

Live-embryo imaging

Embryos were mounted on microscope slides and covered with 10S Voltalef oil (VWR International). Embryonic development was recorded using an Axiophot2 Image (Zeiss) equipped with software for 4D-recording, according to published methods [73]. The videos were created using the image processing software 'ImageJ' together with a macro file (kindly provided by Kai Lindemann and available on request). The macro automatically recognizes sharp areas from the different levels and combines them to form a single 'focused image', after which the stack of focused images is merged to a movie.

SEM imaging

Fresh embryos were fixed for at least four hours on a wheel in Dubosq-Brasil fixative. Freshly made working solution consists of 14 parts stock solution and one part glacial acetic acid. The stock solution consists of 60% ethanol, 8% formaldehyde and 0.5% picric acid. The embryos were washed for at least 12 h in 70% ethanol. They were dehydrated in a graded ethanol series and were either critical point dried in CO2 (BALTEC CPD 030) or air-dried after transfer into hexamethyldisilazane (HMDS) by way of an intermediate step in a 1:1 mix of HMDS and absolute ethanol. The dried samples were mounted on stubs and sputter-coated with gold (BALTEC SCD 005). Images were taken on a LEO 1450 VP and arranged in Adobe Illustrator or Corel Draw.

Fluorescent staining and imaging

Embryos were collected at different stages of development, and fixed according to standard methods [74]. After rehydrating the embryos into PBS supplemented with 0.1% Tween 20, we incubated them for approximately one hour with about one ng/ml of the fluorescent nuclear marker DAPI (Roche) or Sytox Green (Molecular Probes). After several washes they were photographed under a dissecting microscope (Zeiss Lumar) in the z-stack-mode. This allowed us to get a better impression of the three-dimensional composition of embryonic stages. All the pictures in a z-stack were merged in HeliconFocus (Helicon Soft Ltd.). In Adobe Photoshop or Corel Draw, the images were cropped and colour saturation and contrast were optimized.