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Post-ovipositional development of the monocled cobra, Naja kaouthia (Serpentes: Elapidae)
Zoology 105 (2002): 203–214 © by Urban & Fischer Verlag http://www.urbanfischer.de/journals/zoology
Post-ovipositional development of the monocled cobra, Naja kaouthia (Serpentes: Elapidae) Kate Jackson Museum of Comparative Zoology, Harvard University, Cambridge, MA, USA Received April 4, 2002 · Revised version received August 19, 2002 · Accepted August 23, 2002
Summary External morphological development between oviposition and hatching of the monocled cobra, Naja kaouthia, is described. Ten developmental stages are diagnosed according to nine features. These include fusion of the body wall musculature along the ventral midline, appearance of the endolymphatic ducts, formation of the eyelid, and the appearance of scales on the head and body. Additional observations of the developing skull are made at four of these ten stages, based on cleared and stained heads. Key words: development, elapidae, embryology, morphology, Naja, Serpentes, staging
Introduction The establishment of a staging table for a species standardizes developmental information and facilitates future study of the ontogeny of that species. Classic examples include Gosner’s (1960) staging table for anuran tadpoles, Nieuwkoop and Faber’s (1967) staging table for Xenopus, Hamburger and Hamilton’s (1951) staging table for the domestic chicken, and Rugh’s (1977) descriptions of the ontogeny of amphioxus, fish (Oryzias latipes), frog, chick, pig and mouse embryos. More recently staging tables have been published for less commonly studied vertebrates (Townsend and Stewart, 1985; Beggs et al., 2000; Dünker et al., 2000). Squamate reptiles are a large and diverse group, and their development has been reviewed by Hubert (1985). Within Squamata, snakes have been studied less than non-ophidian squamates and compared to most other vertebrate groups, relatively little is known about their development. Two staging tables exist for snakes: one for a colubrid, Thamnophis sirtalis (Zehr, 1962), and one for a viperid, Vipera aspis (Hubert, 1968).
Previous studies (Hubert and Dufauré, 1968; Zehr, 1962) used viviparous species, and the method for obtaining embryos was either to remove them from the bodies of freshly killed females, or to remove embryos at different stages by the multiple caesarian technique. These methods allowed embryos to be obtained at all developmental stages, starting with the initial divisions of the fertilized egg. Naja kaouthia, like most elapids, is an oviparous species. In the following study, embryos were obtained by harvesting eggs of different ages and fixing the embryos. The youngest embryos included in this study were harvested immediately following oviposition, and this staging table covers only postovipositional stages. However, later ontogenetic stages are likely to differ more between species of snakes than the early ones, and therefore to be of greatest interest for comparative studies. There has been much interest in recent years in the role of development in mediating evolutionary change. In snakes, more basic information is still needed about how development unfolds in different taxa before comparative developmental studies can add to our under-
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Corresponding author: Kate Jackson, Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA, phone: ++1-617-496 4065; fax:++1-617-495 5667; e-mail: [email protected] 0944-2006/02/105/03-203 $ 15.00/0
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standing of the evolutionary morphology of this group. The objectives of the following study are to broaden our knowledge of embryological development in snakes by adding data from an elapid snake, and to develop a staging table that may be used by subsequent researchers studying the embryos of elapid snakes.
Materials and methods Specimens examined and preparation
Embryos used in this study were Suphan-phase monocled cobras (Naja kaouthia). Ten clutches, totaling approximately 70 eggs, were obtained from a breeding colony at the Columbus Zoo, Columbus, Ohio. Adult snakes from which these eggs were obtained were collected in Suphan province in Thailand. “Suphan phase” refers to the distinctive coloration of these snakes: embryos lack pigmentation, except for the eyes which are dark. Staff at the Columbus Zoo report that hatchlings and adults appear pale yellow or pale lavender in color (D. Badgley, pers. comm.). In captivity, these snakes mate in late October and early November. Eggs are laid in late December and early January. The incubation period is 60–65 d. Oviposited eggs were kept in damp vermiculite, inside plastic bags, ventilated daily during inspection of the eggs. The bags were kept in plastic boxes with ventilation holes. Room temperature was maintained at 33 °C. Eggs were opened, and embryos removed and fixed, at regular intervals ranging from 2 to 57 d after oviposition. No eggs were allowed to develop until hatching, by agreement with the Columbus Zoo, because even hatchling snakes are venomous and dangerous to handle. To fix each embryo, the egg shell was cut open and the embryo carefully separated from the surrounding membranes. Embryos were fixed in 10% neutral-buffered formalin for 2 wk, and then stored in 70% ethanol. Older embryos (approximately 40 d after oviposition and beyond) were euthanized prior to fixation by intracardiac injection of sodium pentobarbital, and fixation included injection of formalin into the body cavity. Five specimens were cleared and differentially stained for bone and cartilage (Dingerkus and Uhler, 1977). One day-3 specimen was prepared for scanning electron microscopy (SEM). The embryo was critical-point dried, sputter-coated with gold, and examined using an AMR-1000 SEM at an acceleration voltage of 10 kV. All other fixed embryos were examined using a dissecting microscope. To illustrate this study, some fixed embryos were photographed using a Nikon Coolpix 990 digital camera and a Nikon SMZ-U dissecting microscope. All specimens have been deposited in the permanent collection of the Museum of Comparative Zoology, Harvard University. 204
Acquisition of data
All embryos were scored for nine morphological characters. The characters were chosen from those used in other snake staging tables, Hubert and Dufauré’s (1968) table for Vipera aspis, and Zehr’s (1962) table for Thamnophis sirtalis. The reason for choosing characters used by previous authors was to facilitate comparisons between Naja kaouthia and other ophidian species. Because the embryos in this study represent only postovipositional stages, characters were selected from the later stages described by Zehr and by Hubert and Dufauré, viz., stages 28 to 37 for Zehr’s table, and 33 to 43 for that of Hubert and Dufauré. There is little overlap between the morphological characters used by Zehr and those used by Hubert and Dufauré. Thus, wherever possible, characters used in both tables were included here. Characters scored for use in the staging table
Body length Length of the embryos was measured by aligning a thin copper wire along the coiled embryo and then straightening the wire and measuring it. Total body length in all embryos was measured. Tail length in all embryos was measured except the smallest ones, in which the position of the vent could not be discerned. Brain The brain is described as either visible or not visible. In early stages the brain is visible externally because the tissues surrounding it are thin and translucent. Later it becomes obscured as the skull and scales develop. Musculature The musculature of the body wall of embryos is described as open, partially fused, or completely fused. In early embryos the body wall is open along the ventral midline, so that the body cavity is open to the outside and there is nothing to hold the organs inside the body, except for thin mesenteries and the relative immobility of the developing embryo. As development progresses, the two sides of the lateral musculature of the trunk meet and fuse along the ventral midline, closing the body cavity. This fusion does not occur all at once, but rather as a gradual process. The region of the yolk stalk is the last part of the ventral midline to fuse, and the cardiac region fuses second to last. Heart The heart of the embryos is described as invisible, visible, or obscured. In the earliest embryos, the heart is not sufficiently developed to be visible. Later, the heart increases in size and is clearly visible through the thin, translucent body wall. Finally, the body wall thickens, and the heart can no longer be seen through it. Zoology 105 (2002) 3
Development of the cobra, Naja kaouthia
Results Body length and tail length were found to increase with age (Fig. 1). Cervical flexure increased with age from 90º in early embryos to 180º in older embryos (Fig. 2). Number of body coils, a character found by Zehr (1962) to increase with age in Thamnophis, was not found to correlate with age in N. kaouthia (Fig. 3) and was therefore not used as a staging character in the present study. In early embryos the hemipenes have not developed enough to be visible. Hemipenes later appear, and are
Fig. 1. Total body length (closed circles) and tail length (open triangles) of N. kaouthia embryos. Zero days incubated equals oviposition. The result of a bivariate correlation was statistically significant for both body length (R2 = 0.874; P < 0.001), and tail length (R2 = 0.785; P < 0.001).
Scales Scales appear first on the body and later on the head. Cervical flexure As embryos develop, the angle between the head and the body increases. In early embryos the head is bent ventrally at a 90° angle. It later extends to the 180° angle characteristic of adult snakes. Endolymphatic ducts The calcified endolymphatic ducts of the embryos are described as invisible, visible, or obscured. The endolymphatic ducts are invisible at first, but then calcify and become visible as two bright white spots on the occipital region of the head. Before hatching they become invisible again, obscured by scales.
Fig. 2. Cervical flexure in N. kaouthia embryos following oviposition. The result of a bivariate correlation was statistically significant (R2 = 0.716; P < 0.001).
Eyelid The condition of the eyelid fold is either not yet formed, partially closed, or completely formed. The immovable eyelid of the snake develops from a ring of tissue surrounding the edge of the eye, which gradually grows inward until it meets in the middle of the eye and covers the eye. Mandibular process The mandibular process, the lower jaw, at first does not extend as far as the eye, giving the appearance of an overbite, but then grows anteriorly, eventually extending anterior to the eye. The mandibular process is described as extending not as far as the eye, as far as the eye, or anterior to the eye. Zoology 105 (2002) 3
Fig. 3. Number of body coils in N. kaouthia embryos following oviposition. The results of a bivariate correlation were non-significant (R2 = 0.026; P = 0.234).
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Fig. 4. Stage five N. kaouthia head showing partially formed eyelid fold, and paired endolymphatic ducts: ef, eyelid fold; eld, endolymphatic ducts.
everted and clearly visible for much of embryological development. Finally, shortly before hatching, the hemipenes become inverted (folded inside the body). Presence or absence of the hemipenes was not used as a character because of the difficulty of distinguishing between females, which lack hemipenes, and males in which the hemipenes are not yet developed or are already inverted. Ornamentation of the hemipenes, a much used taxonomic character, was not used as a character in the present staging table because its considerable variability among taxa would make it unlikely to be transferable to other species. The appearance of pigmentation on different parts of the body was used extensively by Hubert and Dufauré (1968) and Zehr (1962) as a character for staging. In the present study it was not possible to use the many possible characters related to the appearance of pigmentation in the embryos because the specimens were Suphan phase, and never developed pigmentation, except in the eye. Figure 4 illustrates the appearance of the endolymphatic ducts and the partially formed eyelid fold.
Fig. 5. Scanning electron micrograph of a stage one N. kaouthia embryo, showing tightly coiled body, enlarged fourth ventricle, and mandibular process not reaching the eye.
Stage 2 (Fig. 6) This stage occurs from approximately six to nine days after oviposition. The mandibular process extends to or anterior to the eye. The heart can be seen through the translucent body wall. Body length is between 6 and 9 cm. Body wall musculature is open along the ventral midline. There are no scales on any part of the body. Cervical flexure is 90°. The brain is visible but the endolymphatic ducts are not. The eyelid has not started to form.
Staging table for Naja kaouthia
Stage 1 (Fig. 5) This stage lasts from two to six days after oviposition. Total body length is 5 to 8 cm, and cervical flexure is 90°. The lateral musculature of the body wall is open along the ventral midline. The heart is not yet visible. The brain is visible, but the endolymphatic ducts are not. The eyelid has not yet started to form. The mandibular process does not extend anterior to the eye. There are no scales on any part of the body. 206
Fig. 6. Stage two N. kaouthia embryo, with visceral organs visible, protruding through open ventral midline. Zoology 105 (2002) 3
Development of the cobra, Naja kaouthia
Stage 3 (Fig. 7) This stage occurs approximately 9 to 15 days following oviposition. The eyelid fold has started to form. The endolymphatic ducts are visible as two bright white spots on the occipital region of the head (Figs. 2–14). Scales have started to appear on the body, but not the head. Total body length is 7 to 11 cm, and the tail, measuring 1.5 to 2 cm, can be distinguished from the rest of the body in some specimens. The brain is visible. The body wall musculature is still open along the ventral midline. The heart is visible through the translucent body wall. Cervical flexure is 90°. Stage 4 (Fig. 8) This stage occurs approximately 15 to 20 days following oviposition. Body length has increased to 11 to 13 cm. Tail length is approximately 2 cm, and the tail can be distinguished from the rest of the body in all specimens. The brain is visible. The body wall musculature is open along the ventral midline. The heart is visible through the body wall. Scales are present on the body but not on the head. The brain is visible. Cervical flexure is 90°. Endolymphatic ducts are visible. The eyelid fold is beginning to form.
Fig. 8. Stage four N. kaouthia embryo, showing increased body length.
Stage 5 (Fig. 9) This stage occurs approximately 20 to 23 days following oviposition. Total body length is 14 to 15 cm, and tail length is 2 to 2.5 cm. The lateral musculature of the body wall has started to fuse along the ventral midline, but is still open in the region of the heart and in the region of the yolk stalk. The heart is visible through the body wall musculature. Cervical flexure has increased beyond 90°, but not as much as 180°. The brain is visible. Scales are present on the body but not on the head. Endolymphatic ducts are visible. The eyelid fold is partially formed.
Fig. 7. Stage three N. kaouthia embryo, with endolymphatic ducts visible. Zoology 105 (2002) 3
Stage 6 (Fig. 10) This stage occurs from approximately 22 to 25 days following oviposition. Total body length is 12 to 15 cm, and tail length is 2 to 3 cm. Scales now appear on the head as well as on the body. The brain is still visible through the skull. The heart is still visible through the lateral body 207
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wall musculature. The body wall musculature is fused in some parts of the ventral midline, but remains open in the region of the heart and of the yolk stalk. Cervical flexure is between 90° and 180°. Endolymphatic ducts are visible. The eyelid is partially formed.
Stage 7 (Fig. 11) This stage occurs from approximately 24 to 28 days following oviposition. Total body length is 14 to 18 cm, and tail length is 2.5 to 3.5 cm. The eyelid is now completely formed, as the edges of the developing eyelid have met in the middle and fused. The brain is visible through the skull. The heart is visible through the lateral body wall musculature. The body wall musculature is still not completely fused along the ventral midline. Scales are present on the head and body. Cervical flexure is between 90° and 180°. Endolymphatic ducts are visible. Stage 8 (Fig. 12) This stage occurs from approximately 28 to 38 days following oviposition. Total length is 16 to 25 cm, and tail length is 3 to 4 cm. Body wall musculature is now completely fused along the ventral midline, enclosing the organs of the body cavity inside. The brain is still visible through the skull. The heart is visible through the lateral body wall musculature. Cervical flexure is between 120° and 180°. Endolymphatic ducts are still visible through the skull.
Fig. 9. Stage five N. kaouthia embryo, showing heart protruding through open ventral midline.
Stage 9 (Fig. 13) This stage occurs from approximately 36 to 53 days following oviposition. Total body length is 26 to 29 cm, and tail length is 4 to 5 cm. The brain is no longer visible through the skull, but endolymphatic ducts can still be seen. The heart is still visible through the lateral body wall. The degree of cervical flexure is 180°.
Fig. 10. Stage six N. kaouthia embryo, with scales on head, and body musculature partially fused.
Fig. 11. Stage seven N. kaouthia embryo. Cervical flexure is now approximately 120º.
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Development of the cobra, Naja kaouthia
Fig. 14. Stage ten N. kaouthia embryo with heart and endolymphatic ducts no longer visible.
Stage 10 (Fig. 14) This is the final stage, and occurs from approximately 51 days following oviposition until hatching. Total body length is 28 to 34 cm, and tail length is 4.5 to 6 cm. The embryo closely resembles a neonate. The heart is no longer visible through the body wall, which has thickened. Endolymphatic ducts are no longer visible. Observations of skull development
Fig. 12. Stage eight N. kaouthia embryo with complete fusion of body wall musculature along ventral midline.
Fig. 13. Stage nine N. kaouthia embryo with brain no longer visible. Zoology 105 (2002) 3
In addition to the external morphological observations made for the staging table, the heads of four N. kaouthia at different stages were cleared and stained in order to supplement external data with accounts of the developing cranial skeleton. In the case of the smallest specimen, the stage-3 embryo, the entire body was cleared and stained. Previous accounts of snake cranial development have been done by serial sections rather than by clearing and staining (e.g., Kamal et al., 1970 a, b, c). Stage 3 embryo (Fig. 15) The chondrocranium is well formed. Some bone is visible, but most of it stains poorly with alizarin. Meckel’s and palatoquadrate cartilages are very clearly visible. Paired Meckel’s cartilages are well separated and not fused at the symphysis. Vertebral centra are ossified from just behind the head to nearly the base of the tail. There is a slight gradation in vertebral ossification, with anterior vertebrae being ossified to the greatest extent. A pair of longitudinal hyoid cartilages are joined and fused anteriorly on the midline. A pair of calcified endolymphatic ducts are present dorsomedial to the otic capsules at the posterior end of the skull. Paired trabecular cartilages are fused medially in the nasal region. In the upper jaw, three separate paired bones, the maxilla, ectopterygoid, and pterygoid, can be discerned as greyish, transluscent bones, although none has yet picked up the alizarin stain. The maxillae are paired and pre209
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Fig. 15. Cleared and stained head of a stage 3 embryo: ec, ectopterygoid; eld, endolymphatic ducts; hy, hyoid; mc, Meckel’s cartilage; mx, maxilla; nc, nasal capsule; oc, otic capsule; pq, palatoquadrate; pt, pterygoid; tc, trabecular cartilage.
Fig. 16. Medial view of a cleared and stained, hemisected head of a stage 7 embryo: ar, articular; dn, dentary; tr, tracheal rings; vc, vertebral centra.
sent ventral to the eyes. Each is a longitudinal bone that is dilated and bifurcated anteriorly. A longitudinal ectopterygoid lies in line with, and posterior to, each maxilla. A pair of elongate pterygoids is present. The posterior portion of the bone lies ventral to the otic capsule, whereas the anterior portion extends anteriorly but medial to the ectopterygoid. The premaxilla is visible as a longitudinal, rod-like bone, ventral to the nasal capsule. At least two bones are beginning to ossify in the lower jaw. The dentary invests the anterolateral surface of Meckel’s cartilage. The articular invests the posteromedial surface of Meckel’s cartilage, from the jaw articulation anteriorly to approximately halfway along the length of Meckel’s cartilage. There is a suggestion of additional ossification centers beginning to form in association with Meckel’s cartilage at this stage, but their presence cannot be confirmed from available material.
lage, and extends anteriorly to the middle of Meckel’s cartilage. Dorsal and medial to the palatoquadrate cartilage is a short, rod-like ossification that may correspond to the supratemporal bone. This bone lies between the dorsal process of the palatoquadrate cartilage and the adjacent otic capsule. The long series of tracheal cartilages is clearly visible with the cricoid cartilages at the anterior end.
Stage 7 embryo (Fig. 16) Ossification of the vertebrae is still confined to the centra, which are more ossified than those of the previous specimen. The calcified endolymphatic ducts are still prominent. In this specimen the nasal capsule was damaged during preparation, which displaced the premaxilla. In the upper jaw, the maxilla, ectopterygoid, and pterygoid can be seen, although they stain very faintly with alizarin. The maxilla extends further posteriorly to approach the ectopterygoid. The ectopterygoid is curved medially and, in turn, approaches the middle portion of the pterygoid, which extends anteriorly near the midline. A dentary can be discerned in the lower jaw. The dentary remains confined to the lateral aspect of Meckel’s cartilage, but extends more posteriorly than in the stage 3 specimen. The articular now invests both medial and lateral sides of Meckel’s carti210
Stage 8 embryo (Fig. 17) There is much more ossification than in the previous stage. Vertebral ossification is spreading outward from the centra into the neural arches. The ribs are also starting to ossify. Approximately the posteroventral third of the otic capsule is now ossified. Calcified endolymphatic ducts are still visible. Anterior to the otic capsule, the braincase is partially ossified. The floor of the braincase posterior to the eye, formed by the basioccipital and basisphenoid, is ossified, as is the anterior portion of the braincase dorso-anterior to the eye, formed by the partially-ossified frontal. The parietal is also partially ossified. The supratemporal is well-ossified and lies between the otic process of the quadrate and the otic capsule. The roof and sides of the braincase are still open. The following bones are present in the otic capsule: exoccipital, supraoccipital and prootic. The basioccipital and basisphenoid are present. The nasal has partly ossified. The bones of the upper jaw are well-ossified. Anteriorly on the maxilla, fangs can be seen (these are not yet ankylosed to the maxilla). The posterior end of the maxilla articulates with the ectopterygoid, and the ectopterygoid curves medially to articulate with the pterygoid. Medial to these lie the palatine and the pterygoid. There is still some cartilage at the articulation between the maxilla and the ectopterygoid. Zoology 105 (2002) 3
Development of the cobra, Naja kaouthia
Fig. 17. Medial view of a cleared and stained, hemisected head of a stage 8 embryo: bo, basioccipital; cb, compound bone; fr, frontal; pa, parietal; pal, palatine; pf, postfrontal; pr, prefrontal; sp, splenial; vo, vomer.
The quadrate bone is well ossified, and only a small remnant of cartilage is left at the jaw articulation and at the otic process (i.e., the dorsal part of the quadrate). In the lower jaw, the dentary, splenial and compound bone are visible. The dentary is more extensive than in the previous specimen, surrounding Meckel’s cartilage. Teeth, although unstained, are present on the dentary. A compound bone, immediately posterior to the dentary and splenial, articulates with the quadrate. The compound bone is represented by at least three separate ossification centers: one short bone medially that articulates with the splenial; a second bone that is replacing cartilage at the jaw articulation; and a third much larger bone that invests the remainder of Meckel’s cartilage. At the anterior tip of the dentary, the anteriormost tip of Meckel’s cartilage can be seen. The bones of the lower jaw wrap around Meckel’s cartilage, leaving an opening between the dentary and splenial and the compound bone, and are open dorsally along the anterior end. The dentary is much more extensive than in the stage 7 specimen, and invests Meckel’s cartilage on all sides. A tiny splenial is present, separate from Meckel’s cartilage. Stage 10 embryo (Fig. 18) Ossification of the skull is nearly complete. Vertebrae are completely ossified, except for a small amount of blue-staining cartilage at the point of articulation between some of the vertebrae. The otic capsule, comprising the prootic, supraoccipital, basioccipital, and exoccipital, is completely ossified. In the braincase, the frontal and parietal meet dorsolaterally along the margin of the orbit. Dorsally, paired frontals and parietals are well separated medially, leaving a large fronto-parietal fontanelle in the skull roof. The parietal and frontal on each side are separated ventrally by a wide, unossiZoology 105 (2002) 3
Fig. 18. Medial view of a cleared and stained, hemisected head of a stage 10 embryo: bs, basisphenoid; otoc, otoccipital; pm, premaxilla; pro, prootic; psp, parasphenoid; soc, supraoccipital.
fied area. The basisphenoid and parasphenoid are both well-developed. The exoccipital is well developed and paired. Immediately anterior to them is the supraoccipital, which straddles the midline. In the paired parietals, alizarin red positive areas are well-developed laterally. However, the uncalcified alizarin-negative portions of the bone extend to the midline. The prefrontal and postfrontal delimit the anterior and posterior margins of the orbit respectively. Anterior to the braincase the nasal capsule is partly ossified, but approximately half of the nasal capsule remains cartilaginous. The nasal bones are triangular in shape viewed from above, and each descends ventrally in the midline. The bones of the upper jaw are clearly visible, as they were in the previous specimen, but are more completely ossified. The ossified premaxilla is visible ventral to the nasal capsule, but still only barely in direct contact with it. The maxilla, ectopterygoid, and palatine are present. The maxilla bears prominent fangs, and teeth are also present on the palatine, pterygoid, and in the lower jaw the dentary. In the lower jaw, the dentary, splenial, and compound bone are visible. The blue-stained, cartilaginous tip of Meckel’s cartilage protrudes anteriorly and medially on the lower jaw. The supratemporal is present. The quadrate is fully ossified. The blue-stained, rings of the trachea and the cricoid cartilage are still cartilaginous, as is the hyoid.
Discussion I have documented the timing of embryonic development in Naja kaouthia in ten stages from oviposition to hatching. Key changes that are visible externally include fusion of the body wall musculature along the 211
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ventral midline, the initial appearance of calcified endolymphatic ducts, increase in cervical flexure from 90° in the early embryo to 180° characteristic of neonate and adult snakes, formation of the eyelid, and initial appearance of scales on the body and the head. One goal of the present study was to correlate stages of Naja kaouthia development with previously published staging tables for other snakes: Zehr’s (1962) staging table for a colubrid, Thamnophis sirtalis, and Hubert and Dufauré’s (1968) table for a viperid, Vipera aspis. Unfortunately, correlating stages among the different tables is not possible except in the most general way. One problem is that Zehr and Hubert and Dufauré mostly used different characters; there are very few characters in common that can be used to correlate stages between their two tables. Another problem is that many characters used in these tables are specific to the species being described and not applicable to all snakes. For example, Hubert and Dufauré (1968) use the appearance of the upturned nose characteristic of V. aspis as a character,
and also details of ornamentation of the hemipenes that are unique to this species. Zehr (1962) relies heavily on the number of body coils as a character for determining stages, but in N. kaouthia there is no correlation between number of coils and stage of development (Fig. 3). Finally, efforts to correlate the present table with others were thwarted by heterochrony, and by the discovery that even for characters that overlap between staging tables, the order of appearance of characters is not consistent across ophidian taxa. For example, formation of the eyelid fold is complete at stage 32 for T. sirtalis (Zehr 1962) and stage 7 for N. kaouthia. The scales appear on the head at stage 36 in T. sirtalis and stage 6 in N. kaouthia. Thus, the three existing staging tables for snakes can only be correlated in the most general way: Stage 1 in the present table for N. kaouthia corresponds roughly to stage 25 for T. sirtalis and stage 35 for V. aspis. This correlation is based on the fact that at these stages the maxillary process extends to or anterior to the eye, but the eyelid fold has not yet started to form.
Table 1. Occurence of skull ossifications during post-ovipositional development. bone ar bo bs cb dn ec eld ex fr fg hy mc mx nc oc otoc pa pal pf pm pq pr pro psp pt qu soc sp st tc th tr vc vo
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articular basioccipital basisphenoid compound bone dentary ectopterygoid endolymphatic ducts exocciptial frontal fangs hyoid Meckel’s cartilage maxilla nasal capsule otic capsule otoccipital parietal palatine postfrontal premaxilla palatoquadrate prefrontal prootic parasphenoid pterygoid quadrate supraoccipital splenial supratemporal trabecular cartilage teeth tracheal rings vertebral centra vomer
stage 3
stage 7
stage 8
stage 10
present absent absent absent present present present absent absent absent present present present present present absent absent absent absent present present absent absent absent present absent absent absent absent present absent absent present absent
present absent absent absent present present present absent absent absent present present present present present absent absent absent absent present present absent absent absent present absent absent absent present present absent present present absent
present present present present present present present present present present present present present present present absent present present present present present present present absent present present present present present present present present present present
present present present present present present present present present present present present present present present present present present present present present present present present present present present present present present present present present present Zoology 105 (2002) 3
Development of the cobra, Naja kaouthia
Formation of the maxilla in Naja kaouthia is of particular interest in light of hypotheses concerning how the venom system evolved in colubroid snakes. In colubroid snakes, teeth may occur on the maxilla, dentary, palatine, and pterygoid. Fangs, when they occur, always occur on the maxilla, and for this reason maxillary dentition has been the subject of considerable study. Several authors have noted a morphological difference between the anterior and posterior maxillary teeth of colubroids, and this has led to speculation about the evolutionary origins of fangs. Bogert (1943) derived the tubular front fangs of viperids from the tubular front fangs of elapids, and these in turn from the enlarged, grooved posterior teeth of other colubroid snakes. Hence the anterior maxillary fang had a posterior maxillary origin. Anthony (1955), on the basis of arranging living taxa in a morphocline, derived the elapid anterior maxillary fang from an enlarged anterior maxillary tooth, but the viperid anterior maxillary fang from an enlarged posterior tooth that migrated anteriorly along the maxilla. Kardong (1980, 1982) argued on the basis of functional morphological studies of the maxilla of colubroid snakes, and consideration of the post-orbital position of the venom gland, that the elapid and the viperid front fang both evolved from an enlarged posterior maxillary tooth that migrated anteriorly. Jackson and Fritts (1995) argued on the basis of morphological markers on colubroid teeth, that the elapid front fang evolved from a posterior maxillary tooth. Jackson and Fritts (1996) described an example of an opisthoglyphous colubrid with enlarged and grooved anterior maxillary fangs, and suggested that a front fang could have evolved from an enlarged anterior maxillary tooth by means of such a pathway. Whatever the evolutionary pathway that led to the formation of front fangs of different types, the developmental pathway also remains to be elucidated. Haluska and Alberch (1983) reported that in Elaphe obsoleta, a colubrid snake, the maxilla forms from two separate centers of ossification, which eventually grow together and fuse, forming a single maxillary bone. McDowell (1986) noted the possible evolutionary significance of this observation, noting that in the Bolyeridae, a sistergroup to the Colubridae, the adult maxilla is in two parts and is hinged at the middle. The morphology of the bolyerid maxilla is described in detail by Cundall and Irish (1989). Thus, the distinction between anterior and posterior maxillary teeth and fangs is an important one for colubroid snakes, and may be reflected in the embryological development of the maxilla. In the present study, however, observations of cranial development in Naja kaouthia showed only one center of ossification for the maxilla. However, this could mean one of several things. First, there is the possibility that the specimens Zoology 105 (2002) 3
included in this study happened not to include the crucial stage where the maxilla is in two parts. Secondly, it could be that in elapid snakes, one part of the two-part maxilla has been lost, and only the part that bears venom-conducting teeth has been retained. Finally, the two-part maxilla of developing colubroid snakes may be a peculiarity of Elaphe obsoleta, and not a general feature of colubrid snakes, resting, as it does, on a single observation by Haluska and Alberch (1983). Savitzky (1992) reports only one center of ossification of the maxilla in crotaline snakes. To really resolve the question of whether the colubroid maxilla forms from one or two centers of ossification will require detailed histological studies, on several colubroid taxa, focusing on the stage at which the maxilla appears. The present study finds no evidence for more than one center of ossification. Thus, the post-ovipositional development of Naja koauthia can be divided into ten stages, using characters used in the description of embryological development of other snake taxa, as well as new characters. It is hoped that this staging table will provide a foundation on which future studies of the development of elapid snakes can be based.
Acknowledgements This work is dedicated to the memory of the late Mike Goode, whose cobra breeding program at the Columbus Zoo provided all the eggs used in this study. I thank the staff of the Reptile Section of the Columbus Zoo for logistical assistance and for information about the origin of the snakes in their breeding program. N. Kley took the photographs from which figures 4, and 6 to 18 were made. J. Hanken provided assistance with the identification of bones in the cleared and stained specimens. J. Gross and H. Thompson helped with the statistical analysis. Helpful comments on the manuscript were provided by E. Brainerd, J. Hanken, F. Jenkins and two anonymous reviewers.
References Anthony, J. 1955. Essai sur l’évolution anatomique de l’appareil venimeux des ophidiens. Ann. Sci. Nat. Zool. 11: 7–53. Beggs, K., J. Young, A. Georges and P. West. 2000. Ageing the eggs and embryos of the pig-nosed turtle, Carettochelys insculpta (Chelonia: Carettochelydidae), from northern Australia. Can. J. Zool. 78: 373–392. Bogert, C. M. 1943. Dentitional phenomena in cobras and other elapids with notes on adaptive modifications of the fangs. Bull. Am. Mus. Nat. Hist. 131: 285–360. Cundall, D. and F. Irish. 1989. The function of the intramaxillary joint in the Round Island boa, Casarea dussumieri. J. Zool., Lond. 217: 569–598.
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Dingerkus, G. and D. Uhler. 1977. Enzyme clearing of Alcian blue stained whole small vertebrates for demonstration of cartilage. Stain Technology 52: 229–232. Dünker, N., M.H. Wake and W.M. Olson. 2000. Embryonic and larval development in the caecilian Ichthyophis kohtaoensis (Amphibia, Gymnophiona): A staging table. J. Morphol. 243: 3–34. Gosner, K.L. 1960. A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16: 183–190. Haluska, F. and P. Alberch. 1983. The cranial development of Elaphe obsoleta (Ophidia Colubridae). J. Morphol. 178: 37–55. Hamburger, V. and H.L. Hamilton. 1951. A series of normal stages in the development of the chick embryo. Univ. of Chicago Press, Chicago, pp. 213. Hubert, J. 1985. Embryology of the Squamata. In:. Biology of the Reptilia, Volume 15, Development B (C. Gans and F. Billett, eds.). John Wiley & Sons, New York, pp. 1-713. Hubert, J. and J.P. Dufauré. 1968. Table de developpement de la vipere aspic, Vipera aspis L. Bull. Soc. Zool. France 93: 135–148. Jackson, K. and T.H. Fritts. 1995. Evidence from tooth surface morphology for a posterior maxillary origin of the proteroglyph fang. Amphibia-Reptilia 16: 273–288. Jackson, K. and T.H. Fritts. 1996. Observations of a grooved anterior tooth in Psammodynastes pulverulentus: Does the Mock Viper resemble a protoelapid? J. Herpetol. 30: 128–131. Kamal, A.M., H.G. Hammouda and F.M. Mokhtar. 1970a. The development of the osteocranium of the Egyptian Cobra: I. The embryonic osteocranium. Acta Zoologica 1970: 1–17.
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Kamal, A.M., H.G. Hammouda and F.M. Mokhtar. 1970b. The development of the osteocranium of the Egyptian Cobra: II. The median dorsal bones, bones of the upper jaw, circumorbital series, and occipital ring of the adult osteocranium. Acta Zoologica 1970: 19–30. Kamal, A.M., H.G. Hammouda and F.M. Mokhtar. 1970c. The development of the osteocranium of the Egyptian Cobra: III. The otic capsule, palate, temporal bones, lower jaw and hyoid apparatus of the adult osteocranium. Acta Zoologica 1970: 31–42. Kardong, K.V. 1980. Evolutionary patterns in advanced snakes. Amer. Zool. 20: 269–282. Kardong, K.V. 1982. The evolution of the venom apparatus in snakes from colubrids to viperids and elapids. Mem. Inst. Butantan 46: 105–118. McDowell, S.B. 1986. The architecture of the corner of the mouth of colubroid snakes. J. Herpetol. 20: 353–407. Nieuwkoop, P.D. and J. Faber. 1967. Normal Table of Xenopus laevis (Daudin). Second Edition, North Holland Publ. Co. Amsterdam. Rugh, R. 1977. A Guide to Vertebrate Development. Seventh Edition, Burgess Publishing Co., Minneapolis, Minnesota. Savitzky, A. 1992. Embryonic development of the maxillary and prefrontal bones of crotaline snakes. In Biology of the Pitvipers (J.A. Campbell and E.D. Brodie, Jr., eds.). Selva: Tyler, Texas, pp. 466. Townsend, D.S. and M.M. Stewart. 1985. Direct development in Eleutherodactylus coqui (Anura: Leptodactylidae): A staging table. Copeia 1985: 423–436. Zehr, D.R. 1962. Stages in the normal development of the common garter snake Thamnophis sirtalis. Copeia 1962: 322–329.
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Post-ovipositional development of the monocled cobra, Naja kaouthia (Serpentes: Elapidae) Kate Jackson Museum of Comparative Zoology, Harvard University, Cambridge, MA, USA Received April 4, 2002 · Revised version received August 19, 2002 · Accepted August 23, 2002
Summary External morphological development between oviposition and hatching of the monocled cobra, Naja kaouthia, is described. Ten developmental stages are diagnosed according to nine features. These include fusion of the body wall musculature along the ventral midline, appearance of the endolymphatic ducts, formation of the eyelid, and the appearance of scales on the head and body. Additional observations of the developing skull are made at four of these ten stages, based on cleared and stained heads. Key words: development, elapidae, embryology, morphology, Naja, Serpentes, staging
Introduction The establishment of a staging table for a species standardizes developmental information and facilitates future study of the ontogeny of that species. Classic examples include Gosner’s (1960) staging table for anuran tadpoles, Nieuwkoop and Faber’s (1967) staging table for Xenopus, Hamburger and Hamilton’s (1951) staging table for the domestic chicken, and Rugh’s (1977) descriptions of the ontogeny of amphioxus, fish (Oryzias latipes), frog, chick, pig and mouse embryos. More recently staging tables have been published for less commonly studied vertebrates (Townsend and Stewart, 1985; Beggs et al., 2000; Dünker et al., 2000). Squamate reptiles are a large and diverse group, and their development has been reviewed by Hubert (1985). Within Squamata, snakes have been studied less than non-ophidian squamates and compared to most other vertebrate groups, relatively little is known about their development. Two staging tables exist for snakes: one for a colubrid, Thamnophis sirtalis (Zehr, 1962), and one for a viperid, Vipera aspis (Hubert, 1968).
Previous studies (Hubert and Dufauré, 1968; Zehr, 1962) used viviparous species, and the method for obtaining embryos was either to remove them from the bodies of freshly killed females, or to remove embryos at different stages by the multiple caesarian technique. These methods allowed embryos to be obtained at all developmental stages, starting with the initial divisions of the fertilized egg. Naja kaouthia, like most elapids, is an oviparous species. In the following study, embryos were obtained by harvesting eggs of different ages and fixing the embryos. The youngest embryos included in this study were harvested immediately following oviposition, and this staging table covers only postovipositional stages. However, later ontogenetic stages are likely to differ more between species of snakes than the early ones, and therefore to be of greatest interest for comparative studies. There has been much interest in recent years in the role of development in mediating evolutionary change. In snakes, more basic information is still needed about how development unfolds in different taxa before comparative developmental studies can add to our under-
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Corresponding author: Kate Jackson, Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA, phone: ++1-617-496 4065; fax:++1-617-495 5667; e-mail: [email protected] 0944-2006/02/105/03-203 $ 15.00/0
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standing of the evolutionary morphology of this group. The objectives of the following study are to broaden our knowledge of embryological development in snakes by adding data from an elapid snake, and to develop a staging table that may be used by subsequent researchers studying the embryos of elapid snakes.
Materials and methods Specimens examined and preparation
Embryos used in this study were Suphan-phase monocled cobras (Naja kaouthia). Ten clutches, totaling approximately 70 eggs, were obtained from a breeding colony at the Columbus Zoo, Columbus, Ohio. Adult snakes from which these eggs were obtained were collected in Suphan province in Thailand. “Suphan phase” refers to the distinctive coloration of these snakes: embryos lack pigmentation, except for the eyes which are dark. Staff at the Columbus Zoo report that hatchlings and adults appear pale yellow or pale lavender in color (D. Badgley, pers. comm.). In captivity, these snakes mate in late October and early November. Eggs are laid in late December and early January. The incubation period is 60–65 d. Oviposited eggs were kept in damp vermiculite, inside plastic bags, ventilated daily during inspection of the eggs. The bags were kept in plastic boxes with ventilation holes. Room temperature was maintained at 33 °C. Eggs were opened, and embryos removed and fixed, at regular intervals ranging from 2 to 57 d after oviposition. No eggs were allowed to develop until hatching, by agreement with the Columbus Zoo, because even hatchling snakes are venomous and dangerous to handle. To fix each embryo, the egg shell was cut open and the embryo carefully separated from the surrounding membranes. Embryos were fixed in 10% neutral-buffered formalin for 2 wk, and then stored in 70% ethanol. Older embryos (approximately 40 d after oviposition and beyond) were euthanized prior to fixation by intracardiac injection of sodium pentobarbital, and fixation included injection of formalin into the body cavity. Five specimens were cleared and differentially stained for bone and cartilage (Dingerkus and Uhler, 1977). One day-3 specimen was prepared for scanning electron microscopy (SEM). The embryo was critical-point dried, sputter-coated with gold, and examined using an AMR-1000 SEM at an acceleration voltage of 10 kV. All other fixed embryos were examined using a dissecting microscope. To illustrate this study, some fixed embryos were photographed using a Nikon Coolpix 990 digital camera and a Nikon SMZ-U dissecting microscope. All specimens have been deposited in the permanent collection of the Museum of Comparative Zoology, Harvard University. 204
Acquisition of data
All embryos were scored for nine morphological characters. The characters were chosen from those used in other snake staging tables, Hubert and Dufauré’s (1968) table for Vipera aspis, and Zehr’s (1962) table for Thamnophis sirtalis. The reason for choosing characters used by previous authors was to facilitate comparisons between Naja kaouthia and other ophidian species. Because the embryos in this study represent only postovipositional stages, characters were selected from the later stages described by Zehr and by Hubert and Dufauré, viz., stages 28 to 37 for Zehr’s table, and 33 to 43 for that of Hubert and Dufauré. There is little overlap between the morphological characters used by Zehr and those used by Hubert and Dufauré. Thus, wherever possible, characters used in both tables were included here. Characters scored for use in the staging table
Body length Length of the embryos was measured by aligning a thin copper wire along the coiled embryo and then straightening the wire and measuring it. Total body length in all embryos was measured. Tail length in all embryos was measured except the smallest ones, in which the position of the vent could not be discerned. Brain The brain is described as either visible or not visible. In early stages the brain is visible externally because the tissues surrounding it are thin and translucent. Later it becomes obscured as the skull and scales develop. Musculature The musculature of the body wall of embryos is described as open, partially fused, or completely fused. In early embryos the body wall is open along the ventral midline, so that the body cavity is open to the outside and there is nothing to hold the organs inside the body, except for thin mesenteries and the relative immobility of the developing embryo. As development progresses, the two sides of the lateral musculature of the trunk meet and fuse along the ventral midline, closing the body cavity. This fusion does not occur all at once, but rather as a gradual process. The region of the yolk stalk is the last part of the ventral midline to fuse, and the cardiac region fuses second to last. Heart The heart of the embryos is described as invisible, visible, or obscured. In the earliest embryos, the heart is not sufficiently developed to be visible. Later, the heart increases in size and is clearly visible through the thin, translucent body wall. Finally, the body wall thickens, and the heart can no longer be seen through it. Zoology 105 (2002) 3
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Results Body length and tail length were found to increase with age (Fig. 1). Cervical flexure increased with age from 90º in early embryos to 180º in older embryos (Fig. 2). Number of body coils, a character found by Zehr (1962) to increase with age in Thamnophis, was not found to correlate with age in N. kaouthia (Fig. 3) and was therefore not used as a staging character in the present study. In early embryos the hemipenes have not developed enough to be visible. Hemipenes later appear, and are
Fig. 1. Total body length (closed circles) and tail length (open triangles) of N. kaouthia embryos. Zero days incubated equals oviposition. The result of a bivariate correlation was statistically significant for both body length (R2 = 0.874; P < 0.001), and tail length (R2 = 0.785; P < 0.001).
Scales Scales appear first on the body and later on the head. Cervical flexure As embryos develop, the angle between the head and the body increases. In early embryos the head is bent ventrally at a 90° angle. It later extends to the 180° angle characteristic of adult snakes. Endolymphatic ducts The calcified endolymphatic ducts of the embryos are described as invisible, visible, or obscured. The endolymphatic ducts are invisible at first, but then calcify and become visible as two bright white spots on the occipital region of the head. Before hatching they become invisible again, obscured by scales.
Fig. 2. Cervical flexure in N. kaouthia embryos following oviposition. The result of a bivariate correlation was statistically significant (R2 = 0.716; P < 0.001).
Eyelid The condition of the eyelid fold is either not yet formed, partially closed, or completely formed. The immovable eyelid of the snake develops from a ring of tissue surrounding the edge of the eye, which gradually grows inward until it meets in the middle of the eye and covers the eye. Mandibular process The mandibular process, the lower jaw, at first does not extend as far as the eye, giving the appearance of an overbite, but then grows anteriorly, eventually extending anterior to the eye. The mandibular process is described as extending not as far as the eye, as far as the eye, or anterior to the eye. Zoology 105 (2002) 3
Fig. 3. Number of body coils in N. kaouthia embryos following oviposition. The results of a bivariate correlation were non-significant (R2 = 0.026; P = 0.234).
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Fig. 4. Stage five N. kaouthia head showing partially formed eyelid fold, and paired endolymphatic ducts: ef, eyelid fold; eld, endolymphatic ducts.
everted and clearly visible for much of embryological development. Finally, shortly before hatching, the hemipenes become inverted (folded inside the body). Presence or absence of the hemipenes was not used as a character because of the difficulty of distinguishing between females, which lack hemipenes, and males in which the hemipenes are not yet developed or are already inverted. Ornamentation of the hemipenes, a much used taxonomic character, was not used as a character in the present staging table because its considerable variability among taxa would make it unlikely to be transferable to other species. The appearance of pigmentation on different parts of the body was used extensively by Hubert and Dufauré (1968) and Zehr (1962) as a character for staging. In the present study it was not possible to use the many possible characters related to the appearance of pigmentation in the embryos because the specimens were Suphan phase, and never developed pigmentation, except in the eye. Figure 4 illustrates the appearance of the endolymphatic ducts and the partially formed eyelid fold.
Fig. 5. Scanning electron micrograph of a stage one N. kaouthia embryo, showing tightly coiled body, enlarged fourth ventricle, and mandibular process not reaching the eye.
Stage 2 (Fig. 6) This stage occurs from approximately six to nine days after oviposition. The mandibular process extends to or anterior to the eye. The heart can be seen through the translucent body wall. Body length is between 6 and 9 cm. Body wall musculature is open along the ventral midline. There are no scales on any part of the body. Cervical flexure is 90°. The brain is visible but the endolymphatic ducts are not. The eyelid has not started to form.
Staging table for Naja kaouthia
Stage 1 (Fig. 5) This stage lasts from two to six days after oviposition. Total body length is 5 to 8 cm, and cervical flexure is 90°. The lateral musculature of the body wall is open along the ventral midline. The heart is not yet visible. The brain is visible, but the endolymphatic ducts are not. The eyelid has not yet started to form. The mandibular process does not extend anterior to the eye. There are no scales on any part of the body. 206
Fig. 6. Stage two N. kaouthia embryo, with visceral organs visible, protruding through open ventral midline. Zoology 105 (2002) 3
Development of the cobra, Naja kaouthia
Stage 3 (Fig. 7) This stage occurs approximately 9 to 15 days following oviposition. The eyelid fold has started to form. The endolymphatic ducts are visible as two bright white spots on the occipital region of the head (Figs. 2–14). Scales have started to appear on the body, but not the head. Total body length is 7 to 11 cm, and the tail, measuring 1.5 to 2 cm, can be distinguished from the rest of the body in some specimens. The brain is visible. The body wall musculature is still open along the ventral midline. The heart is visible through the translucent body wall. Cervical flexure is 90°. Stage 4 (Fig. 8) This stage occurs approximately 15 to 20 days following oviposition. Body length has increased to 11 to 13 cm. Tail length is approximately 2 cm, and the tail can be distinguished from the rest of the body in all specimens. The brain is visible. The body wall musculature is open along the ventral midline. The heart is visible through the body wall. Scales are present on the body but not on the head. The brain is visible. Cervical flexure is 90°. Endolymphatic ducts are visible. The eyelid fold is beginning to form.
Fig. 8. Stage four N. kaouthia embryo, showing increased body length.
Stage 5 (Fig. 9) This stage occurs approximately 20 to 23 days following oviposition. Total body length is 14 to 15 cm, and tail length is 2 to 2.5 cm. The lateral musculature of the body wall has started to fuse along the ventral midline, but is still open in the region of the heart and in the region of the yolk stalk. The heart is visible through the body wall musculature. Cervical flexure has increased beyond 90°, but not as much as 180°. The brain is visible. Scales are present on the body but not on the head. Endolymphatic ducts are visible. The eyelid fold is partially formed.
Fig. 7. Stage three N. kaouthia embryo, with endolymphatic ducts visible. Zoology 105 (2002) 3
Stage 6 (Fig. 10) This stage occurs from approximately 22 to 25 days following oviposition. Total body length is 12 to 15 cm, and tail length is 2 to 3 cm. Scales now appear on the head as well as on the body. The brain is still visible through the skull. The heart is still visible through the lateral body 207
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wall musculature. The body wall musculature is fused in some parts of the ventral midline, but remains open in the region of the heart and of the yolk stalk. Cervical flexure is between 90° and 180°. Endolymphatic ducts are visible. The eyelid is partially formed.
Stage 7 (Fig. 11) This stage occurs from approximately 24 to 28 days following oviposition. Total body length is 14 to 18 cm, and tail length is 2.5 to 3.5 cm. The eyelid is now completely formed, as the edges of the developing eyelid have met in the middle and fused. The brain is visible through the skull. The heart is visible through the lateral body wall musculature. The body wall musculature is still not completely fused along the ventral midline. Scales are present on the head and body. Cervical flexure is between 90° and 180°. Endolymphatic ducts are visible. Stage 8 (Fig. 12) This stage occurs from approximately 28 to 38 days following oviposition. Total length is 16 to 25 cm, and tail length is 3 to 4 cm. Body wall musculature is now completely fused along the ventral midline, enclosing the organs of the body cavity inside. The brain is still visible through the skull. The heart is visible through the lateral body wall musculature. Cervical flexure is between 120° and 180°. Endolymphatic ducts are still visible through the skull.
Fig. 9. Stage five N. kaouthia embryo, showing heart protruding through open ventral midline.
Stage 9 (Fig. 13) This stage occurs from approximately 36 to 53 days following oviposition. Total body length is 26 to 29 cm, and tail length is 4 to 5 cm. The brain is no longer visible through the skull, but endolymphatic ducts can still be seen. The heart is still visible through the lateral body wall. The degree of cervical flexure is 180°.
Fig. 10. Stage six N. kaouthia embryo, with scales on head, and body musculature partially fused.
Fig. 11. Stage seven N. kaouthia embryo. Cervical flexure is now approximately 120º.
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Fig. 14. Stage ten N. kaouthia embryo with heart and endolymphatic ducts no longer visible.
Stage 10 (Fig. 14) This is the final stage, and occurs from approximately 51 days following oviposition until hatching. Total body length is 28 to 34 cm, and tail length is 4.5 to 6 cm. The embryo closely resembles a neonate. The heart is no longer visible through the body wall, which has thickened. Endolymphatic ducts are no longer visible. Observations of skull development
Fig. 12. Stage eight N. kaouthia embryo with complete fusion of body wall musculature along ventral midline.
Fig. 13. Stage nine N. kaouthia embryo with brain no longer visible. Zoology 105 (2002) 3
In addition to the external morphological observations made for the staging table, the heads of four N. kaouthia at different stages were cleared and stained in order to supplement external data with accounts of the developing cranial skeleton. In the case of the smallest specimen, the stage-3 embryo, the entire body was cleared and stained. Previous accounts of snake cranial development have been done by serial sections rather than by clearing and staining (e.g., Kamal et al., 1970 a, b, c). Stage 3 embryo (Fig. 15) The chondrocranium is well formed. Some bone is visible, but most of it stains poorly with alizarin. Meckel’s and palatoquadrate cartilages are very clearly visible. Paired Meckel’s cartilages are well separated and not fused at the symphysis. Vertebral centra are ossified from just behind the head to nearly the base of the tail. There is a slight gradation in vertebral ossification, with anterior vertebrae being ossified to the greatest extent. A pair of longitudinal hyoid cartilages are joined and fused anteriorly on the midline. A pair of calcified endolymphatic ducts are present dorsomedial to the otic capsules at the posterior end of the skull. Paired trabecular cartilages are fused medially in the nasal region. In the upper jaw, three separate paired bones, the maxilla, ectopterygoid, and pterygoid, can be discerned as greyish, transluscent bones, although none has yet picked up the alizarin stain. The maxillae are paired and pre209
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Fig. 15. Cleared and stained head of a stage 3 embryo: ec, ectopterygoid; eld, endolymphatic ducts; hy, hyoid; mc, Meckel’s cartilage; mx, maxilla; nc, nasal capsule; oc, otic capsule; pq, palatoquadrate; pt, pterygoid; tc, trabecular cartilage.
Fig. 16. Medial view of a cleared and stained, hemisected head of a stage 7 embryo: ar, articular; dn, dentary; tr, tracheal rings; vc, vertebral centra.
sent ventral to the eyes. Each is a longitudinal bone that is dilated and bifurcated anteriorly. A longitudinal ectopterygoid lies in line with, and posterior to, each maxilla. A pair of elongate pterygoids is present. The posterior portion of the bone lies ventral to the otic capsule, whereas the anterior portion extends anteriorly but medial to the ectopterygoid. The premaxilla is visible as a longitudinal, rod-like bone, ventral to the nasal capsule. At least two bones are beginning to ossify in the lower jaw. The dentary invests the anterolateral surface of Meckel’s cartilage. The articular invests the posteromedial surface of Meckel’s cartilage, from the jaw articulation anteriorly to approximately halfway along the length of Meckel’s cartilage. There is a suggestion of additional ossification centers beginning to form in association with Meckel’s cartilage at this stage, but their presence cannot be confirmed from available material.
lage, and extends anteriorly to the middle of Meckel’s cartilage. Dorsal and medial to the palatoquadrate cartilage is a short, rod-like ossification that may correspond to the supratemporal bone. This bone lies between the dorsal process of the palatoquadrate cartilage and the adjacent otic capsule. The long series of tracheal cartilages is clearly visible with the cricoid cartilages at the anterior end.
Stage 7 embryo (Fig. 16) Ossification of the vertebrae is still confined to the centra, which are more ossified than those of the previous specimen. The calcified endolymphatic ducts are still prominent. In this specimen the nasal capsule was damaged during preparation, which displaced the premaxilla. In the upper jaw, the maxilla, ectopterygoid, and pterygoid can be seen, although they stain very faintly with alizarin. The maxilla extends further posteriorly to approach the ectopterygoid. The ectopterygoid is curved medially and, in turn, approaches the middle portion of the pterygoid, which extends anteriorly near the midline. A dentary can be discerned in the lower jaw. The dentary remains confined to the lateral aspect of Meckel’s cartilage, but extends more posteriorly than in the stage 3 specimen. The articular now invests both medial and lateral sides of Meckel’s carti210
Stage 8 embryo (Fig. 17) There is much more ossification than in the previous stage. Vertebral ossification is spreading outward from the centra into the neural arches. The ribs are also starting to ossify. Approximately the posteroventral third of the otic capsule is now ossified. Calcified endolymphatic ducts are still visible. Anterior to the otic capsule, the braincase is partially ossified. The floor of the braincase posterior to the eye, formed by the basioccipital and basisphenoid, is ossified, as is the anterior portion of the braincase dorso-anterior to the eye, formed by the partially-ossified frontal. The parietal is also partially ossified. The supratemporal is well-ossified and lies between the otic process of the quadrate and the otic capsule. The roof and sides of the braincase are still open. The following bones are present in the otic capsule: exoccipital, supraoccipital and prootic. The basioccipital and basisphenoid are present. The nasal has partly ossified. The bones of the upper jaw are well-ossified. Anteriorly on the maxilla, fangs can be seen (these are not yet ankylosed to the maxilla). The posterior end of the maxilla articulates with the ectopterygoid, and the ectopterygoid curves medially to articulate with the pterygoid. Medial to these lie the palatine and the pterygoid. There is still some cartilage at the articulation between the maxilla and the ectopterygoid. Zoology 105 (2002) 3
Development of the cobra, Naja kaouthia
Fig. 17. Medial view of a cleared and stained, hemisected head of a stage 8 embryo: bo, basioccipital; cb, compound bone; fr, frontal; pa, parietal; pal, palatine; pf, postfrontal; pr, prefrontal; sp, splenial; vo, vomer.
The quadrate bone is well ossified, and only a small remnant of cartilage is left at the jaw articulation and at the otic process (i.e., the dorsal part of the quadrate). In the lower jaw, the dentary, splenial and compound bone are visible. The dentary is more extensive than in the previous specimen, surrounding Meckel’s cartilage. Teeth, although unstained, are present on the dentary. A compound bone, immediately posterior to the dentary and splenial, articulates with the quadrate. The compound bone is represented by at least three separate ossification centers: one short bone medially that articulates with the splenial; a second bone that is replacing cartilage at the jaw articulation; and a third much larger bone that invests the remainder of Meckel’s cartilage. At the anterior tip of the dentary, the anteriormost tip of Meckel’s cartilage can be seen. The bones of the lower jaw wrap around Meckel’s cartilage, leaving an opening between the dentary and splenial and the compound bone, and are open dorsally along the anterior end. The dentary is much more extensive than in the stage 7 specimen, and invests Meckel’s cartilage on all sides. A tiny splenial is present, separate from Meckel’s cartilage. Stage 10 embryo (Fig. 18) Ossification of the skull is nearly complete. Vertebrae are completely ossified, except for a small amount of blue-staining cartilage at the point of articulation between some of the vertebrae. The otic capsule, comprising the prootic, supraoccipital, basioccipital, and exoccipital, is completely ossified. In the braincase, the frontal and parietal meet dorsolaterally along the margin of the orbit. Dorsally, paired frontals and parietals are well separated medially, leaving a large fronto-parietal fontanelle in the skull roof. The parietal and frontal on each side are separated ventrally by a wide, unossiZoology 105 (2002) 3
Fig. 18. Medial view of a cleared and stained, hemisected head of a stage 10 embryo: bs, basisphenoid; otoc, otoccipital; pm, premaxilla; pro, prootic; psp, parasphenoid; soc, supraoccipital.
fied area. The basisphenoid and parasphenoid are both well-developed. The exoccipital is well developed and paired. Immediately anterior to them is the supraoccipital, which straddles the midline. In the paired parietals, alizarin red positive areas are well-developed laterally. However, the uncalcified alizarin-negative portions of the bone extend to the midline. The prefrontal and postfrontal delimit the anterior and posterior margins of the orbit respectively. Anterior to the braincase the nasal capsule is partly ossified, but approximately half of the nasal capsule remains cartilaginous. The nasal bones are triangular in shape viewed from above, and each descends ventrally in the midline. The bones of the upper jaw are clearly visible, as they were in the previous specimen, but are more completely ossified. The ossified premaxilla is visible ventral to the nasal capsule, but still only barely in direct contact with it. The maxilla, ectopterygoid, and palatine are present. The maxilla bears prominent fangs, and teeth are also present on the palatine, pterygoid, and in the lower jaw the dentary. In the lower jaw, the dentary, splenial, and compound bone are visible. The blue-stained, cartilaginous tip of Meckel’s cartilage protrudes anteriorly and medially on the lower jaw. The supratemporal is present. The quadrate is fully ossified. The blue-stained, rings of the trachea and the cricoid cartilage are still cartilaginous, as is the hyoid.
Discussion I have documented the timing of embryonic development in Naja kaouthia in ten stages from oviposition to hatching. Key changes that are visible externally include fusion of the body wall musculature along the 211
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ventral midline, the initial appearance of calcified endolymphatic ducts, increase in cervical flexure from 90° in the early embryo to 180° characteristic of neonate and adult snakes, formation of the eyelid, and initial appearance of scales on the body and the head. One goal of the present study was to correlate stages of Naja kaouthia development with previously published staging tables for other snakes: Zehr’s (1962) staging table for a colubrid, Thamnophis sirtalis, and Hubert and Dufauré’s (1968) table for a viperid, Vipera aspis. Unfortunately, correlating stages among the different tables is not possible except in the most general way. One problem is that Zehr and Hubert and Dufauré mostly used different characters; there are very few characters in common that can be used to correlate stages between their two tables. Another problem is that many characters used in these tables are specific to the species being described and not applicable to all snakes. For example, Hubert and Dufauré (1968) use the appearance of the upturned nose characteristic of V. aspis as a character,
and also details of ornamentation of the hemipenes that are unique to this species. Zehr (1962) relies heavily on the number of body coils as a character for determining stages, but in N. kaouthia there is no correlation between number of coils and stage of development (Fig. 3). Finally, efforts to correlate the present table with others were thwarted by heterochrony, and by the discovery that even for characters that overlap between staging tables, the order of appearance of characters is not consistent across ophidian taxa. For example, formation of the eyelid fold is complete at stage 32 for T. sirtalis (Zehr 1962) and stage 7 for N. kaouthia. The scales appear on the head at stage 36 in T. sirtalis and stage 6 in N. kaouthia. Thus, the three existing staging tables for snakes can only be correlated in the most general way: Stage 1 in the present table for N. kaouthia corresponds roughly to stage 25 for T. sirtalis and stage 35 for V. aspis. This correlation is based on the fact that at these stages the maxillary process extends to or anterior to the eye, but the eyelid fold has not yet started to form.
Table 1. Occurence of skull ossifications during post-ovipositional development. bone ar bo bs cb dn ec eld ex fr fg hy mc mx nc oc otoc pa pal pf pm pq pr pro psp pt qu soc sp st tc th tr vc vo
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articular basioccipital basisphenoid compound bone dentary ectopterygoid endolymphatic ducts exocciptial frontal fangs hyoid Meckel’s cartilage maxilla nasal capsule otic capsule otoccipital parietal palatine postfrontal premaxilla palatoquadrate prefrontal prootic parasphenoid pterygoid quadrate supraoccipital splenial supratemporal trabecular cartilage teeth tracheal rings vertebral centra vomer
stage 3
stage 7
stage 8
stage 10
present absent absent absent present present present absent absent absent present present present present present absent absent absent absent present present absent absent absent present absent absent absent absent present absent absent present absent
present absent absent absent present present present absent absent absent present present present present present absent absent absent absent present present absent absent absent present absent absent absent present present absent present present absent
present present present present present present present present present present present present present present present absent present present present present present present present absent present present present present present present present present present present
present present present present present present present present present present present present present present present present present present present present present present present present present present present present present present present present present present Zoology 105 (2002) 3
Development of the cobra, Naja kaouthia
Formation of the maxilla in Naja kaouthia is of particular interest in light of hypotheses concerning how the venom system evolved in colubroid snakes. In colubroid snakes, teeth may occur on the maxilla, dentary, palatine, and pterygoid. Fangs, when they occur, always occur on the maxilla, and for this reason maxillary dentition has been the subject of considerable study. Several authors have noted a morphological difference between the anterior and posterior maxillary teeth of colubroids, and this has led to speculation about the evolutionary origins of fangs. Bogert (1943) derived the tubular front fangs of viperids from the tubular front fangs of elapids, and these in turn from the enlarged, grooved posterior teeth of other colubroid snakes. Hence the anterior maxillary fang had a posterior maxillary origin. Anthony (1955), on the basis of arranging living taxa in a morphocline, derived the elapid anterior maxillary fang from an enlarged anterior maxillary tooth, but the viperid anterior maxillary fang from an enlarged posterior tooth that migrated anteriorly along the maxilla. Kardong (1980, 1982) argued on the basis of functional morphological studies of the maxilla of colubroid snakes, and consideration of the post-orbital position of the venom gland, that the elapid and the viperid front fang both evolved from an enlarged posterior maxillary tooth that migrated anteriorly. Jackson and Fritts (1995) argued on the basis of morphological markers on colubroid teeth, that the elapid front fang evolved from a posterior maxillary tooth. Jackson and Fritts (1996) described an example of an opisthoglyphous colubrid with enlarged and grooved anterior maxillary fangs, and suggested that a front fang could have evolved from an enlarged anterior maxillary tooth by means of such a pathway. Whatever the evolutionary pathway that led to the formation of front fangs of different types, the developmental pathway also remains to be elucidated. Haluska and Alberch (1983) reported that in Elaphe obsoleta, a colubrid snake, the maxilla forms from two separate centers of ossification, which eventually grow together and fuse, forming a single maxillary bone. McDowell (1986) noted the possible evolutionary significance of this observation, noting that in the Bolyeridae, a sistergroup to the Colubridae, the adult maxilla is in two parts and is hinged at the middle. The morphology of the bolyerid maxilla is described in detail by Cundall and Irish (1989). Thus, the distinction between anterior and posterior maxillary teeth and fangs is an important one for colubroid snakes, and may be reflected in the embryological development of the maxilla. In the present study, however, observations of cranial development in Naja kaouthia showed only one center of ossification for the maxilla. However, this could mean one of several things. First, there is the possibility that the specimens Zoology 105 (2002) 3
included in this study happened not to include the crucial stage where the maxilla is in two parts. Secondly, it could be that in elapid snakes, one part of the two-part maxilla has been lost, and only the part that bears venom-conducting teeth has been retained. Finally, the two-part maxilla of developing colubroid snakes may be a peculiarity of Elaphe obsoleta, and not a general feature of colubrid snakes, resting, as it does, on a single observation by Haluska and Alberch (1983). Savitzky (1992) reports only one center of ossification of the maxilla in crotaline snakes. To really resolve the question of whether the colubroid maxilla forms from one or two centers of ossification will require detailed histological studies, on several colubroid taxa, focusing on the stage at which the maxilla appears. The present study finds no evidence for more than one center of ossification. Thus, the post-ovipositional development of Naja koauthia can be divided into ten stages, using characters used in the description of embryological development of other snake taxa, as well as new characters. It is hoped that this staging table will provide a foundation on which future studies of the development of elapid snakes can be based.
Acknowledgements This work is dedicated to the memory of the late Mike Goode, whose cobra breeding program at the Columbus Zoo provided all the eggs used in this study. I thank the staff of the Reptile Section of the Columbus Zoo for logistical assistance and for information about the origin of the snakes in their breeding program. N. Kley took the photographs from which figures 4, and 6 to 18 were made. J. Hanken provided assistance with the identification of bones in the cleared and stained specimens. J. Gross and H. Thompson helped with the statistical analysis. Helpful comments on the manuscript were provided by E. Brainerd, J. Hanken, F. Jenkins and two anonymous reviewers.
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Kamal, A.M., H.G. Hammouda and F.M. Mokhtar. 1970b. The development of the osteocranium of the Egyptian Cobra: II. The median dorsal bones, bones of the upper jaw, circumorbital series, and occipital ring of the adult osteocranium. Acta Zoologica 1970: 19–30. Kamal, A.M., H.G. Hammouda and F.M. Mokhtar. 1970c. The development of the osteocranium of the Egyptian Cobra: III. The otic capsule, palate, temporal bones, lower jaw and hyoid apparatus of the adult osteocranium. Acta Zoologica 1970: 31–42. Kardong, K.V. 1980. Evolutionary patterns in advanced snakes. Amer. Zool. 20: 269–282. Kardong, K.V. 1982. The evolution of the venom apparatus in snakes from colubrids to viperids and elapids. Mem. Inst. Butantan 46: 105–118. McDowell, S.B. 1986. The architecture of the corner of the mouth of colubroid snakes. J. Herpetol. 20: 353–407. Nieuwkoop, P.D. and J. Faber. 1967. Normal Table of Xenopus laevis (Daudin). Second Edition, North Holland Publ. Co. Amsterdam. Rugh, R. 1977. A Guide to Vertebrate Development. Seventh Edition, Burgess Publishing Co., Minneapolis, Minnesota. Savitzky, A. 1992. Embryonic development of the maxillary and prefrontal bones of crotaline snakes. In Biology of the Pitvipers (J.A. Campbell and E.D. Brodie, Jr., eds.). Selva: Tyler, Texas, pp. 466. Townsend, D.S. and M.M. Stewart. 1985. Direct development in Eleutherodactylus coqui (Anura: Leptodactylidae): A staging table. Copeia 1985: 423–436. Zehr, D.R. 1962. Stages in the normal development of the common garter snake Thamnophis sirtalis. Copeia 1962: 322–329.
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