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 2, Blastoderm
During stage 2, the cleavage energids (nuclei plus its surrounding cytoplasm) reach the egg surface where they are conspicuous with finger-like projections (Figure 2d, at movie frame 80 of Additional file 2). After a few division cycles, their cytoplasm attains a more rounded shape (at movie frame 210 of Additional file 2). This is when the cleavage type changes from superficial to holoblastic, and each nucleus on the egg surface is surrounded by its cell membrane (Figures 2e, f). A layer of early blastodermic cells is now evenly distributed over the egg surface (Figures 2e, f). The yolk mass is composed of compartments shaped like pyramids with the tips pointing towards the egg centre (white dotted line; Figure 2g). Data from nuclear staining and live-embryo imaging identify some nuclei that stay below the blastodermic cell layer (white arrows; Figure 2e). These nuclei probably belong to vitellophages; multi-nucleic cells which phagocytize the intracellular yolk. It is currently unclear whether all of these vitellophages have their origin in the early blastoderm (secondary immigration) or whether some of them derive from inside the egg [30, 43]. However, it is likely that during cell migration from the egg centre to the surface, some cells remain in the yolk mass and do not reach the surface.
Stage 3, Blastopore
The following cell divisions are asynchronous. Because of the frequency of cell division cycles, the egg appears to contract (e.g. at movie frame 320 of Additional file 2). After roughly three cell division cycles, the contractions subside, leaving the blastodermic cells still more or less evenly distributed over the egg surface. There is no formation of a germ disc (a dense aggregation of cells that provides the primordial tissue for the embryo body and is commonly present in arthropod embryos [30]). However, in a particular region of the blastoderm cells begin aggregating to form the blastopore (white arrow; Figure 2h). Cells appear to migrate inwards at the blastopore (Figures 2h, i; at movie frame 200 of Additional file 3) and initiate gastrulation- the developmental process that results in a layer of mesendoderm beneath the surface layer of blastoderm (now ectoderm). As development progresses, the blastopore comprises more and more cells and becomes pore-like in appearance, while the surrounding blastoderm cells are no longer evenly distributed (Figure 2j).
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 6, Dorsal field
This stage is characterised by a major rearrangement of embryonic tissue. Cells between the primary thickening and the cumulus start to migrate laterally, and the dorsal field is formed (DF, Figure 3f). This region is relatively translucent and is made up of far fewer cells than the ventral area where the embryo body will differentiate. Our data does not show whether the dorsal field is formed exclusively by cell migration or whether cell death is also involved. At the same time, the cumulus decreases in size (Figure 3f). As development progresses, the dorsal field expands approximately 100 degrees around the egg surface to form a semicircle, while the cumulus disappears (Figure 3h).
Figures 3g and 3h show that the embryo axes (anterior/posterior and dorsal/ventral) have now been specified as a result of preceding events (compare to [25]). The primary thickening (former blastopore region) will become the posterior end of the embryo body. Figure 3g is a ventral view showing the region of low cell density (extra-embryonic region) at the top of the photo, while the caudal primary thickening is at the bottom. Figure 3h is a posterior view of the dorsal field (DF) that becomes extra-embryonic.
Stage 7, Germ band
At this stage, it is not possible to determine the axis orientation of unstained eggs because of a lack of visible landmarks. When nuclear staining is applied, however, a germ band becomes evident (Figures 4a-d). The germ band is a ventral strip of cells with a convex flexion, bearing the former primary thickening at its posterior end (Figure 4h). This latter region is now called the growth zone (GZ) as in Figures 4a-d. The embryonic tissue expands anteriorly beyond the ventral equator margin (between the white arrows; Figure 4b).
As the germ band becomes visible and gradually lengthens along the ventral curvature, the equator disappears and the dorsal field expands laterally. The equator is evident in Figure 3e (between the white arrow heads) and Figure 4b (between the white arrow heads) but is not evident in Figures 4c, d and later figures. The widening of the dorsal field is evident (white dotted lines) in Figures 4b-d. At the end of this 'Germ band' stage, the embryonic tissue has lengthened anteriorly and covers the entire ventral surface of the embryonic region. In this ventral region of the embryo, the cell density is much greater than in the dorsal field. As a result of cell migration, the cells in the embryonic region become more evenly distributed with only the dorsal field displaying a significantly lower cell density (DF; Figure 4d).
Stage 8, Segmented germ band
Within the spherical eggshell, the embryo now has the shape of a flattened ovoid. The C-shaped germ band invariably lies along the longitudinal egg axis (Figures 4e-h). The developing embryo body (embryo proper) of primordial segments and appendages is very dense, comprising many more cells than the dorsal extra-embryonic tissue (Figures 4e-g). The dense region of cells just anterior to the cheliceres initially has no organized structures (Figures 4e, f) and the border between this region and the extra-embryonic region is less defined than the perimeter of the rest of the embryo body. This precheliceral region (Pc) gradually becomes consolidated into a more distinct structure (Figures 4g, 5a, a'). All future prosomal segments (those of the cheliceres, pedipalps and four walking legs) are visible and distinctly divided by inter-segmental furrows (Figure 4g). The cheliceral segment is slightly smaller than the posterior segments (Figures 4e-g). At its posterior end, the embryo proper has a growth zone (GZ; Figures 4e, g, h) which appears brighter in nuclear staining. The growth zone is rounded posteriorly and has a higher cell density than the remaining embryo proper.
Stage 9, Prosomal limb buds
At this stage (Figure 5a) the border of the embryo proper is more clearly defined than during the previous stage. In the precheliceral region there is often a slight difference in the progression of development of the right and left halves (e.g. Figure 5a'). The lower cell density in the medial precheliceral region marks the start of the formation of the ventral sulcus (VS; Figure 5a') which continues to extend posteriorly into the midline of the anterior segments (cheliceres and pedipalps) of the prosoma (Figures 5b, 6a'). The posterior margins of the precheliceral lobes appear anterior to the cheliceral segment but will gradually extend posteriorly (white arrows; Figures 5a', b). All the prosomal segments are more prominent than in the previous stage, and the prosomal limb buds (cheliceres, pedipalps and four walking legs) bulge outward. The buds are broad and flat and point in a postero-ventral direction (Figures 5a, a', b). The cheliceral buds are smaller and slightly more medial than the buds of the pedipalps and walking legs (Figures 5a, a', b). The opisthosoma has about four visible segment anlagen (Figures 5a, a'', c). The growth zone has a posterior curvature and is less broad than the more anterior segments (Figures 5a'', c).
Stage 10, Prosomal limb bud elongation
For stages 1-9 we adhere to the staging system defined for A. tepidariorum[5, 25, 44]. However, we deviate from this system at stage 10 as this stage is not well described for A. tepidariorum and has not been used extensively by other authors. Furthermore, stage 10 for A. tepidariorum represents too great an advance from stages 1-9. We define a new stage 10 and new subsequent stages for C. salei. In our stage 10, the embryo has reverted from an ovoid to a largely spherical shape (Figure 6a). The precheliceral region is broader than the remaining parts of the germ band (Figures 6a', b). The posterior margins of the precheliceral region have moved anterior to the pedipalpal segment and now enclose the cheliceral segment (Figures 6a', b).
None of the prosomal limb buds are segmented yet, and they vary in their width-to-length ratio (Figures 6a-c). The slightly depressed ventral sulcus (VS) extends from the centre of the precheliceral region to opisthosomal segment three (Figures 6a', c). The first opisthosomal segment is clearly visible but will eventually disappear (Figure 6d). It is at this point considerably smaller than the subsequent opisthosomal segments. In Figure 6a'' a developing sixth opisthosomal segment (white arrow head) is evident anterior to the growth zone. In older embryos of this stage, limb buds appear on opisthosomal segment two (Figure 6d).
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 15, Inversion II
By the second stage of inversion, the lateral/dorsal movement of the body halves has progressed, and the anlagen of tergite plates have extended dorsally (Figures 11a'', d). The opisthosomal body halves have reached the dorsal hemisphere of the egg and form a line when viewed from a caudal perspective (white dotted line; Figure 11a''). The two labral lobes have completely fused and the labrum is now an unpaired structure (Figure 11a'). The labrum and stomodeum have jointly started the posterior migration that will be continued in subsequent stages. By stage 15, the cleft between the two precheliceral lobes has become deeper, and the mouth opening lies between the lateral subdivisions on both head lobes (white dotted line; Figure 11a'). The labrum now partially covers the stomodeum (Figure 11b).
Ten separate opisthosomal segments are evident anterior to the growth zone (Figures 11a'', b). The posterior base of the limb bud on opisthosomal segment two bears two pulmonary furrows (black arrows) and the lateral opening of the pulmonary sac (white arrow; Figure 11c). The tenth and the future eleventh opisthosomal segments are now forming, together with the growth zone of the tail-like post-opisthosoma (Figure 11b). Small bulges of tergite anlagen are evident on the dorsal surface of the opisthosomal segments (Figure 11b).
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
Postembryo
Eclosion marks the end of the embryonic stages. In C. salei, this process includes the rupturing of the egg membranes and moulting from the embryonic cuticle (EC; Figure 18b). The rupturing of the egg membranes invariably starts around the pedipalps, and is likely initiated by the pressure of the egg teeth on the membranes. The embryonic cuticle, which bears the egg teeth, also opens along a predetermined breaking line around the carapace (Figures 18a, b). The resulting stage, which we name the 'postembryo' after [47] is completely immobile. The outer appearance is very similar to late embryonic stage 21, with legs that still bend ventrally. However, once released from the egg membranes, the postembryo is completely unfolded. The spinnerets and anal tubercle become more pronounced (Figures 18c-g) and the first pigments can be seen in the eyes. The pointed fangs have not yet fully extended (Figure 18c) and the endite of the pedipalps does not touch the other mouth parts (Figure 18d). The postembryo has two tiny tarsal claws on the tip of each leg (black arrow heads; Figure 18e). No sensory hairs are visible on the cuticle.
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.
Second instar
The prosoma has not grown substantially from the first instar: the opisthosoma has reduced drastically and is only slightly larger than the prosoma (Figures 19f, g). The second instar may have little or no yolk reserves, as suggested from the reduced size of the opisthosoma (Figure 19f). The walking legs have doubled in length, coinciding with the ability to walk. The cuticle is pigmented, with a grey striped pattern on a light brown background (Figure 19f). At this stage, the mother opens the cocoon and the young spiders leave it to forage and disperse [7].