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32B EMBRYOLOGY OF MARINE TURTLEB

bei den Seeschildkrbten, untersucht an Embryonen von Chelonia viridis. Anat. Anz, 8, 801-803. Voeltzkow, A. (1903). Beitrage zur Entwicklungsgeschichte der Reptilien. VI. Gesichtsbildung und Entwicklung der ausseren Kbrperform bei Chelone imbricala Schweigg. Abh. senckenb. naturf. Ges. 27, 179-190. CHAPTER Wassersug, R. J. (1976). A procedure for differential staining of cartilage and bone in whole formalin-fixed . Stain Tech. 51, 131-134. Wiedersheim, R. (1890a). Uber die Entwicklung des Urogenitalapparates bei Krokodilen und 5 Schildkrbten. Anal. Anz. 5, 337-344. Wiedersheim, R. (1890b). Uber die Entwicklung des Urogenitalapparates bei Krokodilen und Schildkrbten. Arch. mikr. Anal. 36, 410-468. Will, L. (1893). Beitrage zur Entwicklungsgeschichte der Reptilien. 2. Die Anlage der Keirn­ blatter bei der menorquinischen Sumpfschildkrbte (Cistudo lutaria Gesn.). Zool. Jahrb .. Abt. Anal. 6, 529-615. Reproductive Biology and Witzell, W. N. (1983). Synopsis of biological data on the hawksbill turtle, Erelmochelys imbricata (Linnaeus, 1766). FAD Fish. Synop. 137, 1-78. Embryology of the Wood, J. R. and Wood, F. E. (1980). Reproductive biology of captive green sea turtles CIlelonia mydas. Amer. Zool. 20, 499-505. Crocodilians Yamamoto, Y. (1960). Comparative histological studies of the thyroid gland of lower verte­ brates. Folia anat. jap. 34, 353-387. Yntema, C. L. (1964). Procurement and use of turtle embryos for experimental procedures. Anal. Rec. 149, 577-586. Yntema, C. L. (1968). A series of stages in the embryonic development of Chelydra serpentina. f. Morph. 125, 219-251. MARK W. 0. FERGUSON

Yntema, C. L. (1976). Effects of incubation temperatures on sexual differentiation in the turtle, Department of Basic Dental Sciences, Turner Dental School, University of Chelydra serpentina. J. Morph. 150, 453-462. Manchester, Higher Cambridge Street, Manchester, M 1 5 6FH England Yntema, C. L. (1979). Temperature levels and periods of sex determination during incubation of eggs of Chelydra serpentina. f. Morph. 159, 17-28. Yntema, C. and Mrosovsky, N. (1979). Incubation temperature and sex ratio in hatchling loggerhead turtles: a preliminary report. Mar. Turtle Newslet. 11, 9-10. Yntema, C. and Mrosovsky, N. (1980). Sexual differentiation in hatchling loggerheads (Caretta caretta) incubated at different controlled temperatures. Herpetologica 36, 33-36. Yntema, C. L. and Mrosovsky, N. (1982). Critical periods and pivotal temperatures for sexual differentiation in loggerhead sea turtles. Can. f. Zool. 60, 1012-1016. Yoshie, S. and Honma, Y. (1976). Light and scanning electron microscopic studies on the esophageal spines in the Pacific Ridley turtle, Lepidochelys olivacea. Archs. Hislol. Jap. 38, 339-346. Zanger!, R. (1969). The turtle shell. In Biology of the Reptilia (c. Gans, A. d'A. Bellairs, and T. Parsons, eds.). Volume 1, Academic Press, London and New York, pp. 311-339. Zanger!, R. (1980). Patterns of phylogenetic differentiation in the toxochelyid and cheloniid sea turtles. Amer. Zool. 20, 585-596. Zeleny, C. (1901). The ear!y development of the hypophysis in Chelonia. BioI. Bull., Stockh 2, 267-281. Zwinenberg, A. J. (1976). The olive ridley, Lepidochelys olivacea (Eschscholtz, 1829): probablY the most numerous marine turtle today. Bull. Maryland herp. Soc. 12, 75-95. Zwinenberg, A. J. (1977). Kemp's Ridley, Lepidochelys kempii (Garman, 1880), undoubtedly the most endangered marine turtle today (with notes on the current status of Lepidochel.'l' olivacea). Bull. Maryland herp. Soc. 13, 170-192. INTRODUCTION 331

P. Endocrine Glands, 445 CONTENTS Q. Thymus and Immune System, 446 R, Limbs and Tail, 446 I. INTROOUCTION 331 S, Integument and Its Glands, 450 II. REPRODUCTIVE BIOLOGY T. Caruncle, 450 333 A. General, 333 VIII. DEVELOPMENTAL ABNORMALITIES 451 B. Sexual Maturity and Adult Sex Ratios, 333 IX. SHELL·LESS, SEMI.SHELL.LESS, AND IN VITRO C. Courtship, Spermatogenesis, Ovulation, Copulation, CULTURE TECHNIGUES 460 the Breeding Season, Fertilization, and Egg Laying, 336 D. Nesting Biology, 349 X. CONCLUSIONS 462 E. Maternal Behavior, 355 REFERENCES 464 F. Captive Breeding, Egg Collection, and Artificial Incubation, 356 G. Hatching, 360

III. THE EGGSHELL AND SHELL MEMBRANES 363 A. General, 363 B. Egg Banding, 363 C. Structure, 367 D. Chemical Composition, 374 E. Water and Gas Conductance and Embryonic I. INTRODUCTION Metabolism, 376

IV. THE EGG CONTENTS AND EXTRA.EMBRYONIC The living crocodilians, represented by 26 living species placed in three MEMBRANES 377 subfamilies (see Table III later in chapter), are the end products of a rela­ V. tively conservative lineage, which arose from thecodont ancestors approxi­ EARLY EMBRYONIC DEVELOPMENT [BEFORE EGG LAYING) mately 230 million years ago (Carroll, 1969; Walker, 1972). The evolution 381 and of the order have been the subject of considerable debate VI. STAGES OF EMBRYONIC DEVELOPMENT [AFTER EGG involving paleontological (Steel, 1973), neontological (Wermuth and Mer­ LAYING) 390 tens, 1977; Groombridge, 1982), immunological, and biochemical ap­ VII. ORGANOGENESIS proaches (Perutz et al., 1981; Le Clercq et al., 1981; Densmore, 1981; Coul­ 416 son and Hernandez, 1983). Densmore's and Groombridge's classifications A. Branchial Arches, 41 6 are used here. All types of data indicate that are the closest living B. Face and Nose, 41 9 relatives of crocodilians. However, several aspects of crocodilian embryo­ C. Palate and Nasopharyngeal Duct, 421 D. Tongue, 427 genesis, for example, palatogenesis (Section VII, VIII), resemble mamma­ lian phenomena, and variations in hemoglobin amino acid sequences place E. Ear, 428 I" F. Eye, 428 t crocodilians closer to than to snakes (Perutz et al., 1981; Le G. Chondrocranium and Osteocranium, 431 I' Clercq et al., 1981; Densmore, 1981). Thus, further studies of crocodilian H. Teeth, 431 embryogenesis not only may shed light on fundamental aspects of or­ I. Central Nervous System, 432 ganogenesis, but also exploit the potential of these vertebrates as models ..J. Vertebrae and Ribs, 435 for experimental investigations, which are difficult or impossible to per­ K. Respiratory System, 436 form in other amniotes (Ferguson, 1981a, b, 1984a). L. Cardiovascular System, 436 Crocodilian embryology has received very little attention. Some general M. Diaphragm, 438 accounts are available for niloticus (Rathke, 1866; Voeltzkow, N. Gastrointestinal System, 438 O. 1899, 1901, 1903a), mississippiensis (Clarke, 1891; Reese, 1908, Urogenital System and Sex Determination, 440 1910a-c, 1912, 1915a, b, 1921, 1936), C. palustris, and C. porosus (Derani­

330 332 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS REPRODUCTIVE BIOLOGY 333 yagala, 1934, 1936, 1939), whereas Wettstein (1937, 1954) included some factors influencing egg laying. Therefore this chapter begins with a se­ embryological data in his general review of crocodilian biology. The pres­ lected review of relevant data on these topics. This is not intended to be a ent chapter reviews the older, incomplete data and a special effort is made comprehensive review; rather it is intended to highlight areas of interest to correct ambiguous or misleading statements or concepts. My studies on common to ecologists, conservationists, and farmers, as well as to develop­ shell structure (Ferguson, 1981c, 1982a), sex determination (Ferguson and mental biologists. Insights into life history facilitate appreciation of some of Joanen, 1982, 1983), and craniofacial development (Ferguson, 1979a, b, the embryological events described later. They may also stimulate develop­ 1981a, b, 1982b, 1984a) tend to give the chapter a bias, but an attempt has mental biologists to investigate some of the mechanisms which underlie been made to review all available data in the hope that neglected topics will numerous observations of natural history, for example, embryonic dia­ be pursued by others. pause, the relation of maternal age to the incidence of birth defects, the Many of the studies on which the present chapter is based have been gaseous, aqueous, and metabolic physiology of embryos, the hatching carried out on the population of Alligator mississippiensis at the Rockefeller trigger, and the transition from chorioallantoic to pulmonary respiration. Wildlife Refuge in Louisiana, and it might be argued that these are The aim is also to provide the biologist or farmer with a literature review not representative of all alligator populations nor of crocodilians in general. and to stimulate collection of data on numerous unanswered but intriguing However, observations on other populations have revealed no significant questions. The difference of format of this chapter from others in this differences with respect to basic reproductive biology and embryology. volume was dictated by the nature of the available information. My objec­ Furthermore, analyses of protein electrophoretic patterns have shown that tive is to stimulate future studies in those areas for which data are currently A. mississippiensis is one of the most genetically homogeneous unavailable. I hope that progress in the field will permit extensive revision species; protein variations are so low that animals from populations sepa­ of much of what is said here in a few years' time! rated by more than 1000 miles cannot be identified as to their geographic source (Gartside et al. 1977; Menzies et al. 1979; Adams et al. 1980). In addition, I have made limited observations on some aspects of the embry­ II. REPRODUCTIVE BIOLOGY ology of Crocodylus johnsoni, C. porosus, C. niloticus, C. cataphractus, and C. novaeguineae (Ferguson, 1984a). The basic events during the period of or­ A. General ganogenesis (first half of development) are remarkably similar and may be regarded as crocodilian, whereas species-specific differences are more In this section, I review accumulated data on crocodilian natural history geometrical, for example, variations in the relative sizes and proportions of relevant to developmental (sensu lato) phenomena, which are discussed in structures such as the tail, limbs, snout, and pigmentation pattern, and Sections III to X. Whereas only Alligator mississippiensis, Crocodylus they appear principally during the second half of development. Therefore niloticus, C. johnsoni, C. palustris, and C. porosus are known in any detail, this account is likely to be representative of crocodilians in general. some data from other species are available. A recent, dramatic increase in the literature on ecology, behavior, natu­ ral history, management, and farming of crocodilians across the world B. Sexual Maturity and Adult Sex Ratios offers hope not only for the survival of these , but also for the future availability of embryonic study material. Such are particularly exciting Table I summarizes data on the size and age at sexual maturity for several prospects not only for the study of neglected species such as the , species, including information for the same species in different habitats. but also for determining general principles pertaining to crocodilians, and Within distinct populations of the same species, the onset of sexual matu­ for establishing species-specific variations. rity appears to depend principally on size, which reflects environmental Much of this natural history and farming literature is relevant to embry­ factors such as temperature and the availability of food, both of which logical studies. Because it is extremely unlikely that investigations on the influence the growth rate. Because these environmental factors can be "T stages of embryogenesis can be conducted on anything but captive optimized in captivity, the onset of sexual maturity can be accelerated by 1<;, it is necessary to understand the relevant reproductive data and to 50% compared with wild specimens (Table I). However, size is not the only

Sex Age at (M = male Age at Sexual F = female Size Sexual Maturity grouped = no at Sexual Maturity in distinction Wild III Maturity in Captivity III Species reported) (meters) (years) (years) References

" Alligator mississippiensis M 1.8 9-10 6 Joanen (1969), Joanen and Louisiana F 1.8 9-10 6 McNease (1973, 1975a, b, 1978, 1979a, 1980) Alligator mississippiensis M 1.8 15 Klause et al. (1984a, b) North Carolina F 1.8 18 crocodilus crocodilus ~} 1.3 4 Staton and Dixon (1977) Caiman crocodilus fuscus ~} 1.08 Staton and Dixon (1977) gangeticlIs ~} 3 8-12 Acharjyo et al. (1975), Bustard (1979, 1980c, 1984), Singh and Bustard (1977), Singh (1979) 138 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCOOILIANB

tains and with its normal growth rate. Forms that mature at smaller sizes, ~ ..><: til u ? r example, Caiman crocodilus fuscus, are obviously well suited for captive ~ Zu .D r;:) ·2 eeding and developmental study. 00 ~ ~ ~ 0\ til ,...; .§ Although sexual maturity may be reached at the sizes specified in Table fIl "0 U r: r: r: "0 ' ;:j ~ social order favors the mating of larger animals, for example, male Al- .8 cJl til r: &J S (lj r: til 0' 3: Igator mississippiensis that are longer than 2.7 m (McIlhenny, 1935; Giles r; u (lj cJl ~ S <:0'''''; r: r: ~:i~:: calities (Table II). Figures for adult sex ratios are beset with all kinds of ~ <: til ~~..r: til otential errors, such as the effects of differential sexual mortality and the .D r")' ~ 8 ;:j .D..r: '-' o ,.Q .-=: ...... ""0 .-:::: til t'-- "t:: 00 co >-, ...... 0 ..c:: abitat in which the animals are caught (many species have different pre- r: ..r: ~ 0 ~ ~ Qi 6 -~ U~>J:.<~>J:.< ~ 3:Ul 8 (3 ~ 3: lerred habitats for adult males and females)...... (lj For four consecutive years, the sex ratio of hatchling Alligator mississip­ ..... iensis from a wild population averaged 5 ± 0.7 females per male (Fergu­ is .S cJl .-< (lj~ \D on and Joanen, 1982, 1983). However, surveys of the Louisiana open fIl '<:t< t'-­ OOLr)\DO (lj ";;Jt3"ILr) ,...; .-< i?; I N Lr) N .-< \D ~ ~ 00 N ater canals, during April when males and females congregate to mate '0 .-< (lj l:lo oanen and McNease, 1970, 1972), have revealed an adult sex ratio of (/'J pproximately 1: 1 (Joanen, unpublished). This discrepancy may result r: ~ rom sampling error, differential mortality of young females, and the possi­ o~ cJl --0- 00 00 mty that many females remain in the marsh, which is their preferred adult ~ 0 ~-- \D <"l o o 0 m ..q; a .....l , (lj breeding of all species reported to date has been achieved with sex ratios in .8 o .5 'C r: .5 .....l cJl ~ Qi ;:j III "0 ...-.....""2.7 ...... b the order of 80% females:20% males (Yangprapakorn et al., 1971; Joanen ~ :g ~v ­ ";;J E... - ;j bO'C ~ >< u .~ .- "0 r: "'"0 ::::;' ::: and McNease, 1975a, b, 1979a, b; Webb et a!., 1983e). However, there is (lj J;J ~ :a ::: '1i)'" :§ c .;!] "".c....., ;:: ;:: There is detailed information on the reproductive biology of Alligator mis­ E ;:,'" ;::'" ;:: '" ,issippiensis Goanen, 1969; Joanen and McNease, 1970, 1973, 1975a, b, 1978, Qi ~ ~ .-cJl 16<::: 1979a, 1980; Lance, 1983, 1984) and Crocodylus niloticus (Cott, 1961; aJ .~ f 8 f 0... ::::: i2 i2 l.':' Graham, 1968) with fragmentary information available for other species, Ul "( u u u

337 REPRODUCTIVE BIOLOGY 339 338 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS

100 ~ 300 15J6'fl ~I JANUARY 901- ~.\\ 15 I !": (J~I'F ,~I> ~""~ f"s. ) ,.-. 'A Q""(,""~ 801- I I ~ ,,I, / _ 70 ,, " ~ ~.: E ,, ,I 200 ,.;:- $" '; - ~ ~& E g> ,, ~ ~ '6'~'9. '" l'J $5 '" ./ ~ 60 ,, ~!(J i­ .5 ~ 2 ', :E Cl / ,',, .Testisweight t.\l~~\~'t.\'I:i , ~ Jso , A PlasmaTestosterone ' 0'>1. 0\\"" bellowing LOCi' 30 ,, 0:' ~LO mole prefers -a- = - f-­ ~o \'0 ,, U deep open « '" o .oler arees " , I -r,O\c'\I\lI'1QS II'I\\\O\! IttQlflq t , ..... ',. 201­ , I ,,~,t.~\\"'~~ , I ~~~ \l\'i- ~i --~\ " 10 I-- ~/ / \l~ ~~~ i _.- '.,~ / ;,,;. ~ \ \i.­ 't-'rjiJ~,,+\\\l"'/ /' / '1..- ' ~~ ('q,~-& ~J'" 4.0' ""," ,,/ ff J'~v('~o~ ~ ~ '0 Y', t'; ...,,; 0...----- _ ...... ,,"" I ~ 0""-%",; ~o" ~I' ~ ~ ~~ ~0'::>, g +~"- §~ \ ':i:, ,~ ~~~ "';\ the year. The dotted lines represent months for which no data are currently available. (Cour­ \\ \~ -<1(;, IS \"'~~ tesy of Dr. V. Lance.) sJ\~ GUS!, 18?S ,,) ,t::,,'1 15 \~~ JULY During February, the testes start to enlarge (Fig. 2) so that they are 11-20 'fl times larger by April/May than in the previous September, having dimen­ Fig. 1. Alligator mississippiensis. Chronology of its reproductive biology in Louisiana. (From sions of approximately 14 X 6 ems and weights of 300 g (Joanen and Joanen and McNease, 1975a.) McNease, 1980; Lance, 1983). The right testis is generally larger than the left (Lance, 1983). Testicular regression begins in mid-June and is complete by the end of July (Fig. 2); testicular weights probably remain low for the for example, C. johnsoni (Webb et al., 1983e) and Caiman crocodilus rest of the year. Active testes have a pinkish-cream color, a smooth texture, crocodilus (Staton and Dixon, 1977). Figure 1 summarizes the chronology of and a wide (1-2 em) heavily convoluted ductus deferens; mature but inac­ reproductive events in A. mississippiensis. Unless otherwise indicated, all tive testes are purplish-pink in color; immature testes are small (weight less data reported hereafter refer to this species in Louisiana. Detailed descrip­ than 2 g), ribbon-like, liver colored, granular, and attenuated with a small tions of courtship behavior are available for several species (Cott, 1961, ribbon-like ductus deferens (Joanen and McNease, 1980). Immature ani­ 1975; Joanen and McNease, 1970, 1971, 1972, 1973, 1975a, b, 1979a, 1980; mals show no discernible seasonal increase in testicular weight (compare Guggisberg, 1972; Campbell, 1972; Garrick and Lang, 1977; Garrick et al., Fig. 2) and no increase in the size, thickness, and convolution of the ductus 1978; Staton and Dixon, 1977; Tryon, 1980; Compton, 1981). Shortly after deferens and have extremely low levels of plasma testosterone (Lance, copulation, females move into small isolated ponds in the shallower waters 1983). of the open marsh, initiate nesting, and remain there (with their hatch­ Histological examination reveals that spermatogenesis is advanced even lings) until the following spring. By contrast, males and nonbreeding fe­ by March (Lance, 1983). Active spermatogonial division, secondary sper­ males move extensively within the deep water canals and lagoons through­ matocytes, spermatids, and Leydig cells containing the enzyme ~5-3f3­ out the summer and return to their winter habitat in late October. hydroxysteroid dehydrogenase (~5-3f3-HSD; a key enzyme in steroid hor­ REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCDDILIANS REPRODUCTIVE BIOLOGY 341 340 mone biosynthesis) are present (Lance, 1983, 1984). However, mature The reproductive system of females shows seasonal variation. Each sperm do not appear in the seminiferous tubules until late April/early May. ovary contains at least three size classes of follicles: <1 mm, 1-4 mm, and Living sperm first appear in the penial groove in early May and are present the crop of preovulatory follicles of the current year, which range from 5 to for only a 6-week period, with maximum output (coincident with female 45 mm in diameter (Joanen and McNease, 1980; Lance, 1983). The right ovulation) during a 2-week period in late May/early June (Joanen and ovary (and oviduct) is usually larger (approximately 20 cm x 20 cm) and McNease, 1970, 1972, 1973, 1975a, b, 1979a, 1980). Jenkinson (1913) states contains more follicles of preovulatory diameter than the left (Lance, 1983). that crocodilian sperm are small (20-30 jJ-m), but Lance (1983) reported a Vitellogenesis begins in early spring: the 4-5-mm follicles grow to about 20 mean length of 60 to 70 jJ-m for . Mature males have sperm con­ mm by early April, reach 30 mm by early May, and reach a preovulatory centrations of 0.5 to 1 billion per ml of semen (Larsen, 1981). Sperm storage diameter of 40-45 mm by late May/early June (Lance, 1983; Joanen and occurs principally in the ductus deferens as the epididymis is poorly devel­ McNease, 1980; Giles and Childs, 1949). Developing follicles protrude from oped (Lance, 1983). Dead, immotile sperm were first noted in mid-June, the surface of the ovary and are shed through a small opening directly into and by June 20th, 90% of spermatozoa were dead (Joanen and McNease, the fimbria of the oviduct (Lance, 1983). The time of ovulation varies from 1970, 1973, 1975a, b, 1980). Sperm production varies significantly amongst year to year depending upon ambient temperatures during March-May reproductive size classes (i.e., 1.8 m and above), and large males (over 2.7 (Joanen and McNease, 1979b). Ovulation usually occurs during the first m) produce living spermatozoa for a longer time period than do smaller week in June and shelled eggs appear in the lower end of the oviduct from males (Joanen and McNease, 1980). This may explain the reproductive the second through the last week in June (Joanen and McNease, 1975a,b, superiority of the large bulls, and why social order favors breeding by 1980; Giles and Childs, 1949). Oviposition usually occurs between June 11 males over 2.8 m in length. During testicular regression, the diameter of to 28 (range June 5 to July 5), and normally it takes two weeks for the entire the seminiferous tubules decreases, they contain progressively fewer sper­ female population to oviposit (Joanen and McNease, 1980). Following ovu­ matozoa, ~5-3J3-HSD activity is minimal and the interstitium is composed lation, the follicle forms a corpus luteum composed largely of connective of connective tissue (Lance, 1983). There is no evidence for over winter tissue with only a thin layer of granulosa-Iutea cells forming a steroido­ sperm storage in the males; indeed, the epididymides (the usual site of genic core (Lance, 1983). Whether these corpora lutea are functional is sperm storage in postnuptial reptiles, Lofts, 1977; Moll, 1979) are not en­ unknown (Lance, 1983). It probably takes 3 years for a differentiated oocyte larged even at the height of the breeding season (Lance, 1983). to grow to a preovulatory diameter. The numbers undergoing atresia dur­ Plasma testosterone levels show a similar seasonal fluctuation to that ing this period are unknown, but most follicles that reach preovulatory size observed for testicular weight (Fig. 2) with a peak of 90 ng/ml in April/May are ovulated (Lance, 1983). Loyez (1906) described the developing oocyte, (Lance, 1983, 1984) and rapidly decreasing thereafter. The association of vitelline membrane, and follicular epithelium of immature follicles that elevated testosterone levels with the peak of testicular weight, spermato­ were <4 mm in diameter from the ovaries of Crocodylus niloticus, and Lance genic activity, bellowing intensity, and mating behavior (Fig. 2) suggests (1983) provided a more complete description for Alligator mississippiensis. that these functions are androgen dependent, although this remains to be Plasma estradiol levels in females mirror the trends seen for testosterone tested experimentally. The mechanism initiating gonadal regression is un­ in males (Fig. 2) with a peak of approximately 700 pg/ml in early April-the known although Lance (1983) discusses several possibilities. Clearly the time of preovulatory follicular development and vitellogenesis (Lance, topic is worthy of investigation not only for its biological interest, as it 1983). Plasma from reproducing females in April is chalky white in color (as occurs at a time when temperature and photoperiod appear to be optimal, compared to the light straw-colored plasma of males and nonbreeding but also for its importance in alligator farming. The plasma testosterone females) and has elevated levels of calcium, magnesium, zinc, iron, total rises slightly in September, when the alligator testes are fully regressed protein, cholesterol, vitamin E, and estradiol-17J3 (Lance et al., 1983). (Lance, 1983). This is difficult to explain, but it does not coincide with These levels correlate with the time of preovulatory follicular development mating behavior, spermiogenesis or renewed spermatogenesis. The profile and vitellogenesis, and they return to normal by July (Lance et al., 1983). is, however, typical of postnuptial spermatogenic cycles (Lance, 1983) and Plasma testosterone levels in vitellogenic females are also elevated during may represent the curtailing of another breeding cycle imposed by the the follicular growth period, but concentrations are less than one-tenth of climatic habitat of Alligator mississippiensis, the most northerly ranging those in males (Lance, 1983). The function of this female testosterone is crocodilian. In this regard, comparison between annual testosterone levels unknown. As in other oviparous vertebrates, alligator vitellogenesis is in this species with those in more tropically located crocodilians (particu­ probably oestrogen dependent. Estradiol, secreted by the growing ovarian larly those suspected of having two breeding seasons, e.g., Crocodylus follicles, stimulates the liver to synthesize and secrete vitellogenin (yolk niloticus, C. palustris, C. porosus, and C. johnsoni) would be profitable. precursor protein), which passes via the blood to the ovary where it is REPROOUCTIVE BIOLOGY 343 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIANS 342 optimal environment (in terms of temperature and food) may pursue this taken up by the follicles (Lance, 1983). Immature alligators of either sex can strategy. be induced to produce vitellogenin by injection of estradiol (Dessauer, The data on the peak nesting times of various species presented in Table 1974; Lance, 1983). The oviduct is also stimulated by ovarian oestrogen and III generate many unanswered questions: What environmental factors trig­ hypertrophies during the follicular growth phase (Lance, 1983). After ger ovulation, spermatogenesis, reproductive behavior, and egg laying, oviposition, plasma estradiol levels remain low to nondetectable, and there and how do they operate? is no evidence for a prehibernation onset of vitellogenesis (Lance, 1983). Numerous reports for various species imply a possible correlation Nonbreeding females have low to nondetectable estradiol levels (Lance, among breeding times and changing water levels in the various habitats. 1983) and show nO evidence of follicular growth (Joanen and McNease, The data do not reveal consistent trends across all species and the inter­ 1980). Likewise, immature females have low estradiol levels and small ested reader is referred to the original publications (Cott, 1961, 1975; ovaries that consist of clear cortical tissue with numerous clusters of 00­ Modha, 1967; Graham, 1968; Pooley, 1969a; Staton and Dixon, 1977; De­ cytes distributed evenly throughout the organ. Reproductively senescent raniyagala, 1939; Webb et al., 1977, 1983e; Joanen, 1969; Joanen and or "barren" females (usually over 2.7 m in length) have few growing 00­ McNease, 1975a, 1979b, 1980; Kushlan and Kushlan, 1979). cytes, no corpora lutea, black hemoglobin deposits, and their ovaries fre­ A strong relationship exists between peak nesting times in Alligator quently contain medium-sized resorbing ovarian follicles and retained mississippiensis and average air temperatures. An analysis of nesting data oviducal eggs undergoing resorption (both in Alligator mississippiensis, for ten years reveals a highly significant correlation between the time of Joanen and McNease, 1980; Lance, 1983 and in Crocodylus niloticus, nesting and the ambient temperatures for March-May (Joanen and Graham, 1968). The occurrence of senescence in the suggests McNease, 1979b). Nesting occurs earliest when these temperatures are that there is a limit on the number of oocytes a female can produce, despite highest. Rainfall is significant only when nesting effort is reduced during the fact that oogonial proliferation continues throughout much of adult­ extremes of accrued surface water levels; females apparently respond to higher water levels by laying eggs at higher levels within the nest (Kushlan hood.Macroscopic changes similar to those detailed above have been de­ and Kushlan, 1979). Oviposition occurs on the longest days of the year scribed in the male and female reproductive systems of Crocodylus niloticus (Joanen and McNease, 1979b). Temperature appears to be the major regu­ (Cott, 1961; Graham, 1968). However, the breeding season of Alligator lator of male and female reproductive cycles (Lance, 1983); as in other mississippiensis is shorter and more clearly defined than is that of the more reptiles photoperiod plays only a minor, or at best synergistic, role (Fi­ tropical C. niloticus (Graham, 1968), C. porosuS (Webb et al., 1983b), and C. scher, 1974; Crews and Garrick, 1980). It is known that males in an palustris (R. Whitaker, personal communication). This may reflect environ­ artificially heated pond in Georgia had spermatozoa in the penial groove mental temperature; the cold winters in Louisiana make the alligator inac­ one month earlier than their counterparts inhabiting ponds at ambient tive from October through February, and it must complete its reproductive temperatures (Murphy, 1980). Moreover, motile sperm have been re­ cycle within a restricted time frame. ThuS for C. niloticus in Kenya, sperm covered in January from males kept in artificial hot houses (Cardeilhac, production extends from April through December, and although there is a 1981). Whereas adult A. mississippiensis do respond to experimentally al­ peak breeding season (October-December), follicular development and tered photoperiods (Lang, 1976), such experiments have not been con­ some reproduction occur throughout the year (Graham, 1968). Moreover, ducted in relation to the cueing of reproductive events. Experiments in­ 15% of the sexually active females of C. niloticus have two sets of follicles volving altered photoperiods at constant high temperatures would be developing concurrently (Graham, 1968). This means that maturation of interesting, especially for tropical species in regions with minimal annual one batch of ova may be accompanied by development of a younger batch variations of temperature. that will near maturity soon after oviposition of the first; the second set can It is currently impossible to state categorically which of the potential then be fertilized and two clutches of eggs may be produced in one season. extrinsic regulating factors, such as drought, temperature, water level, This "double clutching" has also been reported for some C. palustris (in photoperiod, or the psychological effect of available breeding grounds, captivity, R. Whitaker, personal communication) and is suspected in some stimulate ovulation and spermatogenesis, and whether these factors are C. porosus (Webb et al., 1983b). The controlling mechanism remains unin­ similar in all crocodilians. Clearly more information is required, not only vestigated. However, given the amount of yolk protein necessary to pro­ on the stimuli themselves, but also on their perception and the way in duce two clutches of approximately 40 eggs weighing apprOXimately 60­ which they achieve their effects. 70 g each, not to mention the energy expended in mating behavior, nest The hormonal regulation of the reproductive cycle is poorly understood. building, and maternal care, it may be assumed that only females in an TABLE III Comparative Data on the Nests, Eggs, and Hatchlings of the Crocodilians"'C

Average SizC' Average Range Average Average Range Type of Nest of Nest Average Range of Average Range of Average Range Average Range Temperature of Egg Humidity of Range of Total of Total Average Range of (from Greer (Breadth x Peak Times Incubation Number Breadth Egg Length Egg of Egg Weight of Egg of Egg Cavity Egg Cavity in Humidities Length of Hatchling Weight of Hatchling 1970, 1971; Height/ of Egg Period of Eggs Numbers of Egg Lengths of Egg Breadths of Egg Weights Cavity in Temperatures Nest (c/o ReI. (% Rel. Hatchling Lengths Hatchling Weights ~cies Campbell 1972) Dep'h) (em) Laying (days) per Clutch per Clutch (em) (em) (em) (em) (g) (g) Nest ('C) (0C) Humidity) Humidity) (em) (cm) (g) Ig) ligator lineage Alligator missis­ Mound Av = 181.6 June 63-65 38.9 2-68 7.4 6.1-88 4.3 3.1-5.0 84 51-108 30 233-35.7 972 94-99.8 d 26.4 22-29 676 58-77 sippiensis x 60.2 (45-799, Range = moisture 112-304.8 content) x 33-121.9 A. sinensise Mound 106 x 29.2 June-Aug 70-80 26 10-40 68 5.6-75 3.4 3.1-3.6 51.7 40.3-56 21 30.2 Caiman CTOCQdi/us Mound ? x 30 75-90 30 20-50 6.2 3.8 apapoTjen~·s-f C. cTocodilus Mound 117 x 44 Aug.-Oct 73 28 17-40 6.5 4.3-7.2 4.0 2.5-43 59 48.7-774 30 28-32 90 85-95 21.5 191-23.8 41.5 31-51.2 rrocodilusg C. crocodilus Mound 150 x 40 Dec.-April 84 31 21-40 6.8 5.6-7.5 4.2 3.3-4.7 75.3 59-83 28 25-32 24 20-25 492 46-62 yacare" C. CTocodilus } fusnd and Mound April-July 75-80 15-30 C. crocodilus chiapasius' C. 1oliTastris} Mound 163 x 41 Aug.-Mar. 63-86 40 20-60 6.6 4.6 84 .\Vlanosucnus Mound 210 x 100 Sept.-Jan. 40-90 40 18-75 8.0 4.4-9.7 5.0 3.5-5.6 100+ nigeri: Pa/eosuchu5 Mound 125 x 39 Aug 90-92 13 6.6 6.1-7.1 4.2 4.1-5.1 687 66.1-74.5 28-31 95 90-96 23.6 202-24.5 45.5 palpebrosus l 39.8-50.0 p, trigonafus Mound ;aviallineage Gavialis Hole ? x 5.1 April 71-94 40 16-61 8.6 8.5-10.2 6.7 6.5-7.0 31 25-37 35 32-40 gangt'ficusm 75 Tomistoma Mound ? x 60 75-90 20-60 10.1 9.7-12.0 7.6 6.4-8.0 30 28-33 St'hlegelii!f :rocodHe lineage Osteolaemus tetra­ Mound spis osborn; Osteolaemus tetra­ Mound 75 x 41 June 82-120 13 6-19 6.3 5.5-8.0 3.7 3.4-4.4 51.9 38.5-70.7 28.8 spis tetraspiso 22.6 19.7-25 34.3 262-445 Crocodylus Hole/mound 230 x 33 April-May 85-111 44 19-81 72 6.3-·7.6 4.4 4.2-5.1 30 265-341 acutusP of sand 24.1 C. cataphracfu;;q Mound 50-80 x Mar.-July 90-98 19 13-27 8.3 8.1-8.5 5.2 305 27-34 100 120-220 303 28.3-35.6 (37% mois­

r ture content) C. intermedius Hole Jan.-Feb. 60 1.5-70 s C. ;ohnsoni Hole 19 x 13 August 63-98 13 10-24 6.6 6.1-7.3 4.2 3.7-4.6 68.2 498-85.8 29.4 28-34 24.4 21-26 C. mindormsis1 42 36-54 Mound 150 x 40 April-July 85 12 7-14 6.5 61-7.2 4.0 3.6-42 U C. moreletii Mound 300 x 100 July 78-89 20-45 10 30 26-31 87 85-90 21.5 16-23 C niloticus F Hole 60 x 40 Jan.-Dec. 84-98 55 25-95 7.5 5.5-9.0 4.8 40-5.5 110 85-125 31 22-34 depending 28 27-37 80 on rains, sometimes two seasons C. Hovaeguineae Mound 122 x 61 Nov.-Jan. 83-87 26 12-40 7.6 6.4-8.8 4.3 3.6-5.8 85 63-111 novaeguineaei.l' 32 30-38 77% H20 28 24-33 by weight C pa/ustri~ Hole;n sand 30 x 50 Feb.-Aug. 60-80 26 6-41 7.5 60-8.8 4.6 3.7-5.2 83.7 47-120 32 or? mound 26-41 70 25 22-30 70 60-110 C. porosus Ceylon; Mound 170 x 53 Dry season 70-90 42 25-72 8.2 61-9.6 5.1 4.2-5.7 113 105-135 32 25-37 July-Aug. 30 28-32 61.7 82-95 C. porosus Mound 200-350 x Wet season 80-98 50 16-71 7.7 6.6-89 5.2 4.2-5.7 113 65-143 30.1 25-37 AustraliaY 80-100 Nov.-Mar. 32 31-33 92 82-95 C. rhomb~rerz Hole/mound April 68 20 7.6 51 C. siLlmensis llll Mound April-July 68-80 20-48 7.6 5.1 ~able Footnotes: see page 346.

344 346 348 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY DF CRDCODILIANS REPRODUCTIVE BIOLOGY 347

No available data indicate that the hypothalamus can respond to changing Hormonal studies may reveal the mechanisms and causes behind sev­ blood temperatures by the secretion of releasing hormones. However, two eral field observations. What keeps female alligators from ovulating under distinct glycoproteins similar in molecular weight and amino acid composi­ confined conditions? Handling stress effects the levels of corticosteroids tion to mammalian follicle stimulating hormone (FSH) and luteinizing hor­ and sex hormones (Lance, 1984). Why are only 63 to 68% of sexually mone (LH) have been isolated from the pituitaries of Alligator mississip­ mature female alligators productive in one season, whereas 34% are quies­ piensis (Licht et al., 1974, 1976). Both of these gonadotropins stimulate cent (inactive for one breeding season) and 3% are barren (Chabreck, 1966; testosterone production by ovarian and testicular tissues, with LH being Joanen and McNease, 1975a, b, 1980)? Comparable data for Crocodylus slightly more effective (Licht and Crews, 1976; Tsui and Licht, 1977). There niloticus indicate that 12% of females are quiescent and 5% are barren (Cott, are no data on circulating gonadotropin levels in any crocodilian. Follicular 1961; Graham, 1968). Female alligators may not reproduce every year, and development can be stimulated artificially with pregnant mare serum the intervals between successful breeding seasons are often variable, but gonadotropin, but artificial induction of ovulation has not been achieved the reasons for this are unclear (Joanen and McNease, 1971, 1975a). Turtles (Cardeilhac et al., 1982). Alligators (unlike turtles) show a marked response also exhibit comparable irregular reproductive cycles (e.g., Hughes, 1976). to the synthetic decapeptide luteinizing hormone releasing hormone (LH­ Female crocodilians do invest considerable resources and energy in viable RH) (Lance, 1983, 1984). Alligator LH-RH is similar to that of mammals but hatchlings; sometimes they may have insufficient reserves to reproduce in differs from it in having at least one amino acid substitution in the 8­ a given year. However, the operant mechanism is unclear. Do metabolic position of the molecule (Lance, 1983, 1984). This is similar, if not identical, end products stimulate or inhibit the endocrine system, is it a behavioral to avian LH-RH (King and Millar, 1982). Tissue levels of between 816 and response, or are females genetically determined to ovulate with a certain 2780 pg/mg protein have been estimated for the alligator hypothalamus periodicity? (Lance, 1983, 1984). The mechanics of copulation have been described in Alligator mississip­ piensis (McIlhenny, 1935; Burrage, 1965; Legge, 1967; Joanen and McNease, 1971; Neill, 1971), Caiman crocodilus crocodilus (Staton and Dixon, 1977), Footnotes for Table IfI Crocodylus niloticus (Cott, 1961; Modha, 1967, 1968), C. palustris (Whitaker UThc figures quoted are poolt'd maximum, minimum, ilnd i'l\Trage villul's frpm Ihe \'i1riou~ publications cited Older accounts \,",'ith obViously exaggerated figures, e.g., Boake 11870) have been omitted. and Whitaker, 1977b; Yadav, 1979), C. johnsoni (Compton, 1981), and Osteo­ 'Species classification b.:lscc! on Densmore (1981) and Groombridge (1982) laemus tetraspis (Tryon, 1980). The time interval between copulation and dClarke (I88Ba, b, 1891); Reese (1915b); McIlhenny 0934, 1935); Giles and Childs (1949): POpt> (1956); Jnolnen (196'J\; Ch,lDred< (197:3, 19(5); Joanen dlHj McNease (1975a, 1978, 1979f, :980); GoodWin .'lnd :\1anon (1978). fertilization is unknown, as is the mode of sperm transport within the loanen (personal communication); Chu (1957); Chu-Chcn (1982); Behler dnd Joanen (19tl2). female genital tract and the exact location within the latter where fertiliza­ tCasas and Guzman (1970); Del Toro (l974). tion occurs. The literature suggests that the interval between insemination gHagmdnn (1906, 1909); Schmidt (1928); Del Taro (1969); Hunt (1969); Neill (1971); Staton dnd Dixon (1977); Corzula (197R) hFort Worth Zoo (personal communication); Mcdem (1960); Cuy;gisber~ (l\i72); Crawshay.,' and Schaller (980). and egg laying varies from between one and five months in different 'Neill (1971); Del Taro (1974) species. The best data are available for A. mississippiensis, in which the 'Neill (l q71); Groombridge (1982). interval is three to four weeks (Joanen and McNease, 1975a, 1979a, 1980; kHagmann (1902a, h, 1909); Recse (1923); Mellem (1963); Croornbridge (19~2). IRec5e (1931 a); Medcm (1971, 1972). Lance, 1983). For C. niloticus in Kenya (Modha, 1967; Graham, 1968) and C.

m Ander50n (lB7S); Butler (1905); Werner (1933); Singh and Bustard (1977); Singh (1979); Bu"tard (1lJ79, 1980a, 1984); Acharjyo et cd (1975). cataphractus in the Ivory Coast (Waitkuwait, 1982), the interval between TlMiil1er (1838); De Rooij (1915); Butler (1905): Sudharfna (1976) copulation (ovulation) and egg laying is approximately one month. These °Guib(' (19701); Neill (1971); Tr"'on (980) figures derive from field observations and the state of ovarian follicular PDescourtilz (1809); Schmidt (1924); Werner (143.1); Rand (196H); Neill (1971); Carrick and Lln/; (1977); Ogden (1978); CasaS' .'lnd Cuzrn,1n (1970). development and the presence of eggs in the oviducts of autopsied fe­ QWaitkuwail (1982). males. However, studies based solely on field observations (with no au­ TBlohm (1982); Medem (1976). topsies) report that the time interval between copulation/ovulation and egg 'Dunn (1977); Webb (1977); Webb e, al. (1983e). I Croombridge (1982). laying is five months for C. niloticus in Zululand (Pooley and Gans, 1976), UHunt (1973,1975); Del Toro (1975); Pcrcz-IJigdredd (1%0). two months for C. palustris in India (Whitaker and Whitaker, 1976a, b), and l'VoeHzkow (1899); Bigalke (1931); Cott (1961, 1969, 1(75); Pooley 0962, 19fil}il, 1971. 1977); Modh

b!l OJ I-< ..c OJ shelled eggs are stored longer than the last. However, they all appear to be 'O.S .... 0 ~ ~ c"1:S III en Lf) N c OJ at comparable stages of development at the time of laying (see Section VI). If': .- III 0... 0'"-< £! 6 '" .-< .-< o .-< N ~"' ..... When first laid, the eggs are coated with a slimy gel (Goodwin and Marion, i3b c .... 0 +1 +1 1 'y; til 00» +1 +1 500 1978). On average, 3 to 15% of eggshells are cracked during the laying "" ~u::;a15..c: o Lf) Lf) t .-< Ei x .~ >"'~6.B o OJ 0 process (Joanen, 1969; Pooley, 1969a; Webb et al., 1977; Goodwin and ~ ~..2 I-< I-< .~ -< ::l D­ ~ '" D­ 'J> u Marion, 1978). Cracked eggs develop normally if the shell membrane re­ OJ~ 'J> '" '" 'y; Ei '" mains intact, but if it is torn, the eggs rot or are eaten by ants (Joanen, 1969; 'J> OJ .~ Ei..c ~ Goodwin and Marion, 1978). The number of eggs present in each nest '"~ If) r--.. o If': {3 0 o I-< ::l 1lI~;:;en .-< c:i o '"-<­ varies both within and among species (Table III). Species size and clutch .. "0 u .2 (::; I-< :;,o~O .-< +1 +1 +1 size may be positively correlated (Greer, 1975). l-l '.,c "'~ CJ .... ~ +1 o N Lf) ",til C Ei Within a particular species there is a significant, but highly variable, & ~] 0... """ .-< ':t:: :§'" '">. correlation between the number of eggs per clutch and body length (and Q,j ::l-"l ;; o ~ hence age) of the laying female [e.g., Alligator mississippiensis (Table IV), ....l ;:l S oo~ '"-<..0 Q,j o '" Caiman crocodilus crocodilus (Medem, 1958; Staton and Dixon, 1977), ... 'O.S C..c: Ul"O ...... D 0 OJ C " .S .B "! ..c OJ Crocodylus novaeguineae (Graham, 1981), C. niloticus (Cott, 1961; Graham, o I:;" til 6 ;::J en 00 N '" ~I-< Q,j CJI:~O.-< I-< 1968), and C. johnsoni (Webb et al., 1983e)]. Because of the variability, 00 +1 +1 +1 '" I-< :;,0 e.5 til .... +1 Ei-"l < o 00 OJ .§ Ul earlier studies, with small sample sizes, indicated no such relationships for Q,j ~ ~ Lf) 0"\ ~ > '"00 6...... c_ ~ A. mississippiensis (Joanen, 1969; Joanen and McNease, 1975a; Deitz and -< 00 0 .....-= ~z .S ~ ~ Hines, 1980) and C. porosus (Webb et al., 1977, 1983b). The contention that o 2.2'(; c '" '" ~ under different circumstances females may lay either many small eggs or OJ '" ~ '" .~ 6 C I-< fewer large eggs with the same total clutch weight (Deitz and Hines, 1980) <.J ;::J rl.8:6 ;> C 6 CJ «")"'"0 Lf) Ei 1?'~ is unconfirmed. ~ ., 'x -E E ~ - 0 <'") ~ ... til ..... 6 N 2 c An accurate method for age determination involving counting growth >-l III ~ 0 ~en -- '"d ...... Ei Ei ;; "8_ '[("lj lr) maternal age and clutch size, egg size, and egg quality in Alligator mississip­ ., -<> piensis (Table IV). "Middle-aged" females are obViously the most "success­ QI ~~~ 00 'w;:n "0 ful" breeders in terms of both quantity, size, and quality of the eggs pro­ 00 ONe ~ 6 O~ ;::J CJ - 00 '" duced. Interestingly, old females are nearly as successful, although their '1:l ~ C· ..... -£ 6..c: ..0 'w C .- 6 Lf) III ~ Ei § c clutch size is usually smaller and more malformed embryos result, whereas 6 0 N <'") """<'") Cl.l [f'; ~ Q,j "'" 0 en N ~..r::-..--l "0 5b c young females with small clutches of smaller eggs are qualitatively inferior. +1 +1 +1 C I-< .­ [;) CJ .... '" Thus even if the laying female is unseen (and its age therefore unknown), :;,0 ~ ~+I .-< o 00 -,""",'" OJ '" 00 'D r--.. r--.. ~ 00 .... CJ ~ B Ei it may be possible to group her into one of the three reproductive age ~ ~.....J 'w 0 Ei oJ::: Ei classes by noting the number and size of the eggs in her clutch (Table IV). oJ -< N 8 ;;- 00 OJ OJ 1 Moreover older females tend to lay their eggs earlier in the breeding season [;) ~CLD ~'" .­ ..c: Lf) 19 x than middle-aged females, which in turn lay them earlier than young fe­ '5 i3b~ ~.BO '" C 0 .... 'D Lf) "0 0 I-< males. This is consistent with a social hierarchy during the mating season ., ~.D 00..2 en cuD­ CJ 6~u.-< +1 +1 """+1 o '" D­ o ~ > ;:l ..... "O"'~ whereby larger females mate earlier (with large males) than smaller fe­ 'D o '" -< Z 0 ~ +1 ~ OJ OJ '" .5 0... N N '" c w males (which may mate with either large or small males). Similar relation­ v til ~ :.2 E ..... 0... Lf) CJ .- C .... CJ ~ eggs early in the nesting season and light eggs toward the end of the ..... CJ .8 ~_..--lO-,;Ul'tSQ)>"" a '" I:I.. til ~ ~ ~W"'"O season. 15 o~ bO~~_'""Ol-log '" I ;:l 6"1:S gg c OJ OJ '" For several species, weight of eggs correlates positively with that of §~:g~8..8:;c ~ &3 en- ~:< o .- ro 0-,; bJ ...... rc E5 gg hatchlings (Staton and Dixon, 1977; Deitz and Hines, 1980; Webb et al., ><~~~~o...O.s " OJ 3152 REPROOUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS REPRODUCTIVE BIOLOGY 3153

1983b, e). In mammals, fetuses with a low birth weight are frequently dutch containing living embryos. The assumption (Staton and Dixon, 1977; malformed, and the same is true of the small light eggs laid by young Webb et a1., 1983b) that the rate of heating increases up to the end of female alligators (Table IV). The alligator is an excellent model in incubation implies that heating relates more closely to the size of the em­ which to study such malformations. The eggs of middle-aged females are bryo (Drent, 1967) than to its metabolic rate. Indeed, the peak weight­ also useful for teratogenic studies as they show a lower basal incidence of specific metabolic rate in C. porosus occurs during the period of or­ spontaneous malformations than avian eggs (Ferguson, 1981a). ganogenesis (i.e., during early/middle incubation; G. Grigg, personal Possible factors that determine the relationships of egg fertility and rate communication). This assumption correlates with the fact that nests of A. of embryonic malformation with maternal age include the following: (1) mississippiensis are often warmer during the second to fourth week of incu­ reduced ovulation of young and old females, (2) the immaturity of young bation than later in their 9-week incubation period (Chabreck, 1973). More­ ova, (3) slight asynchrony between the times of ovulation and copulation over the hole nests of C. acutus (Lutz and Dunbar Cooper, 1982, 1983) and in young females, (4) the lower sperm numbers and quality in males near C. joJmsoni (Webb and Smith, 1984) become progressively warmer during the end of the breeding cycle (when young females mate), and (5) the incubation, and this temperature rise may accelerate development and poorer quality of the large ova released by old females. There is some improve survivorship (Webb and Smith, 1984). evidence that prior to senescence old females either resorb their ova or Although critical for successful embryonic development, nest humidity their eggs, or both. Possibly, young and very old females contribute little to and the water relations of crocodilian eggs are poorly understood. ,All population recruitment. crocodilian nests studied to date have exhibited very high humidities Nest temperature and humidity are important factors for embryonic (Table III). Precise data on the mechanisms of humidity maintenance are survival, development, growth, and sex determination (Table III). Only lacking, but there have been reports of maternal behavior involving urina­ Alligator mississippiensis (McIlhenny, 1934, 1935; Joanen, 1969; Chabreck, tion and splashing of the nest (e.g., Whitaker and Whitaker, 1976b, 1977b). 1973), Crocodylus porosus (Webb, 1977a; Webb et a1., 1977, 1983b; Magnus­ If this is deliberate, it would imply that the mother can monitor nest tem­ son, 1979c), Caiman crocodilus (Staton and Dixon, 1977) and Crocodylus perature and/or humidity, and it has been suggested that this might be the acutus (Lutz and Dunbar Cooper, 1982, 1984) have been studied in detail. function of sensory receptors on the jaws (Ferguson, 1979b, 1981b, 1982b; In general, the positioning and construction of nests ensure fairly stable see also Section II.E). However, during abnormally hot dry years, the thermal and humidity conditions within the egg cavity. humidity within nests of A. mississippiensis decreases, and the eggs begin to Crocodilian nests may obtain three sources of heat: solar heating, de­ form abnormal air spaces (Ferguson and Joanen, 1983) due to cleavage of composition, and metabolism. The temperature of the nest cavity is the shell membrane away from the calcified eggshell. If the air space is equilibrated; the outer layers of the nest absorb solar heat at times of large, it causes embryonic death, representing a significant source of mor­ maximum incidence, but slow its passage to the central cavity; some of this tality, particularly among eggs near the top of alligator nests during hot dry heat later dissipates to the central cavity. However, solar energy is unlikely years. to be the sole source of heat, as crocodilian mean nest temperatures are Nest temperatures and humidities outside the normal range lead to often 1° to 5°C above the mean air temperature (McIlhenny, 1935; Joanen, malformed or dead embryos (Modha, 1967; Bustard, 1969, 1980a, c; Pooley, 1969; Chabreck, 1973; Modha, 1967; Webb et a1., 1977, 1983b; Staton and 1962, 1969a, h; Whitaker and Whitaker, 1976a, b, 1977b; Joanen and Dixon, 1977). McNease, 1975a, b, 1977, 1979a, 1980, 1981; Staton and Dixon, 1977; Og­ The temperature of mound nests may be raised by the decomposition of den, 1978; Ferguson and Joanen, 1983; Webb et a1., 1983a,b,e). Within the the nesting vegetation, particularly during the early stages of incubation range of viable temperatures, temperature and incubation time are in­ (McIlhenny, 1935; Joanen, 1969; Chabreck, 1973; Webb et a1., 1977a, 1983b; versely correlated (Reese, 1915a; McIlhenny, 1934, 1935; Pooley, 1962, Staton and Dixon, 1977; Magnusson, 1979c). Such decomposition may be 1969a,b; Joanen and McNease, 1975a, b, 1977, 1979a, 1980; Packard et a1., enhanced by females urinating onto the nest during and after its construc­ 1977; Webb et a1., 1977, 1983a,b,e; Ferguson, 1982b; Ferguson and Joanen, tion (Pooley, 1962, 1969a, b; Chabreck, 1975; McIlhenny, 1934, 1935; Reese, 1982, 1983). Thus, there are only mean incubation periods (Table III), and 1915a; Deraniyagala, 1936, 1939; Whitaker and Whitaker, 1976b, 1977b; field incubation times are difficult to interpret even with scant information Tryon, 1980; Webb et a1., 1983b). However, decomposition should be on nest temperatures. Consequently, developmental stages must be re­ minimal during the latter stages of incubation in mound nests and nonexis­ lated to an arbitrary (normal) time scale calculated for controlled environ­ tent in hole nests. Embryonic metabolism has also been postulated to rep­ mental conditions (Ferguson, 1982b; Webb et a1., 1983a). resent a source of heat (Modha, 1967; Magnusson, 1979c; Webb et a1., The gaseous conditions within crocodilian nests have been assayed 1983b). Clearly, the extent of such heating varies with the proportion of the twice, once for the hole nests of Crocodylus acutus (Lutz and Dunbar 354 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCODILIANS REPROOUCTIVE BIOLOGY 355

Cooper, 1982, 1984) and again for mound nests of C. porosus (Grigg, un­ shed light on the role of incubation temperature in determining the sex published data). The data do not differ significantly. Shortly after egg ratios of hatchlings (Section VILO). laying, the pOz in the nest is 147 torr (ambient =: 150), and the pC02is 5.0 torr (ambient =: 0.3). These values reflect the properties of the nest as the E. Maternal Behavior same values are obtained whether or not eggs are present (Grigg, unpub­ lished data). Embryonic arterial blood is approximately 86% saturated and Those behavioral repertoires of female crocodilians that improve reproduc­ venous blood about 40%: arterial pOz is 52 torr (range 42-57), arterial pC02 tive efficiency are not restricted to nest selection and construction (Section is 12 torr (range 8-14), and venous pOz is 22 torr (Grigg, unpublished 11.0), but include subsequent nest protection and opening, egg opening, data). Clearly, the eggshell and shell membrane represent a significant transport of hatchlings, and parental care. They have been described for barrier to gaseous diffusion (see Section III.E). As incubation proceeds, Crocodylus acutus (Campbell, 1973; Garrick and Lang, 1977), C. cataphractus nest p02 falls and pCOz rises (Lutz and Dunbar Cooper, 1982, 1984; Grigg, (Waitkuwait, 1982), C. johnsoni (Dunn, 1981; Webb et aI., 1983e), C. unpublished data). Crocodilian embryos can tolerate extremely low p02 moreletii (Hunt, 1975, 1980), C. niloticus (Cott, 1961, 1909, 1971, 1975; Gar­ levels and recover; furthermore, they are very resistant to raised pC02 rick and Lang, 1977; Pooley, 1962, 1969a, 1974a, b, 1976, 1977; Pooley and levels. Even pCOz levels as high as 60 torr do not inhibit the embryonic Gans, 1976; Voeltzkow, 1891, 1892, 1893, 1899; Bohme, 1977; Guggisberg, metabolic rate (Grigg, unpublished data). 1972; Neill, 1971), C. novaeguineae (Neill, 1971), C. palustris (Symons, 1918; Flooding of crocodilian nests has long been known to cause embryonic Deraniyagala, 1939; Whitaker and Whitaker, 1977a, b), C. porosus (Derani­ death (Reese, 1915a; McIlhenny, 1935; Joanen, 1969; Joanen et aI., 1977; yagala, 1936, 1939; Webb, 1977a; Webb et aI., 1977; Magnusson, 1980; Fleming et aI., 1976; Hines et aI., 1968; Goodwin and Marion, 1978; Nichols Bustard and Kar, 1981), Alligator mississippiensis (Reese, 1915a, 1931a; et aI., 1976; Voeltzkow, 1891, 1892, 1893, 1899; Webb, 1977a; Webb et al., McIlhenny, 1935; Giles and Childs, 1949; Lee, 1968; Joanen, 1969; Camp­ 1977, 1983b and e; Magnusson, 1982; Deraniyagala, 1939; Cott, 1961, 1969; bell, 1973; Kushlan, 1973; Herzog, 1975; Garrick and Lang, 1977; Garrick et Pooley, 1962, 1969a). Joanen et al. (1977) tested the effects of simulated aI., 1978; Kushlan and Kushlan, 1980; Kushlan and Simon, 1981; Deitz and flooding on hatchability by immersing the eggs of Alligator mississippiensis Hines, 1980; Hunt and Watanabe, 1982), Caiman crocodilus crocodilus (Del for single periods of two, six, 12, and 48 hours at different stages of devel­ Toro, 1969; Campbell, 1973; Garrick and Garrick, 1978; Staton and Dixon, opment. Immersing eggs of any age for two to six hours had little effect on 1975, 1977), Osteolaemus tetraspis (Tryon, 1980), and Cavialis gangeticus subsequent hatchability, nor did submerging eggs for 12 hours if done (Singh and Bustard, 1977; Bustard, 1980b,c,d; Bosu and Bustard, 1981). before day 30 (after laying). Thereafter, 12 hour submergence killed all Maternal and prehatching vocalization of crocodilians are reminiscent of embryos. Submergence of the eggs for 48 hours killed all the embryos at all avian behavior (Vince, 1969, 1973; Gottlieb, 1973; Oppenheim, 1973). In ages. Similar results are recorded for experiments on Crocodylus porosus birds, they are supposed to accelerate and synchronize late embryonic eggs (Magnusson, 1982). Any moisture on the surfaces of crocodilian development in the clutch of eggs. The same effects have been suggested eggshells drastically reduces their gas conductance and may totally inhibit for crocodilians (Lee, 1968). However, a single experimental study (Mag­ oxygen diffusion. It therefore seems likely that flooding inhibits gaseous nusson, 1980) showed that advanced embryos of Crocodylus porosus called diffusion, and thus causes embryonic death: a similar mechanism operates in response to a tape recording of wild hatchling vocalizations, but neither in chicken eggs (Kutchai and Stean, 1971). All embryos could utilize air developed faster nor hatched more nearly synchronously than did control within the eggshell and so withstand short periods of flooding; whereas eggs. How embryos, enclosed within the fluid environment of an egg, emit younger embryos would have a lower O 2 requirement and relatively more such sounds is unknown. Vocalizations are also involved whenever the air within the eggshell, so that they could withstand longer periods of females excavate nests, open eggs, or transport hatchlings in their cavern­ flooding than older embryos. ous jaws. All these activities require a high degree of oral sensitivity and Factors that influence the site selected by females for nest construction muscular control. Numerous domed sensory receptors on the palate and are complex and incompletely understood but probably include social in­ jaw margins (Figs. 3A and B) may facilitate these and other (e.g., courtship teractions with other animals, vegetation type at site, proximity of the site and feeding) behaviors (Ferguson, 1981b, 1982b). All available reports are to water, its temperature, degree of exposure to sunlight, and height above phenomenologic, but it is obvious that further investigations of the neural­ water level. Whereas investigations of these parameters are, by definition, endocrine mechanisms underlying these complex behaviors are war­ ecological and behavioral, there can be no doubt that these factors affect ranted, not merely for their intrinsic interest, but also for their potential fecundity within a population. Further studies are likely to contribute not contribution to our understanding of the evolution of parental care in onlv to our understanding of crocodilian reproductive strategies, but may birds and mammals. 356 I REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS REPRODUCTIVE alOLOGY 357

1974; Blake and Loveridge, 1975). Other reports describe laboratory main­ tenance of alligators (Coulson et al., 1973, Coulson and Hernandez, 1964) and give detailed information on the construction of egg incubators, con­ trolled environmental chambers and suitable breeding enclosures (Joanen and McNease, 1974a, b, 1976, 1979a; Pooley, 1971). Successful reprOduc­ tion has now been achieved for many species (Chaffee, 1969; Del Toro, 1969; Hunt, 1969, 1973, 1975; Legge, 1969; Yadav, 1969, 1979; David, 1970; Yangprapakorn et al., 1971; Joanen and McNease, 1971, 1975a, 1979a, 1980, 1981; Honegger, 1971, 1975; King and Dobbs, 1975; Dunn, 1977, 1981; Whitaker, 1979; Whitaker and Basu, 1983; Bustard, 1980c, 1984). Thus far, artificial insemination has proved difficult (Joanen and McNease, 1973, 1975b) and has only once been achieved, the major problem being induc­ tion of ovulation in the female (Cardeilhac et al., 1982). In most management (and embryological) programs, eggs are collected from wild nests and artificially incubated. Documentation of recom­ mended collecting methods and their effect on embryonic viability is avail­ able for several species (Blake and Loveridge, 1975; Chabreck, 1977, 1978; Joanen and McNease, 1977, 1979a, 1980, 1981; Pooley, 1969a, b, 1971; Whitaker and Whitaker, 1976a). The exact time of collection and possible deleterious effects of inversion are particularly important; similar problems have been reported with respect to testudinian eggs (Ewert, 1979; Par­ menter, 1980; Blanck and Sawyer, 1981). At oviposition, the embryonic disk, covered by a thin layer of albumen, floats freely on top of the yolk. Its precise location depends upon the position of the egg in the nest. If the egg is moved during the first 24 hours after laying, the embryoniC disk moves to the new highest point of the yolk without detrimental effects. However, after 24 hours, the disk attaches to the inner aspect of the shell membrane, and the egg shows an opaque spot in this area (see Section III. B). The embryo remains attached, hence move­ ment or turning may kill it. After inversion, attachment to the shell keeps the embryos from rising above the yolk maSSi they may be crushed or --:-""J;:;;iF'P:'" drowned by the superjacent heavy yolk. Moreover, in the first few days Fig. 3. Alligator mississippiellsis. (A) The palate of an adult photographed under oblique after laying, the embryo has not undergone torsion (Section VI) and so lies incident illumination to highlight the numerous low sensory papillae. (B) Histological section at apprOXimately right angles to the shell surface, whereas small blood of the sensory papillae on the palate. Note the highly specialized sensory nerve ending and dermal Merkel cells. 292 x. vessels proliferate within both it and the extraembryonic membranes, which are also attached to the shell membrane. Any movement tends to shear the embryo off the embryonic disk and rupture these delicate blood F. Captive Breeding, Egg Collection, and vessels, thereby causing death. Thus eggs should be collected before the Artificial Incubation embryo attaches to the shell membrane (within 24 hours after laying) or much later, after the fourth week of incubation, by which time the embryo Data pertaining to egg collection, artificial incubation, hatchling culture, and extraembryonic membranes are less susceptible to damage. Similar and captive breeding are available for several species (Bustard, 1980c), but considerations apply to turtles (Blanck and Sawyer, 1981). Always mark principally for Alligator mississippiensis (Joanen and McNease, 1971, 1973, the top surface of the egg prior to removal and transport and incubate eggs 1974a, b, 1975a, b, 1976, 1977, 1979a, b, 1980, 1981; Chabreck, 1977, 1978; in their original nest orientation or the nearest horizontal position. Nichols et al., 1976), and Crocodylus niloticus (Pooley, 1969a, b, 1971; Blake, Other reasons for collecting eggs within 24 hours of laying include the 35B REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF' CROCODILIANS REPRODUCTIVE BIOLOGY 3551

following: (1) Reduction of losses from predation, flooding, or physical nite crystals of calcium carbonate by calcite crystals (Solomon and Baird, damage to the nest (e.g., by another nesting ); (2) control and 1979; Baird and Solomon, 1979). Although the eggshells of farmed croco­ optimization of the incubation environment; (3) control of the sex and dilians may be defective, it seems more likely that decreased fecundity and possibly the future growth of hatchlings (Ferguson and Joanen, 1982, increased abnormality may result from maternal dietary deficiencies, e.g., 1983); (4) monitoring development by observing changes in eggshell band­ that of vitamin E (Lance, 1982; Lance et a!., 1983; Elsey and Lance, 1983) or ing (see Section III. B) and detecting (in order to discard) infertile, inappropriate husbandry (Joanen and McNease, 1981; Lance, 1984). malformed, dead, or infected eggs; (5) acceleration or delay of develop­ Incubation of alligator eggs at temperatures ranging from 26° to 34°C, ment by altering the incubation temperature and synchronization of hatch­ produces the best hatching success at 30° to 32°C (Joanen and McNease, ling emergence; (6) correction of the orientation of eggs laid in a detrimen­ 1977, 1979a, 1980, 1981; Ferguson and Joanen, 1982, 1983). Hatchlings from tal position. Thus embryos of eggs laid upright (i.e., with their long axes at eggs incubated below 28°C often twitch, have balance difficulties and swim right angles to the nest base) frequently die or are malformed; they develop with their heads constantly under water; eventually they drown. Eggs of normally if the eggs are reoriented so that the embryonic disk becomes Crocodylus novaeguineae incubated at 23°, 26°, and 38°C (normal approxi­ positioned beneath the top center of the egg. mately 32°C) have a low hatching success (Bustard, 1969, 1971). All sur­ The death of embryos following egg inversion may explain why vivipar­ vivors of the high temperature group showed tail and other abnormalities ity never evolved in the Crocodilia or Testudines. During the evolution of and had to be helped from their shell. Hatchlings of C. palustris recovered viviparity, there is usually an intermediate stage in which the embryo from overheated nests also showed tail abnormalities (Whitaker and develops inside an egg retained within the oviducts of the female. How­ Whitaker, 1976a,b). ever, in taxa in which the embryo attaches to the shell membrane and Crocodilian eggs are susceptible to changes in humidity; at low levels turning or inversion result in embryonic death, this intermediate stage is the shell membrane dries out and shrivels, thereby killing the embryo unlikely to be achieved. The absence of crocodilian viviparity may also be (McIlhenny, 1935; Deraniyagala, 1939; Pooley, 1962, 1969a, b, 1971; Joanen, related to increased dependence of embryos on eggshell calcium as com­ 1969; Staton and Dixon, 1977). The optimum relative humidities for in­ pared to the very slight dependence of snakes and lizards (Packard et aI., cubating Alligator mississippiensis eggs is 90 to 92% (Joanen and McNease, 1977), the absence of biological advantages for prolonged egg retention, 1977, 1979a; Chabreck, 1975). Abnormal air spaces form in alligator eggs and the evolution of nest guarding (Tinkle and Gibbons, 1977). incubated at suboptimal humidities (Ferguson and Joanen, 1983); large air Artificial incubation of the eggs of "hole nesters" has been effected by spaces are lethal. Intact eggs of Crocodylus acutus are resistant to desicca­ reburying the eggs in suitable sites (Pooley, 1969a, b, 1971; Blake and tion, losing water at a rate comparable to the eggs of birds (Rahn and Ar, Loveridge, 1975; Bustard, 1980c). Earlier attempts at incubating the eggs of 1974; Rahn et aI., 1979; Ar and Rahn, 1980; Diamond, 1982), but at a rate mound nesters, e.g., Alligator mississippiensis, involved placing them in much lower than that of the leathery-shelled eggs of Iguana iguana (Rand, buckets of nesting material maintained at the appropriate temperature and 1968). Removal of a 3 x 3 cm piece of shell (leaving the shell membrane humidity (Reese, 1901a, 1931a; Table III). Currently eggs are incubated intact) from the eggs of C. acutus greatly increases the desiccation rate either in incubators or in environmental chambers (Joanen and McNease, (Rand, 1968). The eggs of C. novaeguineae apparently could either lose or 1974a,b, 1976, 1977, 1979a, 1980, 1981). Tests relating to the incubation of gain up to 25% water with no adverse effects on the embryos (Bustard, alligator eggs, including stacked versus nonstacked, wild eggs versus eggs 1971). The soft-shelled eggs of many squamates and turtles can adsorb from captive breeding programs, oxygenated chambers versus nonoxy­ (and then lose) water up to 300% of their initial weight (Bustard, 1971; genated chambers, washed eggs versus nonwashed eggs, exposed eggs Packard et aI., 1977); unlike the eggs of crocodilians (and certain other versus eggs covered with nesting material, and eggs set over water versus turtles) they lack large quantities of hydrophilic albumen, which are likely eggs set over dry concrete (Joanen and McNease, 1977) showed no appre­ to reduce the rate of water loss under adverse conditions. ciable effects, except for a 13% decrease in hatching success from stacked Bustard (1971) has suggested that water uptake is unessential in croco­ eggs, and a lower hatching rate (72%) for captive produced eggs than for dilian eggs, but represents an insurance against lethal levels of water stress those of wild animals (94%). About 15% of embryos from captive produced caused by possible adverse environmental conditions later in develop­ eggs died around hatching, and others had to be assisted from the shell ment. However, Packard et al. (1977) reported that water uptake was criti­ (Joanen and McNease, 1975a, 1977). This may indicate weakness of the cal for normal reptilian embryonic development; prevention of water up­ hatchling (compacted yolks are more common in this group) or abnormal take, particularly in the early stages of incubation, leads to high rates of toughness of the eggshell and its membranes. Decreased hatching success mortality and developmental abnormalities. Indeed, Tracy et a!. (1978) in farmed turtles may be associated with replacement of the normal arago- suggested that the only major difference between the eggs of birds and 361 REPRODUCTIVE BIOLOGY 360 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS those of crocodilians (and certain turtles) is the site where they are laid; avian nests favor water loss and crocodilian nests favor water gain. How­ ever, these authors assume that cracks in the alligator eggshell under natu­ ral nesting conditions indicate water uptake and egg swelling. Alternative explanations for such cracks are enlargement and movement of the embryo and weakening of the shell to facilitate hatching (Ferguson, 1981c, 1982a). Indeed, Lutz and Dunbar Cooper (1982, 1984) reported a net water loss from eggs of Crocodyllis aClltlls; the birth weights of hatchlings were 0.64 ± 0.01 those of the initial egg masses, a value almost identical to that of many birds (0.65), (Rahn, 1982; Diamond, 1982). During incubation of avian eggs, dry matter is metabolized and metabolic water produced, but water concentration is maintained by losing a fixed portion of the water (Ar and Rahn, 1980). The fractional loss (F = total weight loss/initial weight) for eggs of 81 species of birds was remarkably constant (F = 0.150 ± 0.02580) and similar to that of C. aClltlis (F = 0.154) (Lutz and Dunbar Cooper, 1982, 1984). Preliminary data therefore indicate that crocodilian eggs are struc­ turally (Ferguson, 1982a) and functionally (Lutz et al., 1980; Lutz and Dun­ bar Cooper, 1984) similar to avian eggs but dissimilar to those of squamates and some testudines (Packard et al., 1977). Their water vapor conductance is treated below (Section ULE). The water relations of crocodilian eggs require further investigations both in the wild and experimentally. In some turtles, the size of hatchlings (and hence their survivorship) and the length of egg incubation are both related to the hydric environments of egg incubation (Packard et al., 1981, 1982, 1983). Moreover, eggs of Chrysemys picta exhibit normal embryonic metabolism until the pool of egg water is reduced to a threshold level, at which time death occurs (in unfavorable hydric conditions) or hatching 31 ensues (in favorable conditions), depending on the stage of embryonic development (Packard et al., 1983). Thus the water potential of eggs may provide the cue for hatching and an explanation of late embryonic deaths (often during the week of expected hatching). Moreover, preliminary data indicate that the hydric environment of Chrysemys picta eggs influences sexual differentiation (Gutzke and Paukstis, 1983); this may also be true for crocodilians.

G. Hatching illustrati]l~ Fig. 4. Alligator mississippiensis. Sequence of photographs hatching. The mechanics of hatching have been described for Alligator mississippiensis (McIlhenny, 1934, 1935; ]oanen, 1969), CrocodylliS niloticliS (Voeltzkow, week of incubation (Joanen, 1969), diagonal cracks begin several days later, 1891, 1892, 1893, 1899; Pooley, 1962, 1969a), C. POroSliS (Deraniyagala, 1936, and shell flaking begins prior to hatching. The embryo is thuS surrounded 1939; Webb, 1977a), and C. pailistris (Deraniyagala, 1939); they appear to be by extraembryonic and shell membranes. These membranes are later punc­ T similar in all crocodilians (Neill, 1971). The growing and moving embryo tured by the sharp "caruncle" located at the tip of the snout (Section vn. , strains the eggshell, which has already been weakened by mobilization of Figs. 26A to Hand 36A to D), thereby pippipg the egg (Fig. 4). 8za) calcium and extrinsic degradation (see Section lII.C), so that it eventually Because crocodilian eggs normally lack an airspace (Ferguson, 19 , cracks. In A. mississippiensis, longitudinal cracks develop about the seventh It ~ 3SE! REPRODUCTIVE alOLOGY ANO EMBRYOLOGY OF CROCODILIANS THE EGGSHELL AND SHELL MEMBRANES 363 there is no internal pipping, as seen in birds (Rahn et a1., 1979). Hence, crocodilian hatchlings are unlikely to breathe until the shell and mem­ The precise hatching trigger remains unknown. Mechanical stimuli and branes are broken and the flUids are drained from around the head. The vocalizations may playa role (see Section II.E). Growth and movement of time interval between this event and emergence from the egg is relatively the embryo may burst the shell. Increasing levels of waste products, de­ short (ranging from a few minutes to 24 hours), so that the transition from creasing egg water, and the progressive inadequacy of gas exchange may chorioallantoic to pulmonary respiration must be rapid, as in mammals also stimulate hatching. Temperature seems to be important; field reports and megapode birds (Seymour and Ackerman, 1980), but it is contrary to note that an inadvertent rise in egg temperature stimulates premature the slow transition in other birds (Rahn et a1., 1979). Details of this transi­ hatching, and, conversely, decreases in egg temperature delay hatching tion (e.g., the extent of chorioallantoic perfusion during the time between (McIlhenny, 1934, 1935; Deraniyagala, 1936, 1939; Pooley, 1962, 1969a, first breathing and hatching) are unknown but of considerable interest. 1971). Hatchlings are sufficiently well developed to be viable if they hatch The problem is even more intriguing when one recalls that for many hours several days prematurely (McIlhenny, 1934, 1935) so that completion of prior to pipping embryos vocalize in response to certain stimuli (see Sec­ development is not the final trigger for hatching. Lengths and weights of tion 11.E); this suggests that the lungs are functional during this period. As the hatchlings from different species are summarized in Table III. alligator eggs progressively lose water during incubation, an air bubble forms beneath the shell membrane and this moves around in the albumen, so as to always lie beneath the uppermost surface of the egg (Ferguson, III. THE EGGSHELL AND SHELL MEMBRANES 1982a): similar events OCCur in megapode eggs (Seymour and Ackerman, 1980). Perhaps this air is used prior to pipping (analogous to the internal A. General pipping of avian embryos). This area is significant because late embryonic death is common (during both natural and artificial incubation) and could Most crocodilian eggs are elliptical with approximately equivalent ends be caused by improper hydric conditions. Thus, high humidity may be (Figs. 5A to 1). They vary considerably in size and shape, even within required during the early stages of incubation, but lower humidity may be species (Tables III and IV). Normally there is little variation in size and reqUired during the later stages to facilitate water loss and the formation of shape within an individual clutch. However, occasional large, infertile, intra-egg air. This in turn may enable internal breathing, the maturation of double-yolked eggs or very small eggs are seen; these abnormal eggs are the respiratory system, and provide the extra oxygen required for pipping. usually laid at the beginning and end of oviposition and are common in Indeed, Vocalization may represent a signal that the embryo is mature clutches laid by young females (Ferguson and Joanen, 1983). Ferguson enough to survive outside the egg, and it remains to be determined (1982a) described the structure and composition of the eggshell and shell whether it occurs in eggs incubated under conditions of continuous high membranes of Alligator mississippiensis and provided a comprehensive re­ humidity. The changing hydric conditions are likely to be most critical in view of the literature. species with long incubation periods (e.g., Crocodylus porosus). During the hatching process, the embryo first widens the slit in the shell B. Egg Banding membrane (frequently by pushing its snout into the slit and then opening its jaws). Second, it pUShes out the entire head and neck; after a couple of The gradual development of a transverse white band across fertile croco­ minutes, one quick forceful thrust forces the whole bOdy out of the egg dilian eggs has been known for nearly a century (Clarke, 1888a,b, 1891), (Fig. 4). The claws may be used in the hatching process. The ruptured and reports are now available for several species (Ferguson, 1982a; Reese, extraembryonic membranes progressively detach from the shell membrane 1908, 1912, 1915a, 1931a; McIlhenny, 1935; Deraniyagala, 1936, 1939; Webb but remain attached to the umbilical region of the hatchling for about a day; et aI., 1977, 1983a, b, e; Webb, 1977a; Beck, 1978; Hara and Kikuchi, 1978; they eventually shrivel, dry up, and break (Fig. 21). Hatchlings are COvered Deitz and Hines, 1980; Tryon, 1980). with slime, presumably derived from the ruptured allantois, and they Within 24 hours of egg laying, the eggs of Alligator mississippiensis show smell of ammonia. This slime dries after about 24 hours. At the time of a small opaque, chalky white, oval patch on their top surface (Figs. 5A and hatching, the abdomen is distended by absorbed yolk and the umbilical 6). The small embryo has attached itself to the innermost surface of the Scar is evident (Fig. 21). The yolk serves as a food supply for a few weeks shell membrane immediately below this opaque patch. At this and all until the hatchlings begin feeding so that the abdomen becomes less dis­ subsequent stages, the white color is associated with changes both of the ~ended and the umbilicus closes. In hatchlings kept below 210C, the um­ eggshell and shell membrane (which itself appears chalky white in this )ilicus does not heal correctly, yolk is not utilized, and the animals usually He (King and Dobbs, 1975). area). Initially the oval opaque patch expands in width around the shell, extending approximately half-way around the shell on days 3 to 4, three­ quarters way around the shell on days 5 and 6, and completely around the 1

microorganisms in the pores of the eggshell and on the surface of the shell membrane. (D) A completely opaque, cracked, 58-day egg. (E) Attachment of the embryo and chorioallantois to the superior inner surface of the shell membrane in an I8-day egg. Note the two poles of albumen (A) at the ends of the egg. The yolk has been removed. (F) Expansion of the chorioallantois and the regression of albumen seen in the superior inner surface of a 25-day egg. (G) Lateral view. Opaque band in a transilluminated 7-day egg. The band appears black by this technique and appears longer toward the top (right) than the bottom (left) of the egg. Fig. 5. Alligator mississippiensis. (A) Opaque spot on the eggshell and shell membrane of an (H) View of the opaque band in a I2-day egg. (I) View of the opaque band in a 20-day egg. (J) egg 24 hours after deposition. (8) Opaque band on a ten-day eggshell. (C) Opaque band in the View of a 40-day egg, which is almost completely opaque. eggshell (partIy removed) and shell membrane of a 20-day egg. Note the specks of debris and 3615 ...,...

366 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS THE EGGSHELL AND SHELL MEMBRANES 367

malformed), but remain uniformly translucent white (egg contents yellow) throughout incubation. They can easily be distinguished from normal, completely opaque, post day 50 eggs by transillumination (Figs. 5G to J, and 6). Eggs laid at advanced stages of embryonic development, for ex­ ample, by stressed females (see Section II.C), are either banded at the time of laying or become so very rapidly. Band expansion is also more rapid, but oTO T1 T3 8T5 the embryos usually die, at which time the formation ceases. Failure to distinguish between unbanded infertile eggs and either those in which the embryo has died early and become autolyzed or post day 50 normal un­ banded eggs has given rise to erroneous accounts of the relationship be­ tween banding and fertility (Deitz and Hines, 1980; Tryon, 1980). That the appearance and regular development of the opaque band indi­ T7 87 T30 T52 cate normal, healthy development of the embryo is of value not only in aging eggs but also in identifying abnormal development and monitoring Fig. 6. Alligator mississippiensis. Diagram depicting the development of the opaque band in eggs viewed in transmitted light. T, top of egg; B, bottom of egg; numbers, days after the time teratogenic experiments (Ferguson, 1981a, 1982b). The relationship be­ the egg was laid. tween the development of this band and eggshell hydration, albumen metabolism, the formation of the extraembryonic membranes, structural changes in the eggshell and changes in the composition of the shell mem­ shell after day 7 (Figs. SA to C, G to J, and 6). This extension in width is not brane is discussed in Sections III.C through E and IV. matched by a uniform expansion in length, so that the opaque band tapers from a maximum length at the top surface of the shell to a minimum length C. Structure on its bottom surface (Figs. 5 and 6). The embryo always lies beneath the upper expanded part of the fusiform opaque ring (Figs. 5B, E, F, G-J, and 1. GENERAL 6). The opaque band then expands rapidly in length so that it extends over The combined thickness of the eggshell and shell membrane of Alligator approximately 60% of the top surface and 50% of the lower surface of the mississippiensis is approximately 0.5 to 1.0 mm and consists of five layers shell between days 8 and 30 (Figs. 5B to 0, G to J, and 6). The top part of (Fig. 7). From the surface inwards, there is an outer, densely calcified layer the band reaches the ends of the egg around day 40 and the bottom part (100-200-f.Lm thick), a honeycomb layer (300-400-f.Lm thick), an organic around day 50; thereafter the egg is completely opaque and so appears layer (8-12-f.Lm thick), a mammillary layer (20-29-f.Lm thick), and the shell unbanded (Figs. 50, J, and 6). The opaque part of the egg never transmits membrane (150-250-f.Lm thick). Pores run from the egg surface through the light as well as the adjacent translucent regions and is clearly demonstrated calcified layers and end in the shell membrane (Fig. 7). The outer openings as a dark zone if the eggs are transiIIuminated (Figs. 5G to J, and 6). of the pores become modified and numerous erosion craters develop as Eggshell banding represents a useful, external indicator for estimating incubation progresses (Figs. 7 and 8). The different layers of the eggshell the age of alligator eggs (Ferguson, 1982a) up to approximately day 50; consist of varying amounts of calcite crystals and organic matrix. The subsequently the eggs are completely opaque. The normal expansion of freshly laid egg is coated with slimy oviducal secretions, which dry and the opaque band may be retarded in eggs containing malformed or de­ then disappear. velopmentally retarded embryos. Usually, the expansion of the "mini­ mum band length" at the bottom of the egg is the most sensitive indi­ 2. OUTER DENSELY CALCIFIED LAYER cator (Ferguson, 1982a, b). Eggs containing embryos that have died but that are not infected show an arrest of band development at the stage of At low magnification (Fig. 9A), the egg surface appears smooth, but at embryonic death. This may be followed by regression of the band as the higher magnification (Fig. 9B), the granular nature of the numerous calcite embryo autolyzes. Infected eggs have the opaque bands vaguely defined; crystals is evident. There is no detectable organic matrix between the opaque blotchy patches may appear and disappear all over the eggshell, rhombohedral crystals in this layer; their form, size, and orientation are usually in an erratic fashion. Infertile eggs never become banded and never shown in Figs. 8 and 9, and further details are given in Ferguson (1981c, become infected (unless the eggshell or eggshell membrane is damaged or 1982a). The calcite crystals are stacked on their ends (or faces) in this layer. 368 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIANS

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D C C C ·6' . o~-::) ~~L. c_~ H J FillA IIdill 0) SD SD .t,

ODC

'r '~t H :<~,lB .. ,.;.); p .. Fig. 7. Alligator mississippiensis. Diagram of the structure of the eggshell in both the opaque ® CD and nonopaque zones as seen in cross section. Ee, Erosion crater; ESM, shell membrane; H, Fig. 8. Alligator mlsslsslpplensis. Diagrams of the development of erosion craters in an honeycomb layer (crystals oriented with their c-axis horizontal and containing numerous eggshell. (From Ferguson 1981c, 1982a.) (A) A unit of calcite CaC0 Class 3m illustrating the vesicular holes); M, mammillary layer (the mammillary knobs are common in the opaque 3 general shape of the crystal and the direction of its axes a, b, and c. (B) A large (32 molecule) zone, but not so frequent in the nonopaque zone; the eggshell membrane fibers of the latter rhombohedron of calcite based on cleavage rhomb. The true unit cell is the small (two attach directly to a layer of large calcite crystals rather than the mammillary knobs); 0, organic molecule) steep rhombohedron shown inside. (C-J) Series of diagrams illustrating the forma­ layer (a layer immediately superficial to the mammillae containing a higher percentage of tion of the erosion craters (EC) in the outer, densely calcified layer (ODC) of the shell (here the organic matrix); ODe, outer densely calcified layer (crystals oriented. with their c-axis at right calcite crystals are stacked in layers with their coaxes at right angles to the shell surface). angles to the shell surface); P, small pore in the shell membrane; PO, pore (more frequent in Initially there is a surface defect (SD), for example, a crystal missing; acidic erosion spreads opaque zone); PP, pore plug (falling out). (From Ferguson 1981c, 1982a.) from this center dissolving the c-faces of the crystals more rapidly than the a-faces. This process produces regular crater-like stepping of the calcite layers. It stops at the honeycomb layer (H) as its crystals are oriented with their b-faces at right angles to the shell surface and 3. HONEYCOMB LAYER thus are more resistant to acidic erosion. In addition, the fluid at the base of the crater contains more calcium, so that there is an equilibrium balance (diffusion control Dca and open arrows) The honeycomb layer is porous and contains a high percentage of fibrous between dissolution and reprecipitation of calcium (indicated by black areas). However, fluid organic matrix (Figs. 9C and E). When compared to those in the outer loss and replenishment occurs near the mouth of the crater so that dissolution is largely under densely calcified layer, the different (horizontal) stacking of the calcite kinetic control (Kca and solid arrows) with little reprecipitation. Remnants of cuticle (C) or other organic debris may mask layers of calcite from acid attack and so cause irregular step­ crystals in this layer is important in the formation of erosion craters (see ping and a variety of different shapes of crater. (K, L) Diagrams illustrating the fate of pores as Figs. 7 to 9 and text below). The numerous matrix-lined holes make the development progresses. e, Cuticle; CPP, remnants of cuticle pore plug and other organic honeycomb layer similar to the palisade layer of the eggshells of tropical debris; Dca' diffusion control of calcium equilibrium (open arrows); H, honeycomb layer birds (Schmidt, 1957, 1964; Tyler, 1969; Becking, 1975). These holes inter­ (calcite crystals oriented with b-axis at right angles to shell surface); K CM kinetic control of connect extensively with each other and with the intermammillary air calcium equilibrium (solid arrows); ODe, outer densely calcified layer (calcite crystals orien­ tated with coaxes at right angles to shell surface); P, pore; PP, pore plug; SD, surface defect. spaces (Fig. 7). Areas shaded solid black represent the bulges of reprecipitated eggshell minerals.

4. ORGANIC LAYER The high proportion of organic fibers in this layer (Figs. 7 and 9G, H) interweave extensively, and the blebs along them (probably a surface coat of glycosaminoglycan, Ferguson, 1982a) are not only the points of attach­ .,...... ­ i

calcified layer (0), the honeycomb layer (H) beneath it, and the mammillae. (B) Higher power SEM of the boxed edge seen in (A). Note the granular appearance of the densely packed vertical calcite crystals (the diameter of which is 0.5-1 f.Lm) and the absence of any organic matrix. (C) Erosion crater in the nonopaque zone of an IS-day egg. Note the stepped concen­ tric outline of dissolved crystal layers in the outer densely calcified zone, and the openings of numerous vesicular holes in the exposed porous honeycomb layer at the crater base. (D) Pore in the central opaque zone of a 40-day eggshell. Note the crater-like concentric stepping of the outer densely calcified layers at the pore orifice, remnants of the pore plug and microorgan­ isms (bacterial cocci, rods and filamentous fungae). Outer pore diameter approx. 550 f.Lm, inner pore diameter approx. 100 f.Lm. (E) Fractured edge of a 4-day eggshell. Note the shell membrane (E), mammillary layer (M), and honeycomb layer (H). (F) Innermost (egg content side) layer of the shell membrane depicting the numerous blebs caused by the projecting mammillary knobs of the mammillary layer. (G) View of the transversely fractured organic layer in the nonopaque zone of a 2-day egg. Note the smooth fibers of organic matrix inter­ spersed with numerous small calcite crystals. (H) View of the transversely fractured organic layer in the opaque central zone of a 56-day egg. Note the scarcity of small calcite crystals (see Fig. G) and the blebbing on the fibers of organic matrix. (I) High power view of the fibers in the shell membrane of a 3D-day egg. Note the interweaving of the fibers, the numerous blebs on the surfaces of the fibers, and the porous nature of the membrane.

Fig. 9. Alligator mississippiensis. Scanning electron micrographs (SEM). (A) Surface and frac­ tured edge of an IS-day eggshell. Note the smooth shell surface (5), the outer, densely 372 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCODILIANS THE EGGSHELL AND SHELL MEMBRANES 373

ment for the calcite crystals, but may be the templates for crystal growth. 7. EROSION CRATERS Beginning about the third week of incubation, the number of small calcite crystals in the organic layer beneath the opaque band decreases (compare Throughout incubation, the egg surface develops erosion craters, which Fig. 9G with 9H), and this process continues as the band expands. It appear as stepped concentric rings each corresponding to one crystal layer appears that these crystals, in preference to the larger units in the mammil­ of the outer densely calcified zone (Figs. 7, 8, and 9C). They are caused by lary layer, are mobilized for embryonic calcification. This process steadily crystal dissolution, possibly caused by acidic metabolites of nesting mi­ weakens the calcified shell, and horizontal separation usually begins a few croorganisms and/or by the hydration of expired carbon dioxide. These days before hatching, so that only the shell membrane and parts of the craters appear to be present in the eggshells of numerous species (Fergu­ mammillary and organic layers surround the embryo, with the rest of the son, unpublished data), including fossil crocodilians (Hirsch, 1984). Details shell either cleaved off or intact (but weakened). on their mode of formation are summarized in Fig. 8 (Ferguson, 1981c, 1982a). Their development confers possible advantages to the embryo in 5. MAMMILLARY LAYER terms of enhanced exchanges of respiratory gases and water vapor across the shell as well as the facilitation of hatching through eggshell weakening. The mammillary layer (Figs. 7, 9E, and 9F) comprises numerous cones The occurrence of extrinsic degradation is of importance to those in­ ("mammillary knobs") with their bases on the organic layer and their tips cubating eggs in embryological, farming, management, or conservation continuous with the shell membrane at the core of the knobs. The calcite projects. Normally eggs should be left in their natural dirty state and in­ crystals of the mammillary knobs are larger than those elsewhere and are cubated completely surrounded by damp nesting media in order to facili­ arranged into regular prism-like subunits, which are clearly evident in tate the production of bacterial acids. However, clean eggs can be suc­ radial sections of the shell (Fig. 9E). In tangential section, the mammillae cessfully incubated without nesting media by maintaining very high appear as irregular hexagons. Pores (Fig. 7) occur wherever the irregular humidities (circa 90-100%). The high humidity ensures that all of the ex­ faces of one or more mammillae fail to contact each other in the horizontal pired carbon dioxide produces carbonic acid, which degrades the shell. plane as has been described for the avian egg (Tullett, 1975). Pores are most Presumably, this hydration, possibly in combination with mineral acids numerous in the central opaque region of the eggshell. Mammillary out­ from the moist nest sand, forms the erosion craters in hole-nesting crocodil­ lines are much easier to distinguish in the central opaque region than in the ians (Ferguson, 1981c). The relative importance of humidity, or acids pro­ terminal nonopaque ends, where the shell membrane has flat areas of duced by microbial fermentation of nesting vegetation, in normal degrada­ attachment. Shell membrane fibers attach to the central organic core of the tion of the eggs of mound nesters has not been adequately determined mammillary knobs. (Ferguson, 1981c, 1982a). However, embryos from eggs incubated at humidities below 90% without nesting media, frequently die (probably 6. SHELL MEMBRANE from asphyxia or the toxic effects of waste products), whereas the remain­ The shell membrane consists of randomly oriented fibers (Fig. 7) that inter­ ing living hatchlings usually have to be manually helped out of the tough weave extensively in a criss-cross pattern, enclosing air spaces (Fig. 91). eggshell (Ferguson, 1981c, 1982a). Further investigations are also required The fibers are approximately 2 j..Lm in diameter and show the characteristic into which specific microorganisms are involved, whether they derive any surface blebbing of glycosaminoglycans (Fig. 91). The innermost surface of benefit (e.g., calcium) from the process, the changing pH of the eggshell the membrane has a thin, amorphous, nonfibrous, surface-limiting layer of surface in the nest, which acids are involved, and how the system evolved. unknown composition (Fig. 9F). Pores in the latter (Figs. 7 and 9F) are the In other systems, various fungi penetrate and degrade calcareous sub­ innermost end of a complex system of spaces in the more superficial layers, stances such as mollusk shells (Kohlmeyer, 1969, 1972). Although ther­ which connect the contents of the egg (e.g., the chorioallantoic membrane) mophilic fungi have been isolated from alligator nests (Tansey, 1973), their to the external environment and are probably important in respiration and role in eggshell degradation is unknown. water regulation. Contrary to some reports (Bigalke, 1931; Pooley, 1962, 1969a; Bellairs, 1969; Guibe, 1970), crocodilian eggs, unlike those of birds, 8. PORES do not have an air space (Reese, 1915a, 1931a; McIlhenny, 1935; Packard et Single, unbranched pores run vertically upwards from spaces between the al., 1977; Ferguson, 1982a). Abnormal air spaces may form in dehydrated mammillae to end at the shell surface (Figs. 7 and 90). They are more eggs where shrinkage of the shell membrane cleaves it from the shell numerous in the central opaque region of the eggshell, and in freshly laid through the organic layer (Ferguson, 1982a,b; Ferguson and Joanen, 1983). eggs, their openings are capped by an organic plug (Figs. 7 and 90). (Also see Section I.G.) During incubation, the openings of the pores are widened by extrinsic 374 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIANS THE EGGSHELL ANO SHELL MEMBRANES 375 acidic dissolution (Figs. 7, 8, and 90), and the plug becomes dislodged. TABLE V Presumably, this widening of the pore orifices facilitates respiratory and Calcium Contents of the Egg and Hatchlings of Various Species" metabolic exchanges; it also progressively weakens the eggshell, as longi­ tudinal cracks often pass through the pores and the erosion craters. Snake Crocodile Turtle (Vipera (Crocodylus (Dermocllelys (Gallus berus) nouaeguineae) 8. CONCLUDING COMMENTS coriacea) domesticus) The complex structure of the eggshell reflects its adaptation to croco­ Calcium in 25 124 34 23 egg contents, mg dilian nesting biology. At laying, the eggs are strong and not very porous; they are thus protected from physical damage at the time when they are Calcium in 12-16 280 138 120 hatchlings, mg deposited one upon another and whenever the mother treads nesting ma­ b terial on top of them, as well as from dehydration during early develop­ lncrease 0.8x 2.4x 4x 5.2x ment. The complex sequence of changes in the inorganic constituents "From Jenkins, 1975. weakens the shell and makes it more porous throughout incubation, so blndex of a mount of shell calcium used by embryo. that the prehatchling has merely to slit the organic materials with its carun­ cle. Antimicrobial properties of shell or albumen components, known for birds (Board and Fuller, 1974) and turtles (Movchan, 1964, 1966, 1967) have men and extraembryonic membranes also store calcium is unknown for not been reported in crocodilian eggs but may be related to the develop­ crocodilians, but such storage does occur in birds (Simkiss, 1967, 1980). ment of erosion craters and the filter bed arrangement of the porous shell The mechanisms of eggshell decalcification and calcium transport are and shell membrane. Data on such properties would be valuable, not only unknown; however, carbonic acid, formed from respiratory carbon diox­ for their intrinsic interest, but also in view of the practical problems associ­ ide, apparently attacks the shell of birds (Buckner et al., 1925; Romanoff ated with artificial egg incubation. and Romanoff, 1949; Simkiss, 1967, 1980; Rol'nick, 1970). This mechanism is in accord with the assumed respiratory function of the opaque zone in C. Chemical Composition crocodilian eggs. The levels of calcium in egg contents and hatchlings of Crocodylus novaeguineae given in Table V are uncertain due to poor preser­ Relatively little is known about the chemical composition of crocodilian vation and large numbers of infertile eggs (Jenkins, 1975). Because hatch­ eggs. The 6 g of calcium carbonate in the eggs is 99% calcite and less than ling contain 2.4 times more calcium than the egg contents, the 1% aragonite (Erben, 1970; Jenkins, 1975), a ratio far closer to that reported additional calcium must derive from the eggshell. Apparently, the yolk and for birds (Romanoff and Romanoff, 1949; Simkiss, 1967; Rol'nick, 1970) albumen of crocodilians contain more calcium than those of turtles (which than the reported predominance of aragonite in the eggshells of turtles obtain four times as much calcium from the shell as from the egg contents) (Young, 1950; Erben, 1970; Packard et al., 1977; Solomon and Baird, 1979). or birds (which obtain five times as much calcium from the shell), but much Eggshells of Crocodylus novaeguineae contain 82.6% calcium carbonate, less than those of snakes, which obtain no calcium from the eggshell (Jen­ 2.82% magnesium, 0.37% phosphorus, and 3.36% organic protein (Jen­ kins and Simkiss, 1968). This decreased dependence of crocodilian em­ kins, 1975). The total protein content of the eggshell is approximately twice bryos on eggshell calcium permits experimental embryological studies us­ that for the domestic fowl (Jenkins, 1975). The eggshell of Alligator missis­ ing shell-less and semi-sheIl-less culture of alligator embryos; these survive sippiensis also contains calcium, magnesium, and phosphorus, as well as to later developmental stages than their avian counterparts (Ferguson, traces of copper, silicon, sodium, aluminum, iron, zinc, and manganese 1981a, 1982b, 1984a; Fig. 38). (Ferguson, 1982a). Available reports on the biochemical constituents indicate general agree­ The thickness of the shell membranes is reflected in the fact that they ment on the occurrence of various mucopolysaccharides (Neumeister, comprise 21.39% of the total weight of dry shell and membrane (Jenkins, 1895; Simkiss and Tyler, 1959; Kriesten, 1975), but fibrous proteins are less 1975), whereas they only comprise 0.3% in the domestic fowl (Romanoff well-known. A survey of the amino-acid composition of the material of and Romanoff, 1949). The shell membrane acts as an intermediary calcium the shell and shell membranes indicated that the former contained se­ store between that of the eggshell and the calcium in the blood of the quences characteristic of collagen (in contrast to turtles), whereas the latter chorioallantoic vessels (Ferguson, 1982a), a factor which contributes to its contained sequences reminiscent of a keratin-like protein (Kramptiz et al., chalky white coloring beneath the opaque bands. Whether the yolk, albu- 1974), similar to that suspected for birds and turtles (Romanoff and 37B REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIANB THE EGG CONTENTB ANO EXTRAEMBRYONIC MEMBRANEB 377

Romanoff, 1949; Young, 1950; Simkiss and Tyler, 1959; Terepka, 1963a, b; 46.5% ammonium salts, 46.2% urea, and 7.3% uric acid (Clark et al., 1957). Masshoff and Stolpmann, 1961; Simons and Wiertz, 1963; Simkiss, 1967; R. The yolk sac functions as an excretory organ before the allantois develops; Bellairs and Boyde, 1969; Board and Fuller, 1974; Kriesten, 1975). thereafter the urea shifts to the latter. Protein is the preferred energy The kinetics of calcium metabolism of gravid females remains to be source in early development (Clark et al., 1957). studied. Whereas the calcium might be mobilized from specialized medul­ lary limb bones as in birds (Sturkie, 1965; Simkiss, 1967), from the skeleton in general as in turtles (Simkiss, 1967), from specialized stores as in some IV. THE EGG CONTENTS AND EXTRA·EMBRYONIC lizards (Simkiss, 1967), and from the diet (Romanoff and Romanoff, 1949), MEMBRANES it appears to be mobilized from the osteoderms (Ferguson, 1984a, ms) and from the diet. There is no medullary bone. For nearly a century, it has been known that crocodilian albumen is more gelatinous than that of birds (Clarke, 1888a, b, 1891; Voeltzkow, 1892, 1899, E. WBter and Gas Conductance and Embryonic 1901). In Alligator mississippiensis, the albumen is either clear and trans­ Metabolism parent or slightly opaque (Clarke, 1888a, b, 1891; Reese, 1915a, 1931a; McIlhenny, 1935; Ferguson, 1982a). That of Crocodylus niloticus, C. porosus We know far less about the water and gas conductance of crocodilian eggs and palpebrosus has been reported to be green (Voeltzkow, 1892, (Harrison et al., 1978; Packard et al., 1979; Lutz et al., 1980; Lutz and 1899; Reese, 1915a, 1931a; Bigalke, 1931; Deraniyagala, 1936, 1939; Wett­ Dunbar Cooper, 1982, 1983), than about those of turtles (Packard et al., stein, 1954), but these observations may have dealt with abnormal (in­ 1977, 1981, 1983; Ewert, 1979; Ackerman, 1980, 1981a, b; Seymour and fected) eggs. Initially, the albumen completely surrounds the centrally Ackerman, 1980) and birds (Romanoff, 1967; Rol'nick, 1970; Rahn et al., positioned yolk, but, as the volume of albumen decreases during devel­ 1979; Simkiss, 1980; Diamond, 1982). However, these parameters are im­ opment, it is increasingly isolated at the poles of the egg (Fig. 10). , portant for embryonic survivorship, metabolic rate, incubation period, sex Throughout incubation, as water is assimilated in the yolk sac and embry­ determination (see Section VII.O), and for speculations regarding the evo­ onic body (Agassiz, 1857), the viscosity of the albumen increases so that it lutionary constraints on clutch size and nesting ecology (Seymour, 1979; attains a rubbery consistency. Crocodilian eggs lack chalazae (Clarke, Seymour and Ackerman, 1980). The gas-water relations are also dealt with 1888a, b, 1891; Voeltzkow, 1899; Reese, 1915a; Deraniyagala, 1939; Guibe, under Section II. F. 1970m; Ferguson, 1982a). The detailed chemical constitution of crocodilian The vapor conductance of the eggs of Crocodylus acutus is 21.0 mg H 0 1 2 albumen is unknown, as is its precise fate during development, although day-l torr- H 20. This value is approximately two times that for an avian such information is available for birds (Romanoff and Romanoff, 1949; egg of equivalent weight (Lutz et al., 1980), even though the shell of the Romanoff, 1967; Rol'nick, 1970). Recently a protease inhibitor, closely re­ crocodilian egg is two times and the shell membrane ten times as thick. The sembling both the alpha-2 macroglobulin of mammalian serum and avian low resistance of crocodilian eggs probably represents an adaptation to an ovomacroglobulin, has been isolated from the albumen of C. rhombijer, C. environment of high humidity (Lutz et al., 1980; Seymour and Ackerman, siamensis, and Caiman crocodilus apaporiensis (Ikai et al., 1983). 1980; Lutz and Dunbar Cooper, 1982, 1983). At 70% relative humidity, the The spherical yolk, presumably held in position by the viscous albumen, eggsheIl contains 0.92% of its wet weight as water, and the membrane is pale amber/yellow in color and so large that it almost touches the shell contains 22.2%; at saturation, these values increase to 6.8 and 58.6%, re­ membrane at the midline (Clarke, 1888a, b, 1891; Reese, 1915a). In the spectively (Lutz et al., 1980). Permeability to oxygen drops tenfold as freshly laid egg, the yolk is very fluid; it becomes increasingly viscous as humidity rises from 70 to 100%. The oxygen diffusion coefficients (K0 ) development proceeds. It is enclosed within a yolk sac in which numerous 6 3 2 1 -2 1 vary7 between 1.2 x 10- cm STP sec- cm torr- at desiccation to 2.3 x blood vessels develop (Fig. 10). The composition of crocodilian yolk is 3 1 1 10- cm STP sec- cm -2 torr- at saturation (Lutz et al., 1980). Apparently unknown, but it can accumulate organochlorine residues (Hall et al., 1979). drying of the sheIl during the expansion of the opaque band facilitates gas The yolk is the major nutritional source for the embryo and hatchling, and exchange (Ferguson, 1982a). it probably consists of spherical droplets of lipoprotein as does avian yolk Metabolic rate is maximal during organogenesis at which time it is di­ (Romanoff and Romanoff, 1949; Romanoff, 1967; Rol'nick, 1970). Nothing rectly proportional to the incubation temperature (G. Grigg, personal com­ is known of the microscopical, ultrastructural, and biochemical organiza­ munication). During the first two months of development, alligator em­ tion of the crocodilian yolk and yolk sac, or how these change during bryos produce about 15.3 mg waste nitrogen (0.35 mg per g of embryo development (see A. d'A. Bellairs, 1969, and R. Bellairs, 1971, for data on formed). Throughout development, the proportions are approximately other reptiles and birds). 378 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIANS THE EBB CONTENTS AND EXTRAEMBRYONIC MEMBRANES 379 OlA them. Thus the yolk rotates, bringing the embryo up toward the top of the egg regardless of its original position. The vitelline fluid remains beneath the embryo, and its assimilation is responsible for dispersing the albumen to the poles of the egg and also for increasing its viscosity. As a result, the vitelline membrane adheres to the inner surfaces of the shell membrane and produces the opaque spot. The latter keeps expanding as more and

' .. '\. A more fluid crosses the vitelline membrane (Webb, personal communica­ tion). Assimilation of vitelline fluid is an active process, which explains IlA If. I J I", why infertile eggs never become opaquely banded. The opacity reflects '111.'.'....' ALB dehydration of shell and shell membranes, caused by the loss of the central ALB \ 7./////, layer of albumen. Chemical changes occur later and include a more perma­ nent opacity (Ferguson, 1982a). Thus, the length of the opaque band paral­ ( / I - J I SAC lels the regression of albumen early in incubation; later it also reflects the expansion of the chorioallantois and the movement of minerals from the shell to the embryo. Dehydrated or infected eggs (whether fertile or not) may become erratically banded or blotchy as albumen regresses and the yolk sticks to the shell membrane. If an alligator egg is placed with its top surface (as laid in the nest) BV FZ YS vav Y EEC BV uppermost and the widest (blunt) end of the egg pointing toward the Fig. 10. Alligator mississippiensis. Diagrammatic longitudinal section through a 40-day egg. investigator, then the head of the embryo lies distally and its snout points At this age the chorion and related blood vessels extend over the entire inner aspect of the to the right in 98% of cases. This predictable relationship permits eggs to be egg, and the eggshell is entirely opaque. The disposition of the chorionic blood vessels and windowed with accuracy. Because of the advanced stage of development opaque eggshell band illustrated are from an earlier age and for diagrammatic purposes only. at oviposition, it is unknown whether the early embryonic axis of croco­ (A) Amnion (solid black); AC, amniotic cavity; ALC, allantoic cavity; ALB, albumen; BV, chorio-allantoic blood vessels; C, chorion (dotted); CE, calcified eggshell (opaque band in dilians is formed in a constant fashion relative to egg position in the ovi­ cross hatched area); EEC, extraembryonic coelom (potential space between yolk sac mem­ duct as in birds (R. Bellairs, 1971). brane and the chorion, largely occupied by the allantois); ESM, shell membrane (opaque in After Stage 11, the embryonic gut projects through the body wall into cross hatched area); FZ, fusion zone (fusion of chorion, outer and inner layers of allantois and the umbilical stalk and contacts the yolk (Figs. 10, 20, 21, and 341) and the amnion, all of which have a firm attachment to the shell membrane); GL, gut loop (herniated paired vitelline blood vessels (Figs. 20, 21, and 341). Presumably, these out of embryo); ILA, inner layer of the allantois (fused to the amnion); OLA, outer layer of the allantois (fused to the chorion); SAC, seroamniotic caVity (potential space between amniun structures participate in the breakdown of the yolk and its transport to the and chorion, largely occupied by the allantois); VBV, vitelline blood vessels; Y, yulk; YS, yolk embryo. The yolk sac encloses the yolk; the paired vitelline vessels are sac membrane. continuous with the paired intestinal omphalomesenteric vessels at the yolk stalk (Figs. 10, 21, and 341). It is unknown whether there are yolk sac septae as in birds (Patten, 1925; Huettner, 1949), or if there are superficial At the time of laying, eggs have approximately equal volumes of yolk and deep layers of yolk as in other reptiles (R. Bellairs, 1971). Between the and albumen, with the yolk being significantly denser than the albumen. yolk sac and the chorion lies the extra-embryonic coelom, and between the Within the first day, these ratios change as water passes across the vitelline amnion and chorion lies the sero-amniotic cavity (Fig. 10). Both are oc­ membrane from the albumen. The vitelline fluid accounts for approxi­ cupied eventually by the enlarging allantois. The alligator has two bands of mately 3% of the total egg content weight at day 1, 40/0 at day 2, 7% at day firm fusion between the shell membrane, chorion, outer and inner layers of 3, 15% at day 6, and 27% at day 10. During this period, the volume and allantois, and the amnion (where present) (Fig. 10). These two bands are weight of albumen decreases from 50% of the total egg content weight at opposite each other and the lower fusion zone adheres less strongly to the day 1, to 45% at day 2, 37% at day 3, 31 % at day 6, and finally to 18% at day yolk sac (Fig. 10) than does the upper fusion zone to the amnion. The two 10. Concurrently, the volume and weight of yolk remains fairly constant at fusion zones hold the embryo in a fairly constant position and must be cut around 45 to 55%, and the total egg weight changes by only 0.5% (G. to remove an embryo or yolk from a fixed egg (Ferguson, 1982a). Webb, personal communication). The most aqueous vitelline fluid, being Several days before hatching, the embryonic intestines and the small appreciably less dense than the yolk granules, remains separated from yolk sac are withdrawn into the body cavity through the umbilicus (Fig. 380 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS EARLY EMBRYONIC DEVELOPMENT [BEFORE EGG LAYINGJ 381

Ectoderm fibers in the amniotic mesoderm of Alligator mississippiensis may rock the embryo and circulate the amniotic fluid (Schroder and Bautzmann, 1956).

V. EARLV EMBRVONIC DEVELOPMENT [BEFORE EGG LAVING] Entoderm Little is known about the early development, and we must rely upon descriptions nearly 100 years old; Clarke (1891), Voeltzkow (1899) and Reese (1908, 1915a). In these studies, early stages were obtained from the oviducts after killing gravid females because crocodilian eggs are laid at an advanced stage of development (Ferguson, 1984a; see Fig. 18). The earliest stage described for any crocodilian is after the appearance of the embryonic body folds, neural medullary groove, primitive streak, embryonic shield, area opaca, area pellucida, and the early gut (Figs. 12A and B) in Crocodylus niloticus (Voeltzkow, 1899). Currently, it is impossible to describe or dis­ cuss the events that precede this stage. Except for investigations of neural Fig. 11. Alligator mississippiensis. Diagrammatic cross section through the yolk (speckled), crest, palatal, and mandibular development (Ferguson, 1981a, 1982b; Fer­ early embryo and forming extraembryonic membranes to show their composition and the layers which with further development expand and fuse to produce the arrangements shown guson et al., 1982, 1983a, b; Ferguson and Honig, 1984), no experimental or in Fig. 10. (After Huettner, 1949, in the chick.) AL, Allantois; AM, amnion; C, chorion. in vitro studies have been performed on crocodilians; this description relies entirely on old morphological data. The present report offers a concise account of the major events; for 21). At the time of hatching, the abdomen is considerably distended by detailed "catalogue type" descriptions of sectioned materials, the reader is absorbed yolk and yolk sac (Fig. 21), and the umbilicus is often open. The referred to earlier references. The standardized terminology used here absorbed yolk is used as a food supply over the next couple of weeks does not imply homology with synonymous terms for birds and mammals. (traces remain 6 months after hatching, Cott, 1961), and the umbilicus then Shortly after the appearance of the neural folds (Figs. 13A and B), after closes. The mechanisms of yolk digestion and utilization are unknown. the head fold has begun to sink into the underlying yolk, an anterior The arrangement of the extra-embryonic membranes has been described blastodermic fold forms the amniotic head fold (Clarke, 1891; Voeltzkow, in Alligator mississippiensis (Ferguson, 1982a), and other data are available 1899, 1901; Reese, 1915a). Initially the head fold is crescent-shaped, its free on their development in this and other species (Rathke, 1866; Clarke, 1891; edges pointing toward the tail region of the embryo (Figs. 13A, B, 14A, B); Voeltzkow, 1899, 1901; Reese, 1908, 1912, 1915a; Fisk and Tribe, 1949; as development proceeds, the head fold extends craniocaudally. The am­ Ferguson, unpublished). The relevant stages are difficult to obtain as eggs niotic primordium, derived from somatopleure around the trunk (Fig. 11), are laid at a fairly advanced stage of development (Fig. 18). The develop­ arises in continuity with that of the head; whether it does so as a posterior ment of crocodilian extra-embryonic membranes (Fig. 11) is probably simi­ extension of the head fold or as paired lateral amniotic folds (as in the lar to that of the chick (Patten, 1925; Huettner, 1949). chick, Patten, 1925; Huettner, 1949) is unknown. This combined head­ Alligators, like other amniotes, have three extra-embryonic membranes: trunk amnion reaches the level of the blastopore about the time of egg the chorion, amnion and allantois, and a yolk sac (Fig. 10). The chorion laying (Stage I, Fig. 18). lines the inner aspect of the shell membrane, except in the region of the Voeltzkow (1899, 1901) recognizes the development of a tail fold (tube) albumen. As development proceeds, the volume of albumen decreases and of amnion (his Figs. 28A and B), whereas Clarke (1891, 1901), Reese (1908, the chorion increases in size. The general organization and relative rela­ 1912, 1915a), and Fisk and Tribe (1949) claim that such never develops. tions of the amnion, chorion and allantois, and the vitelline and chorioal­ Voeltzkow (1899, 1901) also describes a posterior "amniotic duct," but Fisk lantoic circulations (Figs. 10 and 11) are essentially similar to the avian and Tribe (1949) state that this does not resemble the posterior amniotic condition. No detailed studies are available on the ultrastructure of tube of turtles. Attachment of the amnion to the caudal region between crocodilian extra-embryonic membranes or on the mechanisms of yolk Stages 1 and 2 (Fig. 18) seems to be effected by extension of the trunk folds, mobilization, embryonic excretion, and respiration. The smooth muscle as the membrane becomes well developed laterally and caudally between ~'i ,.,....c~{;.~~:.~,~ \ I ..'t "·.r,"~",,,'1·~J\' /ifjt~c~r:§J~"~i .~' ~ ~jO~:f,~{~~. ~':f'- ~':'m- .'~-, tC'~1;,< " ~ ~.lr~,~" .'

2 ..1 ". , "'~I ',. ~.. "...... ,IllJlIWl1l!llWllIlII\Q'- Y ;.,. 3 ~. -­ 10 .-~. -~."'." MG E.G . '".: iitf:~ ~ • , VI!,t, ,_~ •!e •• .t'. ...,~,. .."~ .,- -.

' .. <, # '.,. .'.!' ,. 4 ,',. ,,, 11 .'" ~ ~. ' .1... . "'::p.:;~,,".,... , = ;---,',' f1I'ff • !'-,',. 5 J...:: ~ ..•• ~VXQP!Ui~ ~IlJlplltJtP-.~ :~., ~ j'" '~l" 'J _'. ·"~~·.~1\:~. IUWI'JI]!- .... _ .. .& _.. •• h ··l~ ~lij' :, ". .. .."'. 12 '1/...... ":."1:~.' • " J ...... ~. ".~.. ".;:~.:•.:~. ·:~:''''~1t.·· 6 "'''i!~'''~ ,*" , .,'.\~...... ~'~~~~" BP ... '.' '~;:;."'i/ .. ·-1~ ':~r~.'''.'' .,,,,~'\' ·_""-',·.1 ;:~_ i-~7.-. ...• 7 ... . ~1I.:'i~~~"pPJIiliU BP "~"~v. ~ .~. h ""~ . ::;.~_~. • '. •••• • til· • ••• ••• • < • ~ ~":' 8 :. " . . .. ,~~. ... -.. .'.. '. y. . ..~ ." -"..... '~ .'. ~~. -.. IllS niloticlls somewhat more advanced than (A) and (B). The lines mark the levels of the '/' . sections depicted in (E). (After Voeltzkow 1899, 1901.) (E) Series of sections of the embryo (, ". .. ~ f / .".. depicted in (C) and (D) at the indicated levels. (After Voeltzkow, 1901.) B, Body folds; BP, y~" E E blastopore; E, extra-embryonic endoderm; Ee, ectoderm; EN, endoderm; ES, embryonic shield; FG, primitive foregut; MG, neural groove; PH, primitive hindgut; PS, primitive streak; 'ig. 12. CrocodylllS niloticllS. (A, B) Dorsal and ventral views respectively of an early embryo. After Voeltzkow, 1899.) (C, D) Dorsal and ventral views respectively of an embryo of Crocody­ Y, yolk. .y

B M MG 6 I I ii" ,~~''-'." "'''''';''~:-'". ",'-",.... ~", ""'l;v{c :.,,) :' '. .:. '--'';'',~ ,,' ;.~~~ y'-, i EN ME .; . .:: I\ ,I "","T . *",'" ,~-, A A. :'·-.1.... v ttl,' . _ .""Ii: "••. 1ft; B ... E ••o.fi, ~',~:.: ·'fe:!i~·~{~~,:·~ "M .-, ., { ,f.~~~::;;;',~'1;'" ,.?; , 2 ~l"-'iffi"~,"*~~~J,it-:.~':'.,,~:... ,~.&~2:::~,,#o~:-~ ; /."<'!'. r~' r'{t;/},. .. ·~'" .~ .?-1":,.. EN ,,~.. 7 .:., :., VB . ".

:- <{s.. :o:~·:·- ,',<:';~. "i "/'~' , .', :ti~ B ..5t'~~ ,~~:;;:~!tl:;Ff~;:~~~,-"" "• 3 .' • -!- ':,:...... " ...· ..1 f l?~rrJ(~i !{}!~1~i!1?:~:1'·J::f.::···~ 'dJ ~Jt ;.,~' a II L. __r;.1Ij ' \,.,. •• •• :••::::. :.f :'< ..,... iP, ", ""'?' .t .~" 4,' :,.r"~, NT .~.- :'..:"'. ','_....,~,~-~ •.. *",. ~:lr.: 41 .V"'" • ',,' "f" '!:' • -,;it:-..,. " ...,.... EN ~~ f" • Be .... c PS 4 '.' "._." J/ ~!t>.. .•~'l'... ~:.,' ~:;:~t~'fY};::'; :~;;~~ '.~~ ~ e'!. .." • EC t, Be ~••_.'-';;~~;l'.:!"-. "J~l!'.~h""4'<1~~~l"";'~~~f4:::mr:~~~::W~~Y:'"''tl'cf~\"l!'~ 5 r ~(~~~..m~. ~...... 'l., {'~f~i}y EN y C·· .~ D -y Fig. 14. Crocodylus niloticus. (After Voeltzkow, 1899, 1901.) (A, B) Dorsal and ventral views of an embryo, Note the clear area in the tail region (T) in which the dorsal wall is visible. The lines indicate the levels of the sections shown in Fig. C. Equivalent to Reese (1908, 1915a) Fig. 13. Crocodylus niloticus. (A, B) Dorsal views of embryo. (A) (After Voeltzkow, 1899.) stage III for Alligator mississippiensis. (C) Series of sections of the embryo. (After Voeltzkow Equivalent to Reese (1908, 1915a) stage II for Alligator mississippiensis. (B) A later stage. (After 1899,1901.) A, Amniotic head fold; B, blastopore; BC, blastoporal canal; DB, dorsal opening of Voeltzkow, 1899.) Equivalent to Reese (1908, 1915a) stage II for Alligator mississippiensis. (C, D) Alligator mississippiensis, (C, D) Sagittal and parasagittal sections of an embryo about the same blastopore canal into the neural groove (MG); E, extraembryonic endoderm; EC, ectoderm; EN, endoderm; ME, mesoderm; M, neural folds; MG, neural groove; PG, primitive groove; stage in development as Fig. 13b. (After Reese 1908, 1915,) A, Amniotic head fold; B, blasto­ PS, primitive streak; S, secondary head fold; T, tail region; VB, ventral opening of blastopore; pore; EC, ectoderm; EN, endoderm; FG, primitive foregut; M, neural folds; MES, mesoderm; MG, neural groove; NT, notochord; PS, primitive streak; S, secondary head fold; Y, yolk. Y, yolk. 386 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIANS

Stages 1 and 10 (Figs. 18-20). Development of the dorsal amniotic fold facilitates craniocaudal separation of the embryo from the blastoderm (Figs. 15B, 16B, 17A-E, and 18), but the process is not completed caudally until Stage 3 (Fig. 18). With further development of the blastoderm consist­ ing of ectoderm and endoderm (Fig. 12E), the neural groove and blasto­ v pore become clearly demarcated (Figs. 12C-E). The endoderm may form "tails" that extend outward and downward into the underlying yolk (Voeltzkow, 1899, 1901; Figs. 12C-E), which probably represent presump­ 50 tive extra-embryonic endoderm. The blastopore is relatively large and pen­ etrates the entire blastoderm (Figs. 12C-E), and the primitive streak lies MG posterior to the blastopore (Figs. 12C-E). r;"F,) With the rapid delineation of the body folds (Figs. 13A and B), the 1',1 l~ boundary between embryonic and extra-embryonic tissues becomes dis­ ~ '.;yJJ..... cernible. Figures 13A-D show the well-defined head fold bounded ante­ ~'" ,.3 riorly by the proamnion. The beginning of the foregut is evident. The ;[~rY loJ notochord extends along the midline from the head fold to the anterior . vA) /'~' .\~.' edge of the blastopore (Fig. 13C). Earlier explanations (Reese, 1908, 1912, \" .. ,_.... F::.)l... jl; P5 ti:..0~\ ~r JifJ27 1915a, 1926) of the origin of the notochord are dubious in the light of IJ:$ ;,.~.• • )!'Y J~J ,r'h,J 1 .:..1 current data from other vertebrates. The primitive streak and primitive A ,)j.:'J.r' ~ " ~ B groove lie posterior to the blastopore (Figs. 13A-D); the primitive groove [Fig. 14C (6)-(7)] is continuous with its posterior end. The ectoderm bor­ MG EC dering this groove is thickened, and its two elevations constitute the primi­ /~i$~,r"l (I) .,;"J(JC'o~Q·\ /~ . Qot::"J~ \ Oo.!, r~ tive streak (Figs. 13A-C, 14A, B, C (6)-(7)). The primitive streak extends o,,~~ig~QO" 6 oQo\f}oat:~ -v.- . about one-third the distance between the head fold and the blastopore EN..;l.{.'1 )o/')~e~~'i.£-,~~t~'~:.-~~, ~1;.~._-tl'l- ~ °ot-qlo'-: .. ~o" ,,~i ~~o: l:'\~:~~ ((D~~e"oc" can only be demarcated arbitrarily from the dorsal opening of the blasto­ :,t'*l'JO~o~~!'i:S'<"'; ~:,~:~o::.,~~i~Cd~~ Q.P.!:::X:£-~ (II) pore (Reese, 1908, 1912, 1915a). This type of gastrulation (blastoporal = .c'O"5r,~t?~~'i..;~.?o"'" canal, etc.) is found in all reptiles, although specific details may differ. EN M,·.. _EP~,~(,~. Neural folds have a double origin in Alligator mississippiensis (Clarke, r;·l;,;,) N __ o"t> ~~_~D c 1891; Reese, 1908, 1912, 1915a) and Crocodlflus niloticus (Voeltzkow, 1899, 1901). The posterior folds arise as ectode~mal ridges extending forward (III) from the blastopore and bounding the neural groove (Figs. 13A,B, and 14A, B). However, a secondary fold occurs anteriorly in the head region (Figs. 13A,B, and 14A,B) and grows posteriorly along the median dorsal (IV) Fig. 15. Crocodylus Ililoticus. (A, B) Dorsal and ventral views of an embryo. Note the point of first fusion (F) of the neural folds and ventral flexion of the cranial end of the embryo (V). (After Voeltzkow, 1899). (C) Series of sections illustrating closure of the neural folds: (i) is from an embryo of a stage shown in Fig. 120, (ii) from Fig. 13A, (iii) from Fig. 14, (iv) from Fig. MY 15 and (v) from Fig. 17. (After Voeltzkow, 1901.) B, Body cavity; EC, ectoderm; EN, en­ doderm; EP, epidermal layer; F, point of first fusion of neural folds; M, mesoderm; MG, (V) neural groove; MY, myocoel; N, nervous layer; NT, notochord; PM, parietal layer of mesoderm; PS, primitive streak; 50, somites; V, ventral flexion of the cranial end of the embryo; VM, visceral layer of mesoderm; Y, yolk. 3BB REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS

v .. ;.;~~~:rm-M ... ·t'~' '.'. (~':?1 '~.\';;:'.(€:,.. y ".{':'.':',.::·m:l:Z;.. ;~,~~:'~ ";' tOY '~'~ 'I~~r~ •..•...& \1(,.'," ·;:~W ~:I

A

Fig. 16. Crocodylus niloticus. Dorsal view of embryos. (After Voeltzkow, 1899). Equivalent to Reese (1908, 1915a) stage IV for Alligator mississippiensis. (A) Ventral flexion (V) of the anterior end of the neural folds (M). (B) Closure of the neural folds (M). A, the amniotic head fold; B, developing brain; PS, primitive streak; Y, yolk. line to form a V-shaped process with the apex pointing toward the blasto­ pore. The apex of the V-shaped secondary head fold later disappears and each of the separate arms becomes continuous with the corresponding posterior neural fold. Thus, the secondary head fold forms the anterior part of the neural folds (Figs. 13A,B and 14A,B). Several stages in neurula­ tion are shown in Figs. 15A-C, 16A,B, and 17A, B). Closure of the folds in

Fig. 17. Crocodylus niloticus. Embryos. (After Voeltzkow, 1899.) (A. B) Dorsal and ventral views illustrating the dorsal fold of amnion and the lateral body folds delimiting the embryo from the overlying blastoderm in a craniocaudal sequence. The primitive head region is flexed ventrally. EqUivalent to Reese (1908, 1915a) stages V and Vll for A. mississippiellsis. (C, D) Dorsal and ventral views illustrating the progressive delineation of the embryo from the blastoderm by the caudal fusion of the edges of the amniotic and body folds. The head is further flexed ventrally. (E, F) Dorsal and ventral views just prior to egg laying. Note that the edge of the dorsal amniotic fold lies at the caudal third of the embryo. The lateral body walls are delimiting the primitive gut tube ventrally and the has developed as a simple tube. The head is flexed ventrally and hangs vertically into the underlying yolk. It is shown rotated to one side for diagrammatic purposes only. A, Dorsal amniotic head fold; H, lateral body fold; BD, blastoderm; E, caudal edge of dorsal amniotic fold; G, primitive gut; H, head region; HT, primitive heart; N, neural tube and notochord; NP, patent posterior neuropore; 0, otic placode; PS, primitive streak; S, somite; Y, yolk.

!:IDCIl 390 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCDDILIANS

C. niloticus occurs first in the middle region of the embryo (Fig. 15A), nearer the posterior end of the neural groove (Voeltzkow, 1899, 1901), but in A. mississippiensis it occurs nearer the cranial end of the neural folds (Reese, 1908, 1915a). The blastoporal or neurenteric canal is visible in all early embryos until after the closure of the neural canal. As in earlier development (Fig. 12E), the blastoporal canal runs through the embryo (Fig. 14C), backward from its cranially located ventral opening [Fig. 14C (3) and (4)], to open dorsally into the neural groove at its caudal limit [Fig. 14C (5)]. Some mammals show this blastoporal canal as the chordal canal (R. Bellairs, 1971). Throughout this period, somitogenesis occurs along the median axis (Figs. 15A and 17A-E), the first pair developing halfway between the anterior and posterior ends. The peripheral somitic cells are compactly arranged, but small myocoels lie in the center of the somites [Figs. 15C(i)-(v)]. The mesodermal layers cleave, forming the somatic and splanchnic compo­ nents [Figs. 15C(i)-(v)] as the foregut enlarges. Early in development the head fold of the embryo projects ventrally into 'the underlying yolk (Figs. l3C and D). This process is accentuated by a ventral bending of the anterior neural folds (Figs. 15B, 16A,B, and 17A-E) and still later by cranial flexure. Thus the entire cranial end of the embryo cannot be seen from above, because it is pushed down into the yolk (Figs. 15B, 16A, and 17A,B). Torsion occurs between Stages 3 and 6 (Section VI), beginning anteriorly and proceeding posteriorly (Figs. 18-20). Despite ear­ lier conflicting reports (Clarke, 1891; Reese, 1908, 1912, 1915a; Derani­ yagala, 1939), the embryos of Alligator mississippiensis, Crocodylus porosus, C. johnsoni, and C. niloticus usually come to lie on their left side (Figs. 18-20). In Crocodilians, torsion occurs at a more advanced developmental stage (Stages 3-6) than in chicks (H. H. Stages 12-15, i.e., 16-20 somites).

VI. STAGES OF EMBRVONIC DEVELOPMENT [AFTER EGG LAVING]

The practical importance of establishing Normal Tables of development for vertebrate embryos, and ectotherms in particular, is discussed elsewhere (Billett, Cans, and Maderson, Chapter 1, this volume). No staging scheme exists for any crocodilian. Clarke (1891), Voeltzkow (1899), and Reese

Fig. 18. Alligator mississippiellsis. Stages 1 to 4 of embryonic development. Numbers indicate the stages. See text for their description. 0, Dorsal view; L, lateral view; V, ventral view. Dorsal (20) and ventral (2V) views of a stage 2 embryo illustrate its attachment and vertical relationship (i.e., no body torsion) to the blastoderm. Body torsion commences at stage 4 (4V) when the cranial end has rotated; it is complete by stage 6 where a ventral view (6V) illustrates its relationship to the overlying chorion. Scale bars = 1 mm. 392 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIANS

Fig. 19. Alligator mississippiensis. Stages 5 to 14 (numbered) of embryonic development. See text for description of stages. Scale bars = 1 mm.

(1908, 1915a) described some embryos in both surface view and section, but the series were incomplete, and chronology and incubation condi­ tions were poorly documented. Random embryos of unknown age and developmental history have been described by some field biologists (De­ raniyagala, 1934, 1936, 1939; Pooley, 1962; Magnusson and Taylor, 1980); such have but limited embryological value. Preliminary information for estimating the ages of Crocodylus porosus and C. johnsoni embryos in the wild, including some data on the effects of varying incubation tempera­ tures is available (Webb et al., 1983a,e). Embryological stages constructed for Alligator mississippiensis equated with the number of days after egg laying, under known incubation conditions (Ferguson, 1982b), proved ! ,. cumbersome to use because staging older embryos concerned mainly the Fig. 20. Alligator mississippiel1sis. Stages 15 to 21 (numbered) of embryonic development. See growth of external structures. Moreover, study of C. johnsoni and C. porosus text for description of stages. Scale bars = 2 mm. has shown that their development is similar to that of A. mississippiensis. Timing is the principal variable, particularly in the later stages. Thus, it is possible to construct a staging scheme applicable to three species, and 384 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS BTAGEB OF EMBRYONIC DEVELOPMENT tAFTER EGG LAYINGJ 385

presumably to crocodilians in general. This is of greater value than species­ velop by enlarging existing somites before they form new ones, whereas specific stages. others may form new somites before enlargement (such variation is often The approach adopted is twofold (Ferguson and Webb, in preparation). temperature dependent). The development of different structures does First, a detailed morphological staging scheme related to specified incuba­ vary independently (e.g., in a particular specimen branchial arch develop­ tion conditions and chronological age is presented. Species-specific char­ ment may lag behind that of the limbs) and appears to be marked at the acters are noted, but the criteria for designating particular stages are extremes of incubation temperatures. The present staging scheme is based "crocodilian" and species-independent. Second, 13 different standard on examinations of approximately 1500 embryos of Alligator mississippiensis measurements, for example, total length, eye length and snout length, and 300 each of Crocodylus porosus and C. johnsoni. All embryos were fixed which were made on embryos of all stages of Alligator mississippiensis, in 10% formal saline and photographed with oblique incident illumination Crocodylus johnsoni, and C. porosus, have been supplemented by data on using a Wild M8 stereophotomicroscope. egg dimensions, weight, and the percentage of opaque eggshell banding. Some incubation ages in days are given for embryos within each stage Such morphometric data permit several analyses: determination of the (see Table VI). For Alligator mississippiensis, these ages, given for all stages, relationship between egg size and embryo size within and among species are based on a number of standard series collected within three hours of (in general, larger embryos come from larger eggs and vice versa); determi­ egg laying and artificially incubated at 30°C and approximately 90-100% nation of the relationship between the percentage of opaque banding and humidity. For Crocodylus johnsoni and C. porosus, the standard series were embryonic stage; determination of diagnostic ratios (e.g., eye length over incubated under similar conditions (Webb et aI., 1983a,e; unpublished). total length ratios are used to correct for the effect of egg size) for each Ages are not ascribed on the basis of the drawings in Webb et al. (1983a,e), stage in each species; interspecific comparison of similar stages to deter­ which were prepared as a field guide, but on reexamination of the original mine species-specific features (e.g., C. porosus embryos are larger and have embryos. The timing of development relative to stages is virtually identical longer tails, C. johnsoni have larger limbs and narrower snouts, A. mississip­ in all three species up to Stage 20 (i.e., during organogenesis); only later, piensis have more abdominal yolk and retarded external genitalia); formula­ when growth is occurring, do species-specific differences appear. Some of tion of regression equations of chronological age against particular mor­ these may be due to differing incubation conditions (e.g., whether com­ phometric ratios (at different incubation temperatures and humidities) to pensation is made for metabolic heat); perhaps the ages given for A. missis­ predict "morphological age" for any embryo of a particular species (d. rat sippiensis up to Stage 24 are similar in all species. embryos, Ferguson, 1978a). This morphological aging is valuable when the Table VI also includes "equivalence values" for each described stage to time interval between stages is long, due either to slow changes in the the figures in Voeltzkow's (1899) report on Crocodylus niloticus and the external form (Stages 20-24) or because the embryo is fully formed and stages in Reese's (1915a) monograph on Alligator mississippiensis, which merely enlarging in size and absorbing yolk (Stages 25-28). The two sys­ latter included Clarke's (1891) illustrations. Some of the data in these older tems are complementary, so that an embryo may be classified as "Stage 24, works are inaccurate, even self-contradictory. In the stage descriptions morphological age 58.5 days." In general, staging by external morpholog­ presented here, judgment of such major errors invoked the premise that ical criteria is most accurate up to Stage 20, because development is fast, new data for A. mississippiensis, C. porosus, and C. johnsoni are more com­ and the time intervals between stages are small. After Stage 20, the mor­ plete and more detailed. Only limited data are available relating chronolog­ phological age based on morphometric ratios is most accurate for aging, ical age and stage at temperatures other than 30°C. In A. lIlississippiensis, because the time intervals between stages are long, and the embryos are increasing the incubation temperature (within viable limits) accelerates de­ large and easily measured. velopment up to Stage 20 (organogenesis) to a much greater extent than in In the follOWing summary of the morphological staging system (Normal later stages. This is similar to observations on birds (Romanoff, 1967), Table), no morphometric data are given. For each stage the most important snakes (Zehr, 1962), and turtles (Yntema, 1968; Ewert, 1979). However, it is diagnostic features are listed first, and the essential features are illustrated difficult to make precise statements regarding the effects of incubation in Figs. 18-22. Details of important regions of various stages are illustrated temperature on developmental rates because of tissue specificity, e.g., in Figs. 23-26. Some stages may be subdivided by reference to certain gonadal development is retarded at 34°C compared to stage by stage events criteria, for instance, Stages 1-6 may be supplemented with a somite count at 30°C. (e.g., Stage 1120 s embryo), Stages 17-19 with the percentage of palatal There are also difficulties in adjusting regression equations for variations closure, and Stages 20-28 by morphometric ratios (e.g., yolk volume, yolk of temperature during incubation in order to predict embryonic age (or scar width, total length) (Ferguson and Webb, in preparation). Initially, the hatching dates) from morphometric ratios (Webb et al., 1983a,e). Gener­ stages have not been separated by criteria such as somite counts, because ally, development of alligator embryos up to Stage 20 proceeds approxi­ these vary in relation to other features, for example, some embryos de­ mately 1.2 to 1.8 times as fast at 34° as at 30°C, and approximately 1.6 to 2.0 TABLE VI STA13ES OF EMBRYONIC OEVELOPMENT [AFTER EGG LAYING] 397 Concordance for Staging/Aging of Crocodilian Embryos

Stage times as fast at 30° as at 28°C. Thereafter, rates of development at different AM" (days) Cr's (days) CP'" (days) V , dl W,h temperatures show less variation; eggs incubated at 34°e hatch after 55-60 o I (after egg 0-1 0-1 0 laying) VI(37a, b) VlII, IX days incubation, at 30 e after 62-67 days and at 28°e after 70-75 days. The 2 2 2 situation may differ in species with longer incubation periods, possibly as a 3 3 VI(38a, b, c) X, XI 3 3 It VI(40), XII result of increased time spent in the egg between Stages 24 and 28. is likely VlII(5Ia) 4 4 4 that these later stages of growth and yolk absorption are accelerated by 4 VII(43), VlII(5Ia) increased temperatures. Field nests of Crocodylus johnsoni increase gradu­ 5 5 5 6 6 XIII ally in temperature throughout incubation (Webb and Smith, 1984), and it 6 6 7 7 would be interesting to know whether this facilitates accelerated develop­ 7 VII(44), VIII(52, 53), ment, yolk absorption, and hatching. Alligator mississippiensis (and some f 8 8 IX(66 , 74) other species) may hatch in the minimum time possible (perhaps as a result 8 8 VII(45), VlII(54), of enhanced development in the later stages, Webb et al., 1983e) as an 9 IX(67', 75, 76) 9 9 9 IX(68, 77) adaptation to their climatic environment. However, species occupying 10 10-11 10 10 VII(46), more tropical environments may incur a selective advantage by staying VlII(551' 11 12 IX(69) longer in the egg. Accurate prediction of hatching date is difficult for any 12-13 12 12 13-14 species because hatching is initiated by factors other than completion of 14 13 VII(47), XIV VlII(55), development; viable embryos incubated under constant conditions hatch IX(69)f 13 IS over a range of 5 to 10 days and show varying degrees of yolk absorption. IS IS IX(70, 79), XV, XVI The problems of identifying the early embryo have beset numerous 14 16-17 XV(134) 16 17 investigators (Clarke, 1888a, 1891; Voeltzkow, 1892, 1899; Reese, 1907, IS 18-20 XVII 18-20 19 VII(48), VIII(56), XVIII 1915a; Wettstein, 1954; Ferguson, 1982b). Statements in the literature, con­ IX(71, 80), X(84A), 16 XV(135) tending that development begins at oviposition, reflect the difficulties of 21 21-22 17 21 IX(72, 81) observing Stage 1 embryos under field conditions (McIlhenny, 1934, 1935; 22-23 23 VIII(57), IX(82), XIX Pooley, 1962). For Alligator mississippiensis, Crocodylus johnsoni, and C. 18 X(85A) 24-26 26 25 VII(49), VIII (58), porosus, numerous embryos that were recovered within a few hours of egg 19 X(86A), XI(105) 27-28 29 laying have always been Stage 1, although with variation in the number of 20 29-30 X(87A,88A) 30-34 29 somite pairs (9-20; mean 12 ± 2) and in the degree of delineation of the VII (50), VlII(59), XX XI(95, 106), XV(136) 21 31-35 35-39 dorsal wall. Because development of the earliest laid embryo to the latest VIII(60), XI(107A, B) 22 36-40 40-45 35 occurs in 24 hours, Stage 1 embryos have been ascribed an age of 0 to 1 VIII(61, 62) 23 41-45 46-54 44 XXI XI(96) days. 24 46-50 55-64 55 XI(97) 25 51-60 65-74 58-70 VIII(63), XI(98A-B) Stage 1 [Fig. 1 Bl 26 ABSENT 75-80 71-75 XXII 27 60-63 81-83 75-80 XI(99, 100) 28 64-70 84-90 81-85 VlII(64), XI(108) XXlII Blastoderm. Blastoderm and embryo lie on top of the yolk and are not

"AM, Alligator mississippiensis. dV, Voeltzkow (1899) stages for C. niloticus. VII(4a) = Tafel VII, Fig. 4a. attached to the overlying shell membrane. Blood vessels are not evident. bq, Crocodylus johnsani. 'R, Reese (1915a) stages for A. mississippiensis. Somites. 9-20 pairs (16-18 are very common at the time of egg laying) 'CP, Crocodylus porasus. fmajor error in the figure. sThe numbers of embryos of Cracodylus johnsoni and C. parasus with definite ages are limited, so that the lie posterior to the otic placode. days given do not inclUde the range per stage, as provided for Alligator mississippiensis. The values repre­ sent the known ages of standard embryos available for the stage. Delineation of the Embryo from the Blastoderm. Approximately the caudal hThe drawings of Reese (1908, 1915a) contain numerous errors, whereas those of VoeItzkow (1899) tend to one-third of the dorsal body wall is not delineated from the blastoderm, for be accurate. Hence, representative figures selected from the latter are used. Some contain major errors, e.g., his Figures 39b and 43; these have either been omitted from this table or, if no other illustration was example, in a ISS embryo the body is delimited to the level of the 7th available are listed with the notation! which indicates a major error. The present cross-reference between somite, in a 16S embryo to the 8th somite, in an 18S embryo to the 12th the drawings of whole embryos and those of regions may differ from the original, but is consistent with the data of the three species for which recent observations are cited above. Voeltzkow's (1899) ages appear somite, and in a 20S embryo to the 16th somite. inaccurate and are inconsistent within his study (e.g., Figs. 49 and 58 show the same embryo, but are stated Branchial Arches. The first branchial arch is just visible. to represent ages of two months and four weeks, respectively). They have been omitted, although the trend suggested is similar to that for the other two species of Cracodylus. Heart. A simple S-shaped tube in the midline. Sensory Placodes, Pits, and Brain. Both the optic and otic placodes are 3BB 39B REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIAN9 BTAGEB OF EMBRYONIC OEVELOPMENT [AFTER EGG LAYING) 399

evident and may be invaginating. The optic and otic vesicles are also obvi­ shape, which extends below the lens vesicle onto the roof of the primitive ous, but the brain is not yet regionalized. The optic pIacodes and vesicles oronasal cavity. are more obvious than the otic. Blastopore and Primitive Streak. Not visible. Flexures and Rotation. The embryo lies at right angles to the yolk sur­ Extra-embryonic Membranes. The amnion is attached ventrally to the lat­ face, that is, body torsion has not yet commenced. eral body walls, cranially to the borders of the pericardium about the level Blastopore and Primitive Streak. Obvious. of the 7th to 8th somite and caudally to the cranial margin of the fold of tail Gut. The gut is incomplete caudally and open ventrally along its entire length. bud. Flexures and Rotation. Head at approximately right angles to the body. Somitomeres. Three pairs of cranial somitomeres are visible anterior to No body torsion. the otic vesicle. Notochord. The notochord is evident. Stage 4 [Fig. 1 S)

Stage 2 [Fig. 1 S) Somiles. 31-35 pairs. The first is beginning to disappear. Tail. Distinct, straight, and contains 3-5 somites in its base; the tip is Blastoderm. The dorsal surface of the blastoderm is attached to the unsegmented. overlying shell membrane, and the embryo remains so tethered through­ Flexures and Rotation. Body torsion has commenced. The cranial half of out subsequent development. Blood vessels are now visible; one pair the embryo is rotated so that its right surface is in contact with the shell emerges from the embryo at the caudal level of the heart, whereas another membrane and its left is parallel to the underlying yolk. The caudal half of (larger) pair runs down the lateral wall of the embryo to emerge at approxi­ the embryo is not rotated and lies at right angles to the shell and yolk. mately the level of the 20th somite. Heart. Displaced to the left side of the embryo and large. Somites. 21-25 pairs (decreasing markedly in size caudally). Allantois. Small elevation is just visible immediately caudal to the cra­ Delineation of the Embryo. Dorsally the embryo is almost completely niallimit of the ventral tail fold, at approximately somite 27. delineated except for a very small circular area at the extreme caudal tip. Branchial Arches. Three branchial arches, the 1st branchial cleft, the 2nd Ventrally the caudal and caudo-Iateral boundaries of the body wall have and 3rd branchial grooves, and the branchial sinus are present. The cranial formed. nerves to branchial arches 1-3 are discernible using oblique or transmitted Branchial Arches. The 1st and 2nd arches and the 1st branchial cleft are illumination. visible. Heart. An extra vertical loop has developed making a total of three Stage 5 [Fig. 1 gJ loops. The heart lies in the midline of the embryo. Sensory Placodes, Pits, etc. The lens placode and optic cup are defined, Somites. 36-40 pairs. Only traces of the first somite can be detected, and the otic pit is distinctly patent. although it is included in this count. Brain. Hindbrain discernible as a clear transparent region. Tail. The tail tip bends ventrally at right angles to the body of the Flexures and Rotation. The cranial end of the embryo is flexed at approx­ embryo; 6-10 somites in its base, tip unsegmented. imately the level of the heart with the head lying at approximately 45° to Flexures and Rotation. Body torsion is complete except for the tail. The the plane of the body. No body torsion has occurred. head is further flexed with the roof of the brain at approximately 25° to the plane of the body. Stage 3 [Fig. 1 S) Allantois. The allantoic bud is distinctly swollen, smaller in height than the tail. Somites. 26-30 pairs. Sensory Placodes, Pits, etc. The otic pit lies dorsal to the junction of the Delineation of the Embryo. Complete. 2nd and 3rd branchial arches, and its external opening is closing. Branchial Arches. Three branchial arches, the 1st branchial cleft, the 2nd branchial groove, and the branchial sinus are all present. Stage S [Figs. 1 S. 1 g. and 23J Tail. Bud present, but lacks somites. Sensory Placodes, Pits, etc. Nasal placodes present. Otic pit closed. Brain. Forebrain, midbrain, and hindbrain are now discernible, the Limbs. Hindlimb buds are just visible on each side, the right hindlimb latter appearing distinctly transparent. ~ bud being marginally advanced over the left, but no forelimb buds are Sensory Placodes, Pits, etc. The optic cup has an elongated horseshoe present (Fig. 23). 400 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS STAGES OF EMBRYONIC DEVELOPMENT (AFTER EGG LAYING) 401

Flexures and Rotation. Body torsion complete. The roof of the brain is at Tail. The tail tip is coiled through two 90° turns and flexion of the tail approximately 45° to the plane of the body, whereas the floor of the brain is base has commenced. Twelve to eighteen somite pairs are present horizontal and parallel to the plane of the body. throughout the tail. Brain. The olfactory bulbs, forebrain, and midbrain are distinct. Four to six neuromeres are discernible in the transparent hind brain area. Stage 9 [Figs. 19, 23, 27C and OJ Tail. The tip is starting to curl but is unsegmented; somites are only present in the proximal region of the vertically oriented tail. Branchial Arches. Four branchial arches, the 1st and 3rd branchial clefts Allantois. The allantoic bud is stilI smaller in height than the tail. and grooves, the 2nd and 4th branchial grooves, and the branchial sinus Gut. The foregut and hindgut are formed, but the midgut is incom­ are visible (Figs. 27C and D). Branchial clefts are present only in the dorsal plete ventrally. halves of the junctions between the 1st-2nd and 3rd-4th arches, the inter­ Extra-embryonic Membranes. Many blood vessels in the vitelline and vening ventral tissue is continuous and forms the branchial grooves. yolk sac membranes; major ones emerge at the level of the 18th somite and Facial Processes. The maxillary process is distinct and extends forward other smaller ones at the 6th and 11th somite levels. to the midpoint of the eye. There are elevated rims of tissue around the nasal pits. A remnant of the ingrowth of Rathke's pouch is visible in the Stage 7 [Figs. 19, 23, and 27AJ roof of the primitive oronasal cavity. Eye. The optic cup and central lens anlage are large and round but Limbs. Distinct hindlimb bud; forelimb bud just visible as a sinusoidal unpigmented. elevation. The forelimb bud extends over approximately somites 12-15; the Limbs. There is a distinct apical ectodermal ridge on the hindlimb bud, smaller hindlimb bud extends over somites 26-28. Differences in size be­ but not on the forelimb bud. The hindlimb bud extends out farther from tween forelimb and hindlimb buds are more marked in C. johnstoni. the body than the forelimb bud (Fig. 23). Sensory Placodes, Pits, etc. Nasal placode begins to invaginate. Tail. The tail tip is curled through three 90° turns, and the tail base is Brain. Midbrain bulge evident. distinctly flexed from the lower lumbar region; the tail contains approxi­ Tail. Tail tip, curled at approximately 90° to the remainder of the tail, mately 20 somites. and contains somites. Allantois. The large allantois is fused to both the amnion and chorion. Flexures and Rotation. At the level of the heart, the cranial region is bent The chorioallantois extends around approximately one-half of the breadth at approximately 90° to the body. The neck region is also flexed, so that the of the eggshell. roof of the brain lies at approximately 60° to the body plane and the floor of Heart. The distinct atria, ventricles, and the lung primordia are visible the brain at 45°. This dichotomy in angulation accentuates the midbrain bulge. through the transparent pericardial sac. Gut and Abdominal Organs. The midgut and body walls are open ven­ Branchial Arches. Three branchial arches, the first branchial cleft and trally from the caudal limit of the pericardial sac to two-thirds the way groove, the second and third branchial grooves, and the branchial sinus down the body. The developing mesonephros and liver are just visible are present (Fig. 27A). through the lateral body walls.

Stage B [Figs. 19, 23, and 27BJ Stage 10 [Figs. 19, 23, 27E, 34A and BJ

Allantois. Now a large ballooning sac, longer than the tail; it contains Eye. Pigment in the iris makes the eye appear light black except for the blood vessels, but is not yet fused with either the amnion or the chorion. central opaque lens. The right eye is usually pigmented earlier and more External Genitalia. A small elevated genital tubercle is present. heavily than the left eye. Sensory Placodes, Pits, etc. Nasal pits are present on each side of the Branchial Arches. Five branchial arches, grooves, and the branchial si­ head external to the swellings of the olfactory bulbs; Rathke's pouch is nus are present. The first cleft lies dorsally, ventral to the otocyst. The third invaginating in the middle of the roof of the primitive oronasal cavity to branchial cleft (between arches 3 and 4) is closing over. Branchial arches 1 approXimate the infundibulum (Fig. 27B). and 2 are merged together in their ventral halves, and arch 2 has started to Limbs. Forelimb and hindlimb buds both distinct and extending over overgrow arch 3 (Fig. 27£). Branchial arches 4 and 5 are very small. Viewed somites 11-16 and 27-32, respectively (Fig. 23). The apical ectodermal from the frontal aspect, the horseshoe-shaped 1st branchial arch is dis­ ridge is developing on the hindlimb bud. tinctly lobulated in the midline. 402 REPRODUCTIVE SIOLOGY AND EMSRYOLOGY OF CROCODILIANS STAGES OF EMSRYONIC DEVELOPMENT tAFTER EGG LAYING) 403

Facial Processes. The medial and lateral nasal processes are distinct ele­ striction for the proximal and distal elements, lies closer to the flank of the vations on either side of the nasal pits. The maxillary processes extend embryo. The elongated hindlimb shows little differentiation into proximal forward as far as the caudal junction of the medial and lateral nasal and distal elements, and although there is still a distinct apical ectodermal processes, and delimits a distinct groove beneath the eye. ridge, foot plate formation is just discernible (Fig. 23). Limbs. The hindlimb bud is fan-shaped with a distinct apical ectoder­ mal ridge (Fig. 23). It extends out further from the body wall than the Stsge 13 [Figs. 1 g, 23, 24A-C, 27H, 34C end 0] forelimb bud. Tail. The tail is coiled through four 90 0 turns. Facial Processes. The nasal pit slits are very distinct (Figs. 24A and B). Gut and Abdominal Organs. The mesonephros and liver are clearly vis­ The prominent maxillary processes are continuous with the lateral nasal ible through the lateral body walls. processes (Figs. 24A-C). Limbs. The forelimb is now distinctly bent towards the pericardium. Stege 11 [Figs. 1 g end 23] The distal hindlimb is flattened and enlarged into a footplate primordium (Fig. 23). Facial Processes. The nasal pit slit is starting to form between the medial Branchial Arches. Arch 3 is almost completely overgrown by arch 2, and lateral nasal processes. The club-shaped maxillary processes extend to which now reaches the pericardium. Arches 4 and 5 are difficult to see. The the junction of the medial and lateral nasal processes and is continuous branchial sinus has closed. The anterior margin of the lower jaw has grown with the lateral nasal process. forward from the merged conglomerate of arches 1 and 2. The 1st branchial Limbs. Both forelimb and hindlimb buds extend out caudally from the cleft is now more horizontally oriented and is hereafter referred to as the body wall, and both have distinct apical ectodermal ridges (Fig. 23). The external auditory meatus. The upper ear flap is sinusoidal with a midline forelimb has a distinct constriction demarcating the proximal and distal bulge formed by the merging of the auricular hillocks (Fig. 27H). A groove elements, but this constriction is less marked in the hindlimb. runs craniocaudally along the basal (dorsal) aspects of the branchial arches Gut and Abdominal Organs. A loop of midgut tube is visible at the level and lower jaw. of the umbilicus. Extra-embryonic Membranes. The chorioallantois now extends as a ring Branchial Arches. The 2nd branchial arch continues to overgrow the around the inner circumference of the central eggshell membrane. 3rd, and arches 4 and 5 are starting to submerge. The 1st branchial cleft is immediately ventral to the otocyst. Stege 14 [Figs. 1 g, 23, 240, 27F end H] Eye. Distinct black pigment present in the iris. Extra-embryonic Membranes. The chorioallantois extends two-thirds the Facial Processes. The nasal pit slit has closed due to the merging of the way around the breadth of the shell membrane. medial nasal, lateral nasal, and maxillary processes (Figs. 240 and 27F). The medial nasal processes are prominent and have anterodorsal external Stage 12 [Figs. 1 g, 23, and 27H] bulges (Figs. 240 and 27F). These will later grow forward to displace the external nares dorsally. The medial nasal processes also have two anterior Branchial Arches. The sinusoidal 1st branchial cleft lies above the oto­ intraoral bulges signifying the onset of primary palate development (Figs. cyst and its margins show condensations for the auricular hillocks (Fig. 240 and 27F). The maxillary processes have sinusoidal intraoral margins 27H). The merged conglomerate of arches 1 and 2 is growing caudally and signifying the onset of secondary palate development. The internal nares has overgrown arch 3 to reach the junction between the 3rd and 4th arches. are distinct. This conglomerate forms the base of the lower jaw, which extends as far Limbs. The foot and hand plates are distinct; the former is advanced forward as the middle of the lens of the eye. Arches 4 and 5 are small but over the latter (Fig. 23). visible. The branchial sinus is patent. Branchial Arches. The 2nd branchial arch has overgrown the 3rd, 4th, Facial Processes. The club-shaped maxillary process extends forward as and 5th, and this merged conglomerate together with the 1st arch forms a large shelf of tissue beneath the eye. The nasal pit slits are deepening as the base of the lower jaw and the neck. A craniocaudal groove is still the medial and lateral nasal processes enlarge. There is a distinct notch and present along the dorsal margins of the merged 1st and 2nd arches. furrow in the midline of the face between the medial nasal processes of Lower Jaw. The lower jaw extends one-fourth the way beneath the each side. upper jaw. It is broad and round in A. mississippiensis but more pointed in Limbs. The forelimb, which is beginning to bend in the region of con- C. johnsoni. REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIANS STAGES OF EMBRYONIC OEVELOPMENT [AFTER EGG LAYING] 404 405 Ear. The upper ear flap is overgrowing the external ear opening. White Facial Processes. The upper jaw is hook-shaped, being moulded around opacities, internal condensations for ear development, are evident in the the pericardial bulge. Small secondary palatal shelves are present im­ region below the ear opening. mediately behind the posterolateral margins of the primary palate. Denticles. One denticle is present on each side of the developing pri­ Caruncle. Two tiny, widely spaced thickenings are just discernible on mary palate (merged medial nasal processes) margins and one on each side the anterior tip of the snout. of the midline of the lower jaw. Flexures and Rotation. The embryonic face rests on the large bulge of the Stage 17 [Figs. 20, 23, 24F, G, 25A, 2SA and B1 thorax (pericardial sac). Gut and Abdominal Viscera. A large loop of gut herniates through the Limbs. Mesodermal condensations for the five forelimb and four hind­ narrow umbilical stalk and touches the underlying yolk. The abdominal limb digits are present (Fig. 23). viscera (e.g., liver and mesonephros) are visible through the body walls. Lower Jaw. The lower jaw lies behind the primary palate bulges (Figs. Tail. This is markedly coiled, and the tip has a terminal kink. 24F and G). The furrows and elevations dorsal to the lower jaw have External Genitalia. In A. mississippiensis, the tubercle is still small, but in disappeared. C. porosus and C. johnsoni it is much larger. Denticles. At this and later stages, the numbers of denticles discernible Embryonic Reflexes. Contralateral withdrawal reflexes occur. macroscopically in the upper jaw varies as some appear and others disap­ pear; consequently, upper jaw denticle number is of little further diag­ Stage 15 [Figs. 20, 23, 24E, and 2711 nostic value. Contrarywise, seven denticles occur on each side of the lower jaw; three are distinct, three less distinct, and one is disappearing and very Lower Jaw. This is one-third to one-half the length of the upper jaw difficult to see. (Fig. 24E). Dorsal to the lower jaw complex, there are three furrows and Caruncle. Two small elevations, widely separated, have appeared on surface elevations, which appear to be dorsal representations of the bran­ the anterior tip of the snout (similar to Fig. 26A). chial arches, which have now coalesced ventrally. Palate. The secondary palatal shelves are distinct. They are closest to Denticles. Two denticles are present on the anterior upper jaw margins each other anteriorly behind the primary palate, but do not contact (Figs. of each side, one on the margin of the primary palate, the other on the 25A, 28A and B). The tectoseptal processes are closing in the posterior half maxillary process behind the closure zone. As before, one mandibular of the oronasal cavity. denticle is present on each side of the midline. Scales, Scutes, etc. The somite pattern is distinct particularly on the Eye. Dark black stria in the iris radiate out from around the lens. The dorsal aspects of the body and tail. This heralds the onset of scale differ­ anlage for the upper eyelid is an elevated rim of tissue above each eye. entiation. Anlagen for the abdominal ribs are visible. Limbs. Distinct proximal and distal regions and hand and foot plates Flexures and Rotation. The head is extended off the bulge of the pericar­ (but no digit rays) are present on both the forelimb and hindlimb (Fig. 23). dial sac due to elongation of the neck. Facial Processes. The anterodorsal (coalesced) bulges of the medial nasal Ear. The external ear flap is distinct as the adult external ear form is processes have displaced the external nares dorsally (Fig. 24E). The pri­ established. mary palate has formed but still retains two anterior bulges (Fig. 24E). There is a distinct hollow in the face beneath the anterior one-third of the Stage 1 S [Figs. 20, 23, 25B, C, 26A, and 2SC-F1 eye. The tectoseptal process is evident in the roof of the primitive oronasal cavity. Limbs. The digit rays on the hand and foot plates are now distinct cartilaginous condensations. The hand and foot plates have a crenellated Stage 1 6 [Figs. 20 and 231 appearance (Fig. 23) caused by the straight margins of the interdigital tissues. Limbs. Faint digital condensations are present in the footplate but not Lower Jaw. The lower jaw lies beneath the anterior primary palate in the handplate (Fig. 23). bulges, and it no longer rests on the pericardial sac. Lower Jaw. This is now two-thirds the length of the upper jaw. Denticles. At the start of this stage, six denticles are visible on each side Denticles. There are four on the upper jaw margins of each side, two on of the mandible, and at the end, eight are discernible. The posterior denti­ the primary palate (that closest to the midline may be difficult to see), and cles are difficult to see. two on the maxillary process. Two denticles lie on each side of the mandi­ Caruncle. Two widely spaced, white blebs on the anterior tip of the ble. snout (Fig. 25A). 406 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS STAGES OF EMBRYONIC DEVELOPMENT [AFTER EGG LAYING) 407 Palate. The secondary palatal shelves are one-fourth closed at the be­ feet, despite the fact that the total number of digits varies between the two ginning of Stage 18 and three-fourths closed at the end (Figs. 25B and C). (Fig. 21). Interdigital clefting now extends along approximately one-fourth This stage can be accurately subdivided by specifying the extent of second­ the length of the digits (Fig. 23). The outer two digits of the hand and the ary palate closure (Figs. 25B and C, and 28C-E). The upper jaw margin is outer digit of the foot never develop nails. straighter and less hooked than previous. Caruncle. The caruncle is now a solid structure due to consolidation of Eye. The margins of the upper eyelid anlage extend over the superior the region between the two initial swellings (Fig. 26C). rim of the iris forming a distinct groove between the eyelids and the eye, Lower Jaw. The lower jaw is in its adult relationship with the upper jaw into which small instruments can be passed. (Figs. 24H and I). Scales, Scutes, etc. Dorsal scalation is now marked. External Genitalia. The external genital primordium is now pointed Pericardia I Sac. The bulge of the transparent pericardial sac is starting to with a distinct elevation of its tip. be submerged into the ventral thoracic wall. Palate. The palate is completely closed but the basihyal valve is not yet present (Fig. 25E). Stage 19 (Figs. 20, 23, 250, 26B, 34E and F] Pericardia I Sac. The pericardial sac is now one-fourth withdrawn into the ventral body cavity. Eye. Upper and lower eyelids are distinct. The anterior nictitating Coloration. White flecks of ossification are present along the margins of membrane anlage is discernible in the anterior corner of the eye (Fig. 26B). the upper and lower jaws, around the external auditory meatus, and in the Caruncle. Two elevations of the caruncle have approximated each other proximal and distal elements of the limbs. at the tip of the snout (Fig. 26B), but the tissue between them is thin, Scales and Scutes. Scale formation is marked dorsally and scutes are appearing transparent under incident illumination. beginning to appear in the neck region behind the skull. Lower Jaw. The lower jaw lies behind the anterior margin of the upper jaw. Consequently, if the premaxillary bulges are large, the mouth opens (as in Fig. 24H). The tongue and floor of the mouth contents sag beneath Stage 21 (Figs. 20, 23, 25F, 260] the margins of the lower jaw. Interdigital clefting extends three-fourths of the way along the Limbs. Interdigital clefting has commenced producing slight marginal Limbs. digits. Phalanges can be distinguished in the digits (Fig. 23). notches particularly in the foot plates (Fig. 23). Scales are now visible on the ventral body wall as well Palate. The palate is almost completely closed (Fig. 250). Scales and Scutes. as dorsally on the snout, neck, body, and tail. The dorsal neck scutes are Coloration. White flecks, representing underlying ossifications, are ob­ clearly defined. vious on the margins of the upper and lower jaws and around the ears. External Genitalia. The end of the external genitalia has developed a Caruncle. The caruncle is a solid mass on the snout tip, but the tissue globular swelling. around the base of the caruncle is not differentiated from the other snout scales (Fig. 260). Denticles. There are eight to nine denticles visible on each side of the The superior basihyal valve flap is present at the posterior mar­ lower jaw. Henceforth, many mandibular denticles appear and disappear Palate. gin of the palate (and the inferior flap at the base of the tongue) and a so that their numbers are too variable to be used in staging. plexus of palatal blood vessels is conspicuous (Fig. 25F). Species Differences. In C. johnsoni and C. porosus, the nostrils are ele­ Pericardial Sac. The pericardial sac is one-half withdrawn into the body vated on a nasal disk. The lateral jaw margins have distinct notches where cavity. the primary and secondary palates closed (as in Figs. 24H and I); these later External Nares. Elevations for the constrictor nares muscles are evident. accommodate the large fourth dentary teeth. Eye. A white ring in the iris surrounds the outline of the lens of the eye and is overlapped by both upper a.nd lower eyelids. Stage 20 (Figs. 20, 23, 24H, I, 25E, and 26C]

Limbs. Nail anlagen develop rapidly in a specific sequence early in this Stage 22 (Figs. 21, 23, 251] stage (Fig. 23). They appear first on the most medial digit of the foot, then on the neighboring two digits, then on the most medial digit of the hand Coloration. Pigmentation is first visible on the margins of the upper and finally on the neighboring two medial hand digits. Consequently, nail jaw, along the ventral aspect of the flank, and on the proximal and distal anlagen are present on the most medial three digits of both the hands and elements of the limbs, but there is little or no dorsal pigmentation. STAGES OF EMBRYONIC DEVELOPMENT [AFTER EGG LAYING) 409

53 55 58 60 65 I 50mm I

12 14 20 25 28 30 35 40 46 48 DAYS AFTER EGG LAYING-

Fig. 22. Alligator mississippiensis. Montage at constant magnification, illustrating the growth and changing bodily proportions of embryos from 12 days after egg laying until hatching. All eggs were collected within 12 hours of egg laying, incubated at a constant 30 Q C and 100% humidity and fixed at indicated number of days after deposition.

Limbs. Interdigital clefting is at its adult level (Fig. 23). Scalation is difficult to see on the limbs. Eye. The eyelids are generally closed at this and subsequent stages. Pericardia I Sac. The pericardial sac is two-thirds withdrawn into the body cavity.

StBge 23 [Figs. 21. 23. 24.... 25G. 26EJ -27 Fig. 21. Alligator mississippiensis. Stages 22 to 28 (numbered) of embryonic development. See Coloration. Pigmentation is more extensive, and the embryos are light text for description of stages. Millimeter scales included. brown. Dorsally, stripes are present, the pattern of which varies within STAGES OF EMBRYONIC OEVELOPMENT [AFTER EGG LAYING) 411

and between species in association with definitive scale and scute patterns. Pigment is also present ventrally and on all limb elements. Limbs. Scales are present on the proximal and distal elements. The nail tips of the feet have a slight distal elevation (Fig. 23). 1ni'm" Jaws. Sensory papillae are present along the lateral jaw margins and STAGE 6 7 8/9 10 scales are evident on the gular skin (Fig. 24J). L:;:::(L~~ Caruncle. The caruncle is located on a smooth white base (Fig. 26£). 0-- Brain. The midbrain is visible as a white bulge at the back of the (\ cranium because the overlying skin is poorly pigmented and the osseous cranial roof is incomplete. Pericardial Sac. The pericardial sac is three-fourths withdrawn into the / 0_ body cavity. ~\ \ RF~ bl:J~ ~ ffrt Palate. An extensive plexus of blood vessels and sensory papillae has 1mm formed (Fig. 25G). STAGE 12 13 14 15 16 17 18 19 Species Differences. In C. johnsoni and C. porosus the scales along the jaw margins are triangular with the apex of the triangle toward the jaw mar­ gins, giving the latter a serrated appearance. The external genitalia are LHf\~>l~~~~~r larger and more differentiated than in A. mississippiensis. St;ags 24 (Figs. 21, 23, and 24KJ

Coloration. Pigmentation is now denser so that embryos appear blacker in color. Various patterns occur both within and between species. Limbs. The nails on the hand also have elevations at their tips (Fig. 23) and these elevations are starting to form the curves at the tip of the nails. Brain. The midbrain, enclosed by bone, is overlain by pigmented skin. Pericardial Sac. The pericardial sac is fully withdrawn into the body cavity and the ventral thoracic wall is closing in the midline. \ Yolk. A large volume of yolk lies outside the body and the ventral rrmn umbilical area is large. STAGER' 20 21 22 23 2mm 24 25 26-28 Scales and Scutes. The scales and scutes are now very evident as eleva­ tions all over the embryo (Fig. 24K).

~ Fig. 23. The typical (diagnostic) appearances of crocodilian right fore and left hind limbs . (including hands, feet and nails) at various stages of development. Stages 6 to 11 are views ~ from the dorsal aspect of the embryo and depict the projection of the limb anlage from the flank. Stages 12 to 17 are lateral views of the sides of the embryo and depict the proximal and distal elements of the limb anlage. Stages 18 to 28 are views of the hands, feet, and nails. Up to Stage 17, no structures except the digital mesodermal condensations (at Stages 16 and 17) are l~ visible macroscopically in the limbs, thereafter anlage for the limb and digit cartilages, for the nails and for the interdigital webbing are evident, hence the change in diagram style at Stage 18. Based on observation of Alligator mississippiensis, Crocodylus porosus, and C. johnsoni, al­ though some (Stages 18 to 28) drawings are adapted from Voeltzkow's (1899) work on C. niloticus. AER, Apical ectodermal ridge; 0, mesodermal condensations for the digits; DC, digit Ai cartilages; N, nail anlage. STAGES OF EMBRYONIC OEVELOPMENT (AFTER EGG LAYING] 413

Stage 25 [Figs. 21, 23, 26F, 34G, and Hl

The embryo is like a miniature version of a hatchling, with a considerable volume of external yolk and a large umbilical region. The hooking of the nails becomes more prevalent toward hatching (Fig. 23). Musk glands are visible along the posterior lateral margins of the intergular floor of the lower jaw. The caruncle is a bifid elevation on a smooth base (Fig. 26F) similar in structure to that at hatching (Figs. 26G, H), although perhaps a little less pigmented (this is variable). As few macroscopic changes are visible at this and later stages, aging is best estimated by morphometric procedures.

Stage 26

Stage 26 was intercalated to designate the time of tooth eruption in C. johnstoni and C. porosus, an event which occurs around the same time as the appearance of sensory elevations on the scales. The teeth of A. mississip­ piensis rarely erupt before hatching, and if they do it is at Stage 28.

Stage 27 [Figs. 21, 23, 26G, and Hl

Stage 27 is characterized by the withdrawal of the remaining yolk into the abdominal cavity. It commences with the outgrowth of the ventral body

Fig. 24. Alligator mississippiellsis. Development of the embryonic face. (A) Stage 13. Note the eye, club-shaped maxillary process, medial nasal process, lateral nasal process, nasal pit, nasal pit slit (arrowed), and notch in the center of the frontonasal process. (B) Stage 13. Ventral view. (C) Stage 13. Lateral view. The maxillary process extends beneath the eye and is continuous with the lateral aspect of the lateral nasal process. The branchial arches are amal­ gamated. (D) Stage 14. Ventral view. The medial nasal, lateral nasal and maxillary processes have merged. Note bulges for the primary palate and the anterior/dorsal extension of the nostrils. (E) Stage 15. Ventral view. The development of the anterior/dorsal bulges has dis­ placed the external nares dorsally. The primary palate bulges and the maxillary processes are evident and the lower jaw is half-way beneath the upper jaw. (F) Early Stage 17 viewed from below. The lower jaw is behind the primary palate bulges. (G) Late Stage 17 viewed from below. Note the forward growth of the lower jaw. (H) Crocodylus Ililoticus. Stage 20. Lateral view illustrating the shift of the lower jaw beneath the primary palate, the notch in the upper jaw (arrowed) in the region of junction of the lateral nasal, medial nasal and maxillary pro­ cesses (which will later become the notch for the large fourth dentary tooth), the elevated nasal disc, denticles, caruncle, lens, iris, eyelids and external ear flap. (I) Alligator mississip­ piellsis. Face-on view of the embryo shown in E. Note the development of the anterior nictitat­ ing membrane of the eye. (J) Stage 23. Oblique lateral view of the snout illustrating the elevations for the sensory papillae on the jaw margins, the ventral scales on the floor of the mouth and the submandibular odoriferous gland. (K) Stage 24. View the top of the head. Note the scalation. 0, denticles; E, caruncle; EN, external naris; G, submandibular odoriferous gland; L, lateral nasal process; M, medial nasal process; MD, mandibular process; MN, man­ dible; MX, maxillary process; N, notch in the frontonasal process; NB, nostril bulges; NO, elevated nasal disc; Nt nictitating membrane; NP, nasal pit; P, primary palate bulges; S, sensory papillae; SC, cervical scutes; V, ventral scales on the floor of the mouth...... "" Fig. 26. Alligator mississippiensis. Views of the snout to show the development of the carun­ cle. (A) Late Stage 18. Frontal view. Caruncle is represented by two widely spaced elevations. The lower jaw has been removed. (B) Stage 19. Ventral view. Note that the two white blobs of Fig. 25. Alligator mississippiensis. (A) Stage 17. View of the inside of the mouth (lower jaw the caruncle have joined in the midline. (C) Stage 20. The caruncle now appears more solid. and tongue removed). Note the closed primary palate, the secondary palatal shelves, which (0) Stage 21. (E) Stage 23. Note the smooth base of the bifid caruncle and elevations for the have grown out from the maxillary processes on each side but which do not yet contact each pigmented sensory papillae (S) on the jaw margins. (F) Stage 25. Note that the smooth base of other, the closing tectoseptal processes, caruncle and denticles. (B) Early Stage 18. Palatal the caruncle is pigmented. (G, H) Ventral and frontal views of Stage 27. E, Caruncle; S, view. The secondary palate is half closed. Note the V-shaped margins of the approximating sensory papillae. palatal shelves. (C) Late Stage 18. Palatal view. The palate is three-quarters closed. (0) Stage 19. Palatal view. The secondary palate and tectoseptal processes are almost completely closed. Note also the distinct anterior nictitating membrane of the eye and lower eyelid. (E) Early Stage 20. Palatal view. The palate is completely closed but the basihyal valve has not yet the paired hypobranchial eminences which will form the lower flap of the basihyal valve. (I) developed. (F) Stage 21. Palatal view. Note the development of the palatal plexus of blood View of the tongue and lower jaw of Stage 22. Note the plexus of blood vessels in the lower vessels and the basihyal valve. (G) Stage 23. Palatal view. Note the extensive palatal plexus of jaw and the lower flap of the basihyal valve. A, Anterior nictitating membrane; B, basihyal blood vessels and the outlines of the sensory papillae along the jaw margins. (H) Stage 19. valve; E, epiglottis; Ee, caruncle; D, denticles; H, hypobranchial eminences; L, lower eyelid; View of the tongue and lower jaw. Note the denticles and teeth, epiglottis and swellings of PP, primary palate; P, secondary palate; T, tectoseptal processes. 416 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS ORGANOGENESIS 417

wall to enclose the embryonic intestines, yolk sac and yolk; it ends with the pouch in mammals; tympanic development in crocodilians has not been absorption of the latter into the abdominal cavity. Ventral skin forms across reported. The significance of the transitory opening of the third cleft is the yolk scar. unknown. The branchial sinus forms at the base of the most caudal bran­ chial arch (Fig. 270); its specialized epithelium invades the underlying Stage 28 [Figs. 21 and 23] pharyngeal mesoderm (Ferguson, 1982b, 1984a). Each branchial arch is covered externally by ectoderm and internally by At Stage 28 the yolk scar diminishes in length and width as the absorbed endoderm, which extends to the outer border of the mandibular arch (Fig. abdominal yolk is utilized. There are considerable variations both among 27G) and consists of a central core of mesoderm surrounded by a mass of and within species as to the volume of absorbed yolk and the size of the mesenchymal cells derived from the neural crest (Ferguson, 1981a). Pre­ yolk scar at hatching (which occurs at the end of this stage). These parame­ liminary experiments, involving surgical excision or chemical destruction ters may be influenced by the temperature of egg incubation (Ferguson and of alligator neural crest cells and heterotypic grafting of quail neural crest Joanen, 1982, 1983). The growth and changing proportions of embryos of cells at different levels of the neuraxis (Ferguson, 1981a, 1982b, 1984a, A. mississippiensis from 12 days after egg laying to hatching are illustrated in unpubl.) suggest that neural crest cells migrate in temporally distinct Fig. 22. waves from different levels of the neuraxis. Crest cells destined for the mandibular arch and its maxillary processes migrate from the rostral and caudal levels of the mesencephalon, respectively (Ferguson, 1981a). It is VII. ORGANOGENESIS therefore possible selectively to block the migration of various waves of neural crest cells to produce alligators with normal maxillary processes and A. Branchial Arches therefore a normal upper jaw and palate, but with virtually no lower jaw or tongue (Fig. 37E; Ferguson, 1981a, 1984a). This technique, combined with In Alligator mississippiensis (Parker, 1883; Clarke, 1891; Reese, 1908, 1912, shell-less, or semi-shell-Iess culture, has enabled palatal development to be 1915a; Ferguson, 1984a), Crocodylus johnsoni (Ferguson, 1984a), C. porosus viewed as it happens; this is the first such longitudinal study in any animal (Ferguson, 1984a), and C. niloticus (Voeltzkow, 1899), the five branchial (Ferguson, 1981a, 1982b, 1984a). arches appear in a cranio-caudal sequence (described in Section VI), begin­ Each branchial arch also contains its own aortic arch artery and appro­ ning as a series of thickened epithelial involutions in the pharynx (Figs. priate cranial nerve (Figs. 18, 19, and 27G). The neural crest cells appear to 27C, E, and G). Initially, these branchial pouches are internal grooves lined give rise to all of the skeletal and connective tissues of the face, apart from by thickened epithelium but they soon approximate the external branchial striated muscle which is derived from the arch mesoderm; these prelimi­ grooves. The pharyngeal endoderm fuses with the surface ectoderm to nary data (Ferguson, 1981a, 1982b) indicate that the general pattern of form true clefts in the dorsal half of the junctions between the 1st and 2nd neural crest derivatives in alligators is comparable to that in birds and the 3rd and 4th arches (Ferguson, 1984a; Figs. 18, 19, 27C, 0, and G). (LeOouarin, 1982). Macroscopic descriptions of branchial arch develop­ In the remaining junctions, thin membranes of ectoderm, mesoderm, and ment and closure of the cervical sinus are given in Section VI and in endoderm separate the external grooves from the internal pouches (Fergu­ Ferguson (1984a). Descriptive accounts of the branchial pouch derivatives son, 1984a; Figs. 18, 19, 27C, 0, and G). and the cartilaginous, bony and muscular derivatives of the branchial The first branchial cleft migrates dorsally until it overlies the otocyst near arches exist for several species and include comparative comments with which it forms the external meatus of the ear (Ferguson, 1984a, Figs. 27H other vertebrates (Parker, 1883; Voeltzkow, 1903a,b; Reese, 1908, 1915a; and I). Auricular hillocks develop along its ventral margin, which becomes Edgeworth, 1907; Goodrich, 1930; Pasteels, 1950; Oalcq and Pasteels, 1954). the superior flap of the external ear (Figs. 27H and I). First branchial arches excised from Stage 10 alligator embryos can be The third branchial cleft is transitory. Its epithelia become specialized cultured in vitro at 30°C and 37°C (Ferguson et al., 1981, 1983). In general, and develop numerous cilia and cell processes, which extend from epithe­ chemically defined, serumless media at 37°C are useful for short-term (up lial cells on one side of the cleft to those on the other (Ferguson, 1982b, to 14 days) studies, whereas serum supplemented media at 30°C are useful 1984a). In this way, the two epithelia oppose one another, fuse, and mi­ for long-term (up to 60 days) studies. Explanted arches consist initially of grate, thus closing the branchial clefts (Ferguson, 1982b, 1984a). In fish, lobulated cylinders of undifferentiated mesenchyme covered by epithe­ patent branchial clefts separate all the arches to form gills, whereas mam­ lium (Fig. 27G). They undergo normal morphogenesis and differentiation mals lack patent branchial clefts (Sperber, 1981). The limiting membrane in culture (Figs. 29A and B; Ferguson et al., 1982, 1983). Paired lingual becomes the tympanum in the remodeling of the first branchial groove and swellings form a tongue-like structure. Blastematae for the Mm. genioglos­ ORGANOGENESIS 419

sus, hyoglossus, and intermandibularis, the dentary, splenial, and angular bones, Meckel's cartilages, lingual lipid, and fibrous tissue appear and are patterned comparable to those seen in vivo (Figs. 29A and B; Ferguson et al., 1982, 1983). However, anlagen for taste buds and teeth are absent. The differentiation of alligator branchial arches in vitro is superior to that for any other vertebrate studied to date, so that they are useful models for investigation of a variety of developmental phenomena.

B. Fses snd Noss

There are accounts of the development of the face and nose for several genera: Alligator (Rathke, 1866; Clarke, 1891; Reese, 1901b, 1908, 1915a, 1925; Bertau, 1935; Wettstein, 1954; Parsons, 1959a, 1970; Ferguson, 1981a, b, 1982b, 1984a), Caiman (Rathke, 1866; Parsons, 1970; Saint Girons, 1976), Melanosuchus (Bertau, 1935; Parsons, 1970) and Crocodylus (Rathke, 1866; Meek, 1893, 1911; Rose, 1893a, b; Seydel, 1896, 1899; Voeltzkow, 1899; Bertau, 1935; Wettstein, 1954; Wegner, 1957; Parsons, 1959a, b, 1970; Guibe, 1970m; Saint Girons, 1976; Bellairs, 1977; Ferguson, 1984a). That of Cavialis is unknown except for a figure in Bruhl (1866), an illustration by Bustard (1980c) and a description of the nasal excrescence (Martin and A. d'A. Bellairs, 1977). Butler (1905) notes that the snout of either Cavialis or Tomistoma is shorter and wider in the embryo than in the adult (but see

( , Fig. 27. Alligator mississippiensis. Embryos. (A-D) and (F-G); Crocodylus lliloticus. (E-I). (A) Stage 7. Scanning electron micrograph (SEM) of the cranial aspect. Note the first and second branchial arches and first branchial cleft (arrowed). (B) Stage 8. Face-on view. Note the midline fissure and facial processes. (C) Stage 9. Scanning electron micrograph. Note the four branchial arches, grooves and clefts, nasal pit, limb buds, and tail. (D) Higher power view of C illustrating the true 1st and 3rd branchial arch clefts (arrowed), the four branchial arches and the branchial sinus. The apparent cleft between the 2nd and 3rd arches is an artifact. (E) Stage 10. Note the nasal pits and surrounding nasal processes, the lens and surrounding optic cup, the second branchial arch starting to overgrow arch 3, the 1st branchial cleft ventral to the otocyst and the brain regions. (After Voeltzkow, 1899.) (F) Stage 14. SEM illustrating closure of the nasal pit slits by the medial nasal, lateral nasal and maxillary processes. Note the intraoral bulges of the club shaped maxillary processes, signifying the onset of secondary palate development, the anterodorsal elevations of the medial nasal processes and the enlarg­ ing mandible. Compare with Figure D. (G) Stage 9. Horizontal hematoxylin and eosin section through the five branchial arches (1-5). Note the thick endoderm, thinner ectoderm, bran­ chial grooves and pouches, aortic arch arteries,and branchiomeric nerves. (H, I) Stages 12 and 15. Two diagrams illustrating the rearrangement of the first branchial cleft to form the external ear and superior ear flap. (After Voeltzkow, 1899.) A, Auricular hillocks; AA, aortic arch arteries; AD, anterodorsal elevations of nasal processes; BN, branchiomeric nerves; BP, bran­ chial pouches; BS, branchial sinus; e, first branchial cleft; 0, denticles; E, eye; Ee, ectoderm; EN, endoderm; EX, external surface. F, ear flap; FL, fore limbbud; G, branchial grooves; H, hindbrain; HL, hind limbbud; LN, lateral nasal process; M, midbrain; MA, mandibular pro­ cess; MD, mandible; MN, medial nasal process; MX, maxillary process; N, nasal placode; NP, nasal pit; 0, optic placode; Oe, optic cup; OT, otocyst; P, palatal shelves; PE, pericardial sac; PH, pharynx; 1, 2, 3, 4, branchial arches 1-4. ORGANOGENESIS 420 REPROOUCTIVE SIOLOGY ANO EMSRYOLOGY OF CROCOOILIANS 421

Bustard, 1980c for a photograph of a Cavialis hatchling with a long thin (Figs. 24A and B). The club-shaped maxillary processes come to lie beneath snout). The literature on crocodilian nasal embryology has been reviewed the nasal pit slits, which are eventually closed as the maxillary processes by Parsons (1959a, 1970). The buccopharyngeal membrane initially sepa­ unite with the lateral and medial nasal processes (Figs. 240 and 27F). This rates the surface ectoderm from the pharyngeal endoderm (Parker, 1883). uniting involves limited cell death and extensive migration of epithelial After its rupture, the primitive mouth is bounded by the forebrain above cells, which exhibit a characteristic cobblestoned, villous morphology. and the pericardial sac below. The differentiation of Rathke's pouch and Throughout this period, differential growth of the head brings the nasal the pituitary is discussed in Sections VII.I and P and for reptiles in general pits and related processes from their early lateral positions (Figs. 27A and by Pearson, Chapter 9, this volume. B) closer to one another in the midline (Figs. 24A-D and 27F). Facial development in Alligator mississippiensis (Ferguson, 1981a, b, The adjacent medial nasal processes develop paired intraoral projec­ 1982b, 1984a), Crocodylus johnsoni and C. porosus (Ferguson, 1984a) may be tions, which merge to form the primary palate and primary nasal septum. summarized as follows (for chronology, see Section VI). The first sign of Differential growth of extraoral caudal elevations of the medial nasal pro­ nasal development is the appearance of bilateral epithelial thickenings on cesses shifts the anterior nasal choanae from the tip of the snout to the the anterolateral aspects of the forebrain bulge (Fig. 27A). These nasal adult dorsal position (Figs. 240 and E, 27F). The posterior nasal choanae placodes invaginate toward the roof of the primitive oronasal cavity and now open behind the primary palate. The development of the secondary form the nasal pits (Fig. 27A). As the nasal pits invaginate, two rims of palate and associated structures is discussed in Section VII. C. tissue are embossed outward around their surface openings to form the Later, cartilage, bone, and muscle differentiate in various parts of the medial nasal and lateral nasal processes (Figs. 24B, 27B and F). The area facial and nasal skeleton, the nasal epithelium becomes regionally special­ between the two nasal pits (including the medial nasal processes of each ized, the anlagen of the nasal glands and ducts appear, and the complex side) is known as the frontonasal process and is divided by a medial groove conchae and recesses, which characterize the crocodilian nose, begin to (Fig. 24B). form (Bertau, 1935; Parsons, 1959a, 1970; Saint Girons, 1976). Bilateral maxillary processes arise from the dorsal ends of the mandibu­ lar arch (Figs. 24A-C, 27A, B, and F) and grow forward from the angles of c. Palate snd Nasopharyngeal Duct the primitive mouth cavity to form progressively more of its cranial borders (Figs. 19, 24A-D, 27A, B, and F). The club-shaped maxillary processes Unlike any other reptiles, adult crocodilians have a -like second­ form the lower boundaries of the developing eye (Figs. 19, 24A-D, and ary palate composed of the sutured palatal processes of the maxillary, 27F). Mesenchyme in the maxillary processes derives principally from the palatine and pterygoid bones (His, 1892; Moll, 1888; Busch, 1898; Gappert, mesencephalic neural crest, whereas that in the medial and lateral nasal 1903; Voeltzkow, 1903b; Hoffman, 1905; Fuchs, 1907, 1908, 1910; Sippel, processes derives principally from the prosencephalic neural crest. The 1907, Fleischmann, 1910; Thater, 1910; Lakjer, 1927; Barge, 1937; Peter, maxillary mesenchymal cells migrate along several routes. Extra-orally 1949; A. d'A. Bellairs and Boyd, 1957; Pasteels, 1950; Guibe, 1970b; Ior­ they migrate forward to form an ever increasing portion of the upper dansky, 1973; Ferguson, 1981a, b, 1982b, 1984a). It is even unlike the avian margin of the stomodeum; also, they pass between the developing eye palate, which is muscular and permanently cleft (Sippel, 1907; Barge, 1937; and ear and between the developing eye and lateral nasal process, demar­ Pasteels, 1950; A. d'A. Bellairs and Jenkin, 1960). Crocodilian embryos are cating the naso-optic furrow between the lateral nasal and maxillary pro­ therefore useful in studies of normal and abnormal craniofacial develop­ cesses. The furrow eventually closes entrapping an epithelial rod, which ment, largely because of their accessibility to experimental manipulations canalizes to form the naso-Iacrimal duct. Intra-orally the maxillary pro­ inside the egg (Ferguson 1979a, 1981a, b, 1982b, 1984; Ferguson and cesses have two principal advancing fronts of migrating mesenchymal Honig, 1984; Ferguson et al., 1981). cells, namely the tectoseptal processes and the palatal shelves (Section Some macroscopic drawings of the palates of embryos appear in His VILC). (1892), Voeltzkow (1899), Gappert (1903), Sippel (1907), and Fleischmann The invaginating nasal pits fuse with epithelium in the roof of the (1910), but no histological details of palatogenesis are given. More detailed oronasal cavity so forming the primitive nasal cavities with anterior and studies of the development of the nasopharyngeal duct by Fuchs (1908) posterior choanae. The posterior choanae open into the roof of the mouth a and Muller (1965, 1967) contain errors (see below; Ferguson, 1981b, 1984a). few millimeters behind the anterior choanae. By a combination of ex­ The following account is based on investigations of Alligator mississippiensis tremely rapid outward proliferation of the medial nasal and lateral nasal (Ferguson 1981a,b,d, 1982b, 1984a; Ferguson et al., 1984; Ferguson and processes and a caudal extension of nasal pit invagination, the nasal pit Honig, 1984) and of Crocodylus johnsoni and C. porosus (Ferguson, 1984a). slits develop between the ipsilateral medial and lateral nasal processes Description of the early development of the alligator face (Section VILB) 4ee REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS indicates that the paired maxillary processes each develop two intra-oral

migratory fronts of mesenchymal cells, namely the tectoseptal processes "~" and the palatal shelves. j~~V F~, ..•••..• .' . The bilateral tectoseptal processes migrate superiorly between the floor ,,»,,~!,...... ,. of the brain and the roof of the oronasal cavity; eventually merging with ~:~fi .~ one another along the midline in a progressive anteroposterior direction (Figs. 25A-O and 28B). Anteriorly, the merged tectoseptal processes grow downward to form the secondary nasal septum (Fig. 28A). The bilateral palatal shelves arise from the maxillary processes as thick, blunt-ended structures (Figs. 25A-O, 28A and B), which grow horizontally from their first appearance (Fig. 28A), except in the posterior one-fifth of the palate where they are more vertically oriented (Fig. 28B). With later development, these posterior shelves gradually flow over the tongue to become truly horizontal (Figs. 28C and 0). The reorientation involves the migration of mesenchymal and epithelial cells, the hydration of palatal glycosaminoglycans, the contraction of microfilaments, and differential mesenchymal proliferation. The shelves first approximate each other ante­ riorly behind the primary palate and from there closure spreads backward (Figs. 25A-O, 28C, and 0). Palatal closure occurs principally during Stage 18 and is characterized macroscopically by a V-shaped gap along which the posterior margins of the opposing shelves approximate each other (Figs. 25B and C, 28E). Palatal closure has been studied in normal cross-sectional sequences and also in ovo in the same embryo (longitudinal sequence) developing in a semi-sheIl-less culture (Ferguson, 1981a, 1982b, 1984). The process of palatal closure involves contact and adherence of the epithelial cells of the two shelves beginning on the oral aspect of the shelf margins and spreading nasally (Figs. 28C and 0). Cell death is limited to the small area of initial contact, after which the epithelial cells of the two shelves migrate nasally and posteriorly out of the region of closure (Figs. 28C, 0, and E). There is never an extensive epithelial seam (Fig. 28C) and epithelial remnants cannot be detected following closure. Numerous small blood vessels in the shelf mesenchyme adjacent to the area of closure represent the earliest development of the palatal vascular plexus that characterizes late embryos (Figs. 24F and G) and adults. Mesenchymal even contacted each other. Epithelial seam absent. The matrix stains positively for gly­ cosaminoglycans and numerous blood vessels are present. (D) Stage 18. Scanning electron continuity is usually established on the oral edges of the palatal shelves micrograph of palate. Note the closing palatal shelves, midline palatal elevation, primary before the nasal edges are in contact (Fig. 28C). Anteriorly, the shelves palatal bulge, denticles, caruncle, and eyes. (E) SEM of the medial edge epithelial cells in the region of palatal closure seen in (D). Note the markedly cobblestoned appearance and numer­ ous microvilli, both characteristic of cell migration. (F) Stage 18. SEM of the developing tongue and lower jaw. Note the paired lingual swellings, tuberculum impar, hypobranchial emi­ Fig. 28. Alligator mississippiensis. (A) Stage 17. Mallory stain. Transverse section through nences, developing larynx and epiglottis, and denticles. The elevated hypobranchial emi­ head. Note the horizontal palatal shelves, bulge from the nasal septum, tongue, Meckel's nences later form from the inferior flaps of the basihyal valve-see Figs. 25H and I. A, cartilages, and intermandibularis muscle. (B) Stage 17. More posterior section of H & E and Anterior pterygoid muscle; B, bulge from the nasal septum; C, caruncle; 0, denticles; E, eye; Alcian Blue stained specimen. Note the vertical secondary palatal shelves, closing tectoseptal G, genioglossus muscle; H, hyoglossus muscle; HB, hypobranchial eminences; I, interman­ processes, anlage of the anterior pterygoid muscle, Meckel's cartilages, anlagen for the inter­ mandibularis, genioglossus, and hyoglossus muscles. (C) Stage 18. Transverse section dibularis muscle; L, lingual swellings; LA, larynx; M, Meckel's cartilages; MP, midline palatal elevation; P, palatal shelves; PP, primary palatal bulge; T, tongue; II, tuberculum impar; TS, through the closing secondary palatal shelves (Mallory stained). Shelf contact and mesenchy­ tectoseptal process. mal continuity are established on the oral edges of the shelves, before the nasal edges have 424 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY 01= CROCOOILIANS merge with each other and with the bulging downgrowth of the secondary nasal septum (Figs. 28A and C) to produce a continuation of the partitioned nasopharyngeal duct (Fig. 28A). Posteriorly the palatal shelves merge to produce an unpartitioned duct (Fig. 28C), which is subsequently par­ titioned by the fusion of internal midline ductal bulges. Thus, the posterior nasal choanae are progressively displaced posteriorly as the secondary palate develops. The epithelial cells of the medial edges of the closing palatal shelves have characteristic surface topographies (Figs. 280 and E). Initially they are flat and rather featureless, but they become progressively more bulbous and develop surface microvilli and distinct cell boundaries so that by the time of shelf contact they are markedly cobblestoned and vil­ lous, an appearance characteristic of migrating epithelia (Figs. 280 and E). Medial edge, oral, and nasal epithelial differentiation, palatal closure, and mesenchymal differentiation occur in palatal shelves cultured in chemically defined or serum supplemented media (Figs. 29C,0, and E; Ferguson et al., 1984). Palatal development in alligators differs from that of mammals and birds. Initially, mammalian palatal shelves grow vertically downward, lat­ eral to the tongue, and suddenly elevate to a horizontal position above the tongue; the opposing medial edge epithelia fuse in a seam and then die, so that mesenchymal continuity is established across the definitive secondary palate (Ferguson, 1977, 1978a, b). Avian palatal shelves, like those of crocodilians, are horizontal from their first appearance. The epithelia of

Fig. 29. Alligator mississippiensis. Explants. (A) Macroscopic view of first branchial arch ex­ planted at Stage 10 and organ cultured for 14 days in serum supplemented media at 30OC. Note the mandibular shape and the ventral aspect of the tongue tip. (B) Transverse section through an explanted first branchial arch after 36 days culture in serum supplemented media at 30°C. Note the tongue, Meckel's cartilages, and osteogenic blastemata for the lower jaw bones. (C) Scanning electron micrograph (SEM) of closing palatal shelves explanted from a 20-day embryo and cultured for three days in chemically defined serumless media at 3TC. (0) Higher power view of the area of closure. Note the cobblestoned, villous, migrating medial edge epithelia, which are similar to those seen in uitro (Fig. 28E.). (E) SEM of the medial edge epithelia of a control recombination of alligator palatal epithelium and alligator palatal mesen­ chyme cultured for three days in media containing serum at 37°C. (F) SEM of a recombination of alligator mandibular epithelium on alligator palatal mesenchyme cultured for 3.5 days in media containing serum at 37OC. Note the differentiation into stratified squamous oral epithelia (0). cobblestoned, villous medial edge epithelia (M) and ciliated columnar nasal epithelia (N). (G) SEM of a recombination of mouse palatal epithelium on alligator palatal mesenchyme with the medial edges of the two opposed. Cultured for 3.5 days in media containing serum at 37°C. Note the oral. nasal. and medial edge epithelia. The latter is cobblestoned, villous and exhibits little cell death, a pattern typical of alligator (Fig. E. above), not mouse (see Fig. H.). (H) SEM of mouse palatal shelf cultured for 3.5 days in media containing serum at 37°C. Note the stratified squamous oral. ciliated columnar nasal epithelia, and the massive epithelial cell death along the medial edge (compare with Figs. E. and G.). M, Medial epithelium; ME, Meckel's cartilage; N, nasal epithelium; 0, oral epithelium; OB, osteogenic blastemata; T, tongue. 426 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS ORGANOGENESIS 427 their medial edges keratinize, the palatal shelves fail to fuse, and thus the palate to seal behind a lower, more rigid flap, that lies posterior to the produce physiological cleft palate (Sippel, 1907; Barge, 1937; Pasteels, 1950; tongue and is supported by the hyoid cartilage (Wood Jones, 1940). The Shah and Crawford, 1980; Koch and Smiley, 1981). superior valve flap is attached anteriorly to the posterior nasal choanae (in The differing characteristics of the medial edge epithelial cells in the the pterygoid bone), runs parallel to the pterygoid palate for a short dis­ alligator (cobblestones, villous, migrating), chicken (keratinized stratified tance, and so forms a small area of nasopharynx between the pterygOid squamous) and mouse (cell death) suggest that palatal differentiation could bony palate and the upper mucosa of the superior basihyal valve flap be regulated by epithelial-mesenchymal interactions. To test this hy­ (Ferguson, 1981a). A failure to recognize the structure of the basihyal valve pothesis, palatal shelves of alligators, chicks, and mice were separated into led Muller (1967) to misinterpret this space as a division of the na­ epithelia and mesenchyme, recombined in various heterotypic, homo­ sopharyngeal duct (by a process of the pterygoid bone) into a "cavum typic, isochronic, and heterochronic combinations, and cultured. Addition­ ventrale" and a "cavum dorsale" (see her Figs. 9 and 10). Muller (1967) also ally, palatal epithelia and mesenchyme were cultured in isolation and in confused the near simultaneous closure of the tectoseptal processes and combination with mandibular epithelia and mesenchyme, respectively secondary palatal shelves in crocodilians (see Ferguson, 1981a, 1984a). (Ferguson and Honig, 1984). Very little is known about the development of maxillary, palatal and If palatal epithelium from alligator is cultured in isolation, it disinte­ salivary glands (Rose, 1893b; Woerdeman, 1920; Reese, 1925; Barge, 1937; grates after approximately three days, whereas palatal mesenchyme cul­ Fahrenholz, 1937; Kochva, 1978). The maxillary glands may arise from tured in isolation differentiates into bony, cartilaginous, and muscular an­ invaginations of the oral epithelium, closely related to the invaginating lagen; control recombinations differentiate normally (Fig. 29E). Palatal dental epithelium (Woerdeman, 1920). A review of the development of oral shelf epithelium recombined with mandibular mesenchyme differentiates glands in Reptilia includes some data on crocodilians (Kochva, 1978). into typical stratified squamous mandibular epithelium. Conversely, man­ dibular epithelium recombined with palatal shelf mesenchyme differ­ D. Tongue entiates into a typical palatal epithelium (Fig. 29F). These results suggest that, in the alligator, differentiation of palatal epithelium is regulated by The tongue develops from three principal anlagen on the pharyngeal as­ an instructive epithelial-mesenchymal interaction. This interpretation is pect of the branchial arches. The paired lingual swellings of the first arch confirmed in recombinations between vertebrate species. Thus, alligator form the anterior two-thirds of the adult tongue, whereas the midline palatal epithelium recombined with a chick palatal mesenchyme exhibits tuberculum impar of the second and the paired hypobranchial eminences the typical avian stratified squamous pattern of medial edge epithelial dif­ of the third form the posterior one-third (Ferguson, 1982b, 1984a; Figs. 25H ferentiation. Equally, alligator palatal epithelium recombined with mouse and I, 27F). The epithelia of these anlagen come together and merge (Fig. palatal mesenchyme exhibits massive medial edge epithelial cell death (Fig. 27F). Behind the tongue lie the developing epiglottis, larynx, and pharynx 29H). Contrariwise, either chick or mouse palatal epithelium recombined (Fig. 27F). The inferior flap of the basihyal valve arises from a superior with alligator palatal mesenchyme shows differentiation characteristic of outgrowth of the paired hypobranchial eminences (Figs. 25H and I, 27F). the alligator (Fig. 29G). These results show that the pattern of epithelial Crocodilian lingual development resembles that of mammals and birds differentiation is regulated by the source of the mesenchyme (Ferguson (portmann, 1950; Scott and Symons, 1974; Sperber, 1981). and Honig, 1984). During palatogenesis, as in the adult, the tongue lies low in the oronasal The alligator oronasal cavity is not as restricted as that of mammals, cavity (Ferguson, 1981a,b, 1982b). The body of the tongue contains fibrous which may explain why alligator palatal shelves grow horizontally (Fergu­ tissue anteriorly and lipid posteriorly, but intrinsic lingual musculature is son, 1981a, b, 1982b). Further development of the palate involves the ap­ absent (Ferguson, 1981a, b, 1982b, 1984a). Anlagen for the Mm. genioglos­ pearance and growth of osseous, muscular, and cartilaginous blastemata, sus, hyoglossus, geniohyoid, and intermandibularis develop, but their ori­ the expansion of the palatal plexus of blood vessels, and the development gins are poorly known. It has been suggested that all the lingual muscula­ of numerous domed tactile receptors (Figs. 3A and B) from subepithelial ture develops from the geniohyoid anlage, which itself has split off from condensations of mesenchymal (Merkel) cells (Fig. 24]). Small out­ the ventral longitudinal muscle anlage formed by the 4th to 8th trunk pouchings of the nasal cavity expand to form the maxillary sinuses, which myotomes (Edgeworth, 1907). Later stages of development of the lingual enlarge laterally and medially to invade the palatal processes of the maxil­ musculature have been illustrated by Humboldt (1807), Rathke (1866), lae after hatching. Voeltzkow (1899), Gappert (1903), Taguchi (1920), Sewertzoff (1929), The fibrous superior flap of the basihyal valve arises by a posteroinferior Wettstein (1954), and Ferguson (1981a, b, 1982b, 1984a). extension of palatal shelf closure (Figs. 25D-F). Crocodilians possess no The lingual glands arise from thickened epithelia on the dorsum of the true soft palate; the superior flap of the basihyal valve descends from tongue. These glands secrete salt in the estuarine crocodile, Crocodylus ORGANOGENESIS 42B REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCOOILIANS 429 porosus (Taplin and Grigg, 1981), and in C. acutus and C. johnsoni but appar­ similar to that of other vertebrates, and several features are illustrated in ently not in Alligator mississippiensis or Caiman crocodilus (Taplin et al., 1982). Figs. 18, 19,20, 27A and F. Numerous taste buds develop on the tongue and in the floor of the mouth The structure and development of the premandibular, mandibular and (Ferguson, 1981a, b, 1982b). As taste buds do not form in tongues which hyoid head cavities associated with the early embryology of the extrinsic develop from excised mandibular arches, cultured in vitro, their induction ocular muscles in Alligator mississippiensis have been described by Wedin and maintenance may be dependent on neural factors (Ferguson et al., (1949, 1953, 1955). These cavities are interpreted as the myocoeles of so­ 1982, 1983a). mites. The origin, migration, and changing disposition of the ocular mus­ cle anlagen are also described (see Table VII, Figs. 30A-D). The anlagen of E. Ear

Bilateral auditory pits arise in the usual vertebrate fashion from ectodermal TABLE VII thickenings which invaginate to form the auditory vesicles (Rathke, 1866; The Origin of the Extrinsic Ocular Muscles in Alligator mississippiensis" Reese, 1908, 1915a). These thick-walled cavities lie close to the lateral walls of the hindbrain and open to the exterior. The development of parts of the Muscle Nerve Supply Origin middle ear has been studied in Alligator mississippiensis and compared with that of other reptiles and birds (Simonetta, 1956). The intertympanic canal The prema ndibular myocoele is apparently a remnant of the vault of the embryonic pharynx, which is cut The superior rectus Oculomotor From the dorso-medial off from the main part of the pharynx by the backward growth of the wall posterior flange of the parasphenoid. A small posterior diverticulum (ap­ The inferior rectus } From a common anlage of Oculomotor parently homologous with the pharyngeal pouches of many mammals) is The medial rectus the caudo-ventra-medial incorporated as a small appendix to the intertympanic canal (Simonetta, wall, above and slightly 1956). The structure and development of the derivatives of the tubotym­ medial to the inferior panic cavity, that is, the Eustachian tubes, the cavum tympani, the man­ oblique dibular recess, and the epitympanic recess have been described by The inferior oblique Oculomotor From the caudo-ventra-lat­ Simonetta (1956). The histogenesis of the crocodilian columella auris and eral wall, below and a lit­ other aspects of otic morphogenesis have been mentioned by Rathke tle lateral to the common (1866), Peter (1868), Hasse (1873), Van Beneden (1882), Parker (1883), Ret­ anlage of the inferior and medial recti zius (1884), Gadow (1888), Versluys (1903), Gaupp (1906), Shiino (1914), Goldby (1925), Kalin (1933), De Beer (1937), Wettstein (1937, 1954), Cordier The mandibular myocoele and Dalcq (1954), Guibe (1970c), and Frank and Smit (1974). The cartilages The superior oblique Trochlear Rostro-dorsal to the pre­ form part of the chondrocranium and their structure and development mandibular myocmere have been reviewed previously in this series (Iordansky, 1973; A. d'A. The hyoid myocoele Bellairs and Kamal, 1981). The structure and function of the crocodilian ear The abducens complex Caudo-dorsal to the pre­ have been reviewed by Baird (1970), A. d'A. Bellairs (1971), and Wever comprising:­ mandibular myocoele, the (1978). common anlage being in a horizontal plane at right F. EYB angles to the median plane The lateral rectus 1 From a postero lateral por­ During early development the eye is a conspicuous feature of the croco­ The r~tr~ctor palpebrae Abducens dilian face (Figs. 18-20). Its development has been described by Rathke supenons tion of the common anlage (1866), Voeltzkow (1899), Reese (1908, 1912, 1915a), Jolk (1923), and Wett­ The retractor bulbi 1 From an antero-medial stein (1954). The reptilian eye was reviewed by Underwood (1970), The retractor membranae Abducens portion of the common an­ whereas Walls (1942), Rochon-Duvigneaud (1954, 1970), and Crescitelli nictitantis lage (1977) reviewed vertebrate visual systems in general. All the available data suggest that the early morphogenesis of the crocodilian eye is essentially "Data from Wedin (1949, 1953); see Fig. 35. ORGANOGENESIS 431 430 REPRODUCTIVE SIOLOGY AND EMBRYOLOGY OF CROCODILIANS

NIV ! paper, the embryology of the crocodilian eye glands is completely un­ T known, although Rathke (1866) and Reese (1925) comment briefly on their structure in late embryos.

G. Chondrocranium and Osteocranium

The development and structure of the crocodilian chondrocranium and ,~i.' !. osteocranium have been reviewed respectively by Bellairs and Kamal I b (1981) and lordansky (1973) in this series. No experimental manipulations (d. Toerien, 1965, 1967; Toerien and Rossouw, 1977) have been performed l_~ on the developing crocodilian skull. A B H. Teeth

The first dental elements to form are rudimentary papillae called denticles (Rose, 1892a, b, 1894; Green, 1930; Deraniyagala, 1936, 1939; Edmund, 1962, 1969; Figs. 25A-I, 280 and F). Green (1930) described them as resem­ bling a selachian denticle and drew attention to the structural similarity between them and the egg tooth of Ornithorhynchus. The denticles arise as outgrowths from the oral epithelium and later differentiate into very small teeth, which make dentine but little or no enamel. They never become functional but sink into the mesenchyme and are resorbed. The dental lamina grows down near the posteromedial aspect of the trough of denticle epithelium, as the latter sinks beneath the oral epithelium (Westergaard and Ferguson, 1984 and in preparation). The sequence of appearance of the C D early tooth germs does not correspond to Zahnreihen sensu Edmund, 1962, Fig. 30. Alligator mississippiensis. (A) Diagrammatic figure of a 9-mm embryo illustrating the 1969 (Westergaard and Ferguson, 1984 and in preparation; also review in spatial disposition of the eye muscle anlagen relative to the eye, the premandibular myocoele Westergaard, 1980). (P.c., dotted) and various other cephalic structures. (After Wedin 1949, 1953.) (B) Schematic Subsequent development of the dentition was reviewed by Edmund figure of the eye muscle anlagen in a 13.5-mm embryo. (After Wedin, 1953.) (C) Sche­ (1962, 1969), and the few papers that have appeared since (Soule, 1967, matic figure of the eye muscle anlagen in a 20-mm embryo. (After Wedin, 1953.) (D) Schematic figure of the eye muscle anlagen in a 34-mm embryo. (After Wedin, 1953.) ABM, Abducens 1970; Miller, 1968; Miller and Radnor, 1970; Berkovitz and Sloan, 1979; musculature; DP!, depressor palpebrae inferioris; G. CIL, ciliary ganglion; GV, trigeminal Slavkin et al., 1982, 1984; Owens and Ferguson, 1982) emphasize the ganglion; 10, inferior oblique; IR, inferior rectus; LR, lateral rectus; MR, medial rectus; NIl, similarities between crocodilians and mammals. The stages of dental devel­ optic nerve; NIII, oculomotor nerve; NIV, trochlear nerve; NV!, abducens nerve; NO, opment are similar (Slavkin et al., 1984) and the enamel protein of Alligator notochord; OTO, otic vesicle; pc, premandibular myocoel; RB, retractor bulbi; RMN, retractor mississippiensis is immunologically cross reactive with mammalian enamel membrane nictitantis; RPS, retractor palpebae superioris; SO, superior oblique; SR, superior rectus. protein (Slavkin et al., 1982). Unlike the teeth of other reptiles, those of crocodilians are attached in the jaws by a periodontal ligament, and both its development and that of root cementum are similar to those in mam­ the muscles appear in the following sequence: (1) abducens, (2) superior mals (Soule, 1967, 1970; Berkovitz and Sloan, 1979; Owens and Ferguson, oblique, (3) inferior oblique, (4) superior rectus, (5) medial and inferior 1.982). Alligator tooth germs, explanted at the bell stage and cultured in recti, and (6) retractor membranae nictitantis (which arises much later than vitro on chemically defined media, develop satisfactorily (Ferguson and the others). Except for the fact that the inferior oblique arises before the Honig, unpublished data). Moreover, if these tooth germs are separated superior rectus, this sequence coincides with that seen in the chick (Adel­ into the outer dental epithelium and inner dental papilla mesenchyme and mann, 1927). The first muscle anlage to appear is not the first to reach its then recombined with the equivalent structures from developing mouse final position (see Fig. 30; Wedin, 1953). Despite the title of Meek's (1893) molars, the recombinants produce enamel and dentine (Ferguson and 432 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS ORGANOGENESIS 433 Honig, unpublished data). The harmonious development of chimaeric al­ ec ligator/mouse teeth indicates that the reciprocal epithelial-mesenchymal interactions involved in amelogenesis and dentinogenesis can operate across vertebrate classes. These results reinforce the contention that al­ ligator embryonic material is promising for the study of general problems in developmental biology.

I. Central Nervous System

The development of the crocodilian central nervous system has been de­ scribed by Rathke (1866), Parker (1883), Voeltzkow (1899, 1903a), Neu­ mayer (1906), Reese (1908, 191Oa, 1915a), Wettstein (1937), and Dalcq and Pasteels (1954). Pasteels (1970) and Senn (1979) review the literature on Reptilia in general, whereas Wettstein (1937), Cordier (1954), and Anthony (1970) describe the structure of the adult central nervous system and com­ j~~'PI1 ment on its development. The early development of the central nervous system, including the .:..i'-~ . closure of the neural folds and the fate of the notochord, was described in 1.ti~ m~ Section V. The anterior end of the neural canal enlarges sequentially to B Fig. 31. Alligator mississippiensis. (A) Sagittal section through the head of a 7-mm embryo form the vesicles of the forebrain, midbrain and hindbrain (Figs. 18-19). illustrating the early appearance of the paraphysis (P), velum (V), cerebellum (C), hypophysis The cranial flexure develops so that the huge midbrain forms a conspicu­ (H) and pharyngeal sac (PS). (After Neumayer, 1906, and Reese, 191Oa.) (B) Sagittal section ous "bulge" at the back of the head (Figs. 18-21, 31A and B). The devel­ through the head of a 13-mm embryo. Insert 1 shows a parasagittal section of the same oping brain cavity is large and retort-shaped with comparatively thick embryo. Note the vesicular appearance of the paraphysis (which lies like a tube between the walls of compactly arranged cells (Reese, 1908, 1915a; Fig. 31A). The walls forebrain and superficial ectoderm) and the forward growth of the velum into the lateral ventricle, where it eventually forms the choroid plexus. Insert 2 shows the hypophysis at this of the forebrain are quite thick, especially anteriorly; the wall of the mid­ stage of development. (After Reese, 191Oa.) C Cerebellum; CH, cerebral hemisphere; CP, brain is somewhat thinner, especially in the region of the cranial flexure, choroid plexus; FB, forebrain; H, hypophysis; HB, hind brain; HS, hypophyseal stalk; I, and it is still thinner posteriorly over the hindbrain (Fig. 31A; Reese, 1908, infundibulum; IP, infundibular pit; LV, lateral ventricle; M, mandible; MB, midbrain; NA, 1915a). Thus the dorsal wall of the hindbrain is reduced to a mere mem­ neuroporus; NT, notochord; 01, 02, 03, lateral diverticula of hypophysis; OC, optic chiasma; brane, whereas the ventral wall is also thin, but the lateral walls are thicker P, paraphysis; PC, posterior commissure; PS, pharyngeal sac; PV, post velar arch; TP, tuber­ culum posterius; TTR, torus transversus; V, velum. (Reese, 1908, 1915a). The hindbrain is wider but not as deep as the fore­ brain (Fig. 31A). In the acute angle caused by cranial flexure is the anterior end of the notochord (Fig. 31A), which is distinctly vacuolated by Stages 2 to 3. Commencing around Stage 5, the walls of the forebrain thicken bilat­ from a fold between mid and hindbrains (Figs. 31A and B). Migration of erally, later forming the cerebral hemispheres (Parker, 1883; Reese, 1908, nerve cells to the outer wall of the neopallium results in the formation of a 191Oa, 1915a; Figs. 31A and B). cerebral cortex. Differentiation in the walls of the brain begins with the formation of an The pineal (epiphysis) is absent (Voeltzkow, 1903a; Reese, 191Oa; inner granular and an outer clear zone, and cranial nerve fibers develop Krabbe, 1939; Hamasaki and Eder, 1977), so that the circulating melatonin from the base of the brain (Reese, 1908, 1915a; Van Campenhout, 1952). is presumably synthesized elsewhere, perhaps in the Harderian glands The median third ventricle has a thick ventral wall and a thin dorsal wall, (Roth et al., 1980). The large paraphysis was mistaken for an epiphysis extended to form a large paraphysis. The two lateral ventricles, the cavities (Fig. 31) by earlier workers. The development of the paraphysis has been of the cerebral hemispheres, have quite thick walls except on the side next described by Voeltzkow (1903a) and Reese (191Oa). In alligator embryos 7 to the third ventricle (Reese, 1908, 1915a). The forming sense organs are mm in length (stage uncertain), it is first seen as a wide evagination of the associated with various regions of the developing brain in a typical verte­ roof of the forebrain, just cephalad to the velum, forming a transverse brate fashion. Crocodilians apparently develop a white fibrillar stratum ventral depression of the dorsal wall of the forebrain (Fig. 31A). Posterior below the grainy layer of the cerebellum (Cordier, 1954), which itself arises to the velum, the roof of the forebrain is slightly arched to form the begin­ 434 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANB ORGANOGENEBIB 435 ning of the postvelar arch, which is limited posteriorly by a thickening of the dorsal wall, the future posterior commissure (Fig. 31A). Shortly after its about Stage 14. At this stage, whole body contralateral withdrawal reflexes appearance, the paraphyseal evagination becomes partially constricted can be elicited by gently touching the lateral flank or jaw margins or limbs from the brain and forms a rounded, hollow diverticulum, connected with of the embryo. As development progresses, such reflexes become more the forebrain cavity by a wide opening (Fig. 31B). Later, the paraphysis marked, more complex, and can be elicited for longer periods of time. At elongates so that in embryos 7-cm long (and older), it is seen as a tubular Stage 20, the body of the embryo may wriggle both away from and toward structure, with thin smooth walls, slightly curved away from the cerebral the stimulus, also the limbs may move. Jaw opening and closing reflexes hemispheres over the top of the diencephalon (Fig. 31B). Simultaneously, can be elicited and the embryo exhibits spontaneous movements including the velum increases in size and projects into the forebrain cavity as a swallowing. In general, the first reflexes or movements to appear embry­ transverse ridge, which ends in an acute posteriorly directed angle and a ologically can be elicited for progressively longer periods of time as devel­ thicker obtuse angle projecting forward under the paraphysis (Fig. 31B). opment progresses. Moreover, when eggs are opened the most recent Subsequently, vesicles lined by cuboidal epithelium appear in the velum reflexes or movements to appear developmentally disappear first as the and form the choroid plexus which projects into the lateral ventricle (Fig. embryo dies, whereas the developmentally earliest movements are the last 31B). In crocodilians, the fate of the cells that form the epiphysis in other ones to disappear. What effects these activities have on the embryology of animals is unknown. certain structures (such as joints and the gastrointestinal system) is un­ The hypophysis (pituitary) originates as an invagination of stomodaeal known for crocodilians, but has been described in other animals (Hum­ ectoderm (Rathke's pouch) beneath the floor of the forebrain (see Section phrey, 1968, 1969a, b, 1971a, b; Gottlieb, 1973; Oppenheim, 1973; Vince, 1973; Freeman and Vince, 1974). VIlA and Figs. 27B, 31A and B). The pharyngeal sac is a smaller, less definite invagination of thickened epithelium (Figs. 31A, -8; Reese, 1910a), that develops caudal to the hypophysis and apparently disappears without oJ. VertebrBe Bnd Ribs trace in older embryos (Reese, 1910a), although it may be associated with the development of the pharyngeal tonsil (Killian, 1888). The original hy­ Several accounts of the morphology and development of the crocodilian pophyseal invagination becomes the stalk of a branched hollow structure, vertebral column and ribs are available (Voeltzkow and D6derlein, 1901; which, by lengthening of the stalk, recedes to some distance from the roof Shipley and McBride, 1904; Shiino, 1914; Higgins, 1923; Goodrich, 1930; of the mouth (Fig. 31B) and loses its connection with the stomoda~al ec­ Emelianov, 1937; Wettstein, 1937; Mookerjee and Bhattacharya, 1948a, b; toderm and eventually loses its stalk (Reese, 1910a). During its develop­ Devillers, 1954a, b; Chiasson, 1962; Guibe, 1970f, g; Seidel, 1978). In the ment, the hypophyseal cavity consists of a central region and three out­ light of current knOWledge, some of these (e.g., Voeltzkow and D6derlein, growths; the largest extending back until it nearly reaches the notochord, 1901; Higgins, 1923) may be misleading, but the most relevant recent re­ the second extending in the same direction from the base of the hypophy­ views are those by Williams (1959) and Hoffstetter and Gasc (1969). seal stalk, and the third extending toward the floor of the infundibulum The ribs arise between two adjoining vertebral segments shortly after (Fig. 31B; Reese, 1910a). These outgrowths become more numerous and the part-sclerotomes have formed a series of paired sclerotomes along the the body of the hypophysis becomes completely solid as development body axis; each rib is said to derive from the scleroblastic cells of both the progresses (Reese, 1910a). original cranial and caudal part-sclerotomes (Higgins, 1923). The devel­ The body of the hypophysis is associated with a downward growth from oping ribs become associated with the neural arches (Higgins, 1923; the floor of the infundibulum (Fig. 31B). The adult crocodilian pituitary has Emelianov, 1937). As development progresses, centers of chondrification three lobes, two of which originate from the ectodermal hypophyseal in­ appear within the membranous ribs and extend throughout their length. vagination and one from the infundibular downgrowth (Guibe, 1970e). Whereas the developing ribs are associated with the vertebral column at o~e end, their main body lies between the epaXial and hypaxial muscles, Baumgartner (1916), Saint Girons (1970a, b), and Pasteels (1970) have de­ ~lth scribed the embryology, morphology, and cytology of the reptilian pitu­ the exception of the first pair (or ribs of the atlas vertebra), which lie itary, and these topics are considered elsewhere (Pearson, Chapter 9, this Internal to the hypaxial muscles and external to the peritoneal lining of the coelom (Mookerjee and Bhattacharya, 1948a, b). volume). The most comprehensive study of the development of the cranial nerves is that of Van Campenhout (1952) on Crocodlflus niloticus. Others The gastralia or abdominal ribs are said to originate by direct ossification (Shiino, 1914; Wettstein, 1937, 1954; Guibe, 1970d; Pasteels, 1970) provide of membranous centers situated in the subcutaneous tissue outside the occasional data on the development of the peripheral nervous system. three abdominal muscles (Voeltzkow and D6derlein, 1901). They insinuate The embryonic crocodilian central nervous system becomes functional t?emselves between these muscles secondarily and apparently do not de­ nve from ossification of the abdominal muscle tendons. 1436 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIANS ORGANOGENESIS 437 K. Respiratory System

The embryology of the upper respiratory tract is little known. Edgeworth (1907) suggests that the crocodilian laryngeal muscles form from an anlage in the second branchial arch, but gives no details. Soller (1931) described the hyoid apparatus, larynx and its musculature in late embryos of Alligator mississippiensis, Caiman crocodilus, and Crocodylus niloticus. The pharynx and trachea in late embryos of A. mississippiensis have been described by Moser (1902), Hesser (1905), Reese (191Oc, 1915a, b, 1926), Hilber (1932), Broman (1939), and Guibe (1970h). v In 30-somite (Stage 4) alligator embryos, the lung primordia arise from the ventral pharynx just caudad to the branchial clefts (Reese, 1915b). Later VI Fig. 32. Heart and aortic arches of a (circa Stage 14), the trachea separates from the esophagus and divides into FP J I . crocodilian embryo, diagrammed from the two bronchial primordia, both surrounded by mesoderm (Reese, 1915b; ventral aspect. I, II, III. IV. V, VI. First to Broman, 1939). When the branchial clefts close, there is a temporary clo­ sixth aortic arches; C, coeliac artery; CC. sure of the anterior end of the esophagus and trachea (Reese, 191Oc, 1915a, common carotid artery; DA, dorsal aorta; DC, dorsal carotid artery (right side nearly b, 1926); both subsequently regain their lumina, the trachea earlier than the atrophied); FP, ; LA, left esophagus. Lung rudiments each consist of an endothelial component di­ aortic arch; P, pulmonary artery; RA, right vided into three main lobes (each subdivided into numerous lobules) and a aortic arch; VC, ventral carotid artery; VS, mesodermal component with a smooth outline (Reese, 1915b). The lobules ventral subclavian artery (Modified from usually consist of a single layer of endoderm surrounded by a thin layer of Shipley and McBride. 1904; Shindo, 1914; Goodrich, 1930; De Beer. 1959; Pasteels. flattened mesoderm in which lie the pulmonary vascular anlagen (Reese, DA 1970.) 1915b). Condensations of mesoderm around the trachea and bronchi dif­ ferentiate into the cartilaginous rings and associated structures (Reese, 1910c, 1915b). considered in detail (Webb, 1979). The septum develops by the inward growth of the edge of the interventricular anlage from behind and above, from the septum aorticum (between the right and left systemic trunks), L. Cardiovascular System from the front, and also from the endocardial rudiment of the right septal valve (Hochstetter, 1906a; Goodrich, 1930). Although manifesting this "ad­ The primary organogenesis of this system is described by Reese (1908, vanced" cardiac condition (similar to that in birds) in its early develop­ 1915a). The heart, lined by a distinct endothelium, appears first as a slight ment, the crocodilian heart still shows the sharp, double flexure of the bulge to the right of the neural canal just anterior to the first somite. conus seen in dipnoans (Hochstetter, 1906a; Kerr, 1919). Likewise, the Vitelline vessels also occur at this time. The initially thin mesodermal wall lateral valve of the right atrioventricular orifice becomes muscular; this increases in thickness as the muscle columns differentiate; they are particu­ feature is absent in mammals, but exaggerated in birds (Shaner, 1924; larly conspicuous ventrally in the presumptive ventricles. Further develop­ GOodrich, 1930). Crocodilians retain the left aortic arch (Fig. 32), which ment of the heart has been described by Rathke (1866). Greil (1903), Ship­ only OCCurs as an abnormality in birds (Goodrich, 1930). Thus, in crocodil­ ley and McBride (1904), Felix (1906a), Hochstetter (1906a, c). Kerr (1919), ians, the right aortic arch (carrying oxygenated blood) arises from the left Goodrich (1930), Stephan (1954), Wettstein (1954), White (1968, 1970), Ventricle, whereas the left aortic arch and pulmonary trunk arise from the Springer (1970), and Guibe (1970i). Hochstetter's (1906a) account is the right ventricle (Fig. 32). The right and left aortic arches communicate via most comprehensive and Goodrich (1930) described crocodilian cardiac the foramen of Panizza (Fig. 32), which is located near the base of the development in a phylogenetic context. Systemic trunks, just anterior to the semilunar valves (Shaner, 1924; Kerr, Although the heart of most reptiles is three chambered, that of croco­ 1919; Goodrich, 1930; Webb, 1979). This foramen apparently arises as a dilians is four chambered, due to the development of a complete interven­ tricular septum (Beddard and Mitchell, 1895; Shaner, 1924; Goodrich, 1930; secondary perforation of the aortic septum comparatively late in develop­ Wettstein, 1954; Foxon, 1955; Bellairs and Attridge, 1975; Webb, 1979). The ment, just before the closing of the interventricular foramen (Grei!, 1903). evolution of the crocodilian and avian interventricular septum have been The development of the arterial and venous systems has been described by Balfour (1881), Zuckerkandl (1895a, b), Voeltzkow (1901), Shipley and 143S REPRODUCTIVE BIOLOGY AND EMSRYOLOGY OF CROCODILIANS ORGANOGENESIS 439 cBride (1904), Hochstetter (1906a, c), Reese (1908, 1915a), Shiino (1914), _...... - ...... \ Shindo (1914), Kerr (1919), Goodrich (1930), Stephan (1954), Wettstein --- \ (1954), DeBeer (1959), Guibe (1970i), and Bellairs and Attridge (1975), The ,/ , fates of the embryonic aortic arches are illustrated in Fig. 32. The right ,,/ , ,, " , I I dorsal carotids atrophy almost completely, and frequently the left common « I P carotid artery gives rise to the dorsal and ventral carotids of both sides. The right and left 4th aortic arches fuse to form the dorsal aorta, whereas the °n/Nf))';:'"~ ('.~'--"', .: T Fig. 33. Alligator mississippicllsis. Diagram 6th aortic arches become the pulmonary arteries (Fig, 32). The anterior : \ )' of a reconstruction of the embryonic gastro­ : " . "'"J .••' ./ cardinal veins lie adjacent to the notochord and unite with the posterior I intestinal system approXimately 3 weeks af­ cardinal veins to form the Ducti Cuvieri, which in turn connects with the ter egg deposition. Digestive canal shown , •.••• ' \ D in solid black, glands in solid lines and em. meatus venosus of the liver (Reese, 1908, 1910c, 1915a). The adult lym­ LU~.!.e.;.. ,.\ \ PG 5 : ..... ,..::..;';, ....•. bryo outline in dotted lines (after Reese phatic system was described by Ottaviani and Tazzi (1977), but its develop­ , . 1910). A, Allantois; C, cloaca; D, ment is poorly understood. \ --~ ,--:-, Pl duodenum; E, eye; H, hindgut; L, liver; H \ 1M .=-,' A LU, lung; M, mouth, O. oesophagus; P, \ ~'/ C M. Diaphragm '.... , ,. , // pharynx; PA, pancreas; PC, post-anal gut , ~ , ~ of Reese (1910); PL, posterior limb bud; S, ... _------­ stomach; T, trachea; Y, yolk sac. Crocodilians are unique among reptiles in having thoracic and abdominal cavities separated by a structure analogous to the mammalian diaphragm, but which has been little studied (Butler, 1889; Hochstetter, 1906b; Good­ rich, 1930). The diaphragm apparently develops from the secondary fusion of many parts: the posthepatic septum (including the ventral oblique he­ patic ligament), the ventral pulmonary fold, and parts of the gastric mesen­ tery. Along its edge, the diaphragm is associated with many of the back muscles. The large diaphragmaticus muscle runs from the liver to the pelvis and assists the costal breathing mechanism by retracting the liver and expanding the pleural cavity by stretching the diaphragm thus power­ ing inspiration (Gans and Clark, 1976). The diaphragm also separates off eight coelomic recesses and cavities (Hochstetter, 1906b).

N. Gastrointestinal System

The most extensive accounts of the development of the gastrointestinal system are those of Reese (191Oc, 1913c) but some comments are given by Rathke (1866), Clarke (1891), Voeltzkow (1899), Reese (1908, 1912, 1915a, 1926), Grunwald (1931), Wettstein (1954), Elias (1955), Arvy and Bonichon (1958), Guibe (1970b), Gabe (1970b), Miller and Lagios (1970), and Skozylas (1978). After early development (Section V), the foregut is a shallow enclosure of the anterior endoderm (Figs. l3C and D) and the blastopore connects the hindgut region with the exterior. Subsequently (Figs. 14 and 15), the fore­ gut becomes a cavity wider laterally than dorsoventrally and extends only to about the cranial third of the trunk, opening onto the yolk sac (Reese, 191Oc). With the appearance of the definitive hindgut, the anterior and posterior intestinal portals are identifiable where the unenclosed midgut joins the foregut and hindgut (Reese, 191Oc). 440 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS ORGANOGENESIS 44'1 The liver first appears as an endodermal diverticulum from the ventral nephric duct arises from a mesodermal blastema closely related to the foregut surrounded by splanchnic mesoderm (Fig. 33; Reese, 1910c; Elias, pronephros (Reese, 1908, 1912, 1915a) and grows posteriorly receiving 1955). A small projection from the wall of the duodenum differentiates into mesonephric tubules as they form. The same coelomic epithelium, that the bile duct, which ends blindly on the anteroventral edge of the liver and gave rise to the mesonephros, also forms the metanephros. The meta­ is closely related to the pancreatic duct. The pancreas derives from one nephros develops convoluted tubules, glomeruli, and gives origin to the dorsal and two ventral diverticula of the foregut, which evaginate just ureters, which join the mesonephric ducts before the latter open into anterior to the hepatic rudiments (Reese, 1910c; Gabe, 1970b; Miller and the cloaca (Forbes, 1940a; Fox, 1977). Lagios, 1970). Their subsequent fate is unknown, but in the late embryo Forbes (1940a) described three phases in gonadal development in Al­ the pancreas lies between the posterior end of the stomach and the anterior ligator mississippiensis: the period of genital ridge formation, the period of end of the duodenum and opens into the latter by two or more short ducts bisexuality, and the period of visible sex differentiation. The following (Reese, 1910c). summary derives primarily from that study. No information is available on the origin and migration of the germ cells in crocodilians. The genital ridge o. UrogenitBI System and Sex Determination is seen first as a thickening of the coelomic epithelium on the ventral and ventromedial surface of the mesonephros, continuous medially with the The literature on the embryology and structure of crocodilian urogenital primordium of the adrenal cortex. This epithelium becomes organized into systems is large (Remak, 1845, 1855; Rathke, 1866; Wiedersheim, 1890a, b, primary sex cords, rete cords, and germ cells. Embryos of 75-88-mm head­ 1891, 1897; VoeItzkow, 1892, 1899; Wilson, 1896, 1900; Szakall, 1899; Felix, trunk length (exact stages uncertain) show a period of bisexuality when 1906b; Loyez, 1905, 1906; Reese, 1908, 1912, 1915c, 1924; Moens, 1912; both male and female primitive gonadal components are present (Forbes, Taguchi, 1920; Forbes, 1934, 1937a, b, 1938a, b, c, 1939, 1940a, b, 1961; 1940a, 1964). The gonad has a well-defined outer layer of simple germinal Gerrard, 1954; Wettstein, 1954; Godet, 1961; Forsburg and Olivecrona, epithelium and a subjacent zone of medullary cords. Subsequently, visible 1963; Ramaswami and Jacobi, 1965; Guibe, 1970j; Pasteels, 1970; Fox, 1977; sex differentiation takes place. In ovaries, a well-defined germinal epi­ Ferguson and ]oanen, 1982, 1983; Webb and Smith, 1984) and has been thelium develops in which the germ cells are fairly numerous, whereas the reviewed by Fox (1977). Here follows a more detailed account of events in medullary cords regress and are distended by large irregular cavities. The crocodilians with particular emphasis on data related to temperature de­ testes retain limited cortical areas external to the tunica albuginea, whereas pendent sex determination. It is based on a number of publications and on the medullary cords differentiate as seminiferous tubules containing germ my observations on Alligator mississippiensis, Crocodylus johnsoni, and C. cells. Both males and females exhibit a degree of embryonic bisexuality; porosus. Good descriptions and illustrations of the developing gonads and immature females retain Wolffian ducts and a small mass of testis-like Mullerian ducts in A. mississippiensis are available (Forbes, 1940a) as are medullary tissue (the medullary rest) in the ovary; immature males may data on the histology of hatchling gonads and reproductive ducts (Fergu­ retain variable testicular cortical areas and occasional Mullerian duct seg­ son and ]oanen, 1983). Histological data are also available for hatchling C. ments (Forbes, 1934, 1937a, b, 1938a, b, c, 1939, 1940a, b, 1961, 1964; johnsoni (Webb and Smith, 1984). Ferguson and ]oanen, 1983). The first indication (at approximately Stage 1) of the development of the Controversy regarding the origin of the Mullerian ducts was reviewed excretory system is the appearance of approximately six ciliated tubules by Fox (1977: 52); the generally accepted view is that of Forbes (1940a) and opening into the anterior coelom (Wiedersheim, 1890a, b, 1891). On either Forsburg and Olivecrona (1963). In the female, the Mullerian ducts become side of this pronephros a large vascular coil covered by coelomic epi­ the OViducts, whereas in the male they remain until around the time of thelium appears and forms the muItilobate glomus. Later, the pronephros hatching when they disintegrate except for short rudimentary segments, degenerates as the mesonephros originates from nephrotomes 11-23 which may persist throughout life (Forbes, 1940a, Ferguson and ]oanen, (which begin posterior to the pronephros and extend to the anterior clo­ 1983). Vestiges at the anterior poles of the testes are common and appar­ acal border). There is no clear demarcation between pronephros and ently homologous with the mammalian appendix testis (Forbes, 1940a). mesonephros (Wiedersheim, 1890a, b, 1891). The mesonephros develops There is no information available on the development of oviducal glands branched tubules (with internal Malpighian corpuscles) which do not open (Such as albumen secreting and shell glands). into the coelom (Wiedersheim, 1890a, b, 1891; Reese, 1908, 1912, 1915a). With degeneration of most of the embryonic mesonephros, the Wolffian They function as collecting ducts through embryogeny and for some time duct has little role in excretion, but it persists in both sexes (Ferguson and posthatching (Forbes, 1940a), but they ultimately become transformed into Joanen, 1983). In males, the ends of the Wolffian ducts closest to the testes epididymal structures in the male or disappear in the female. The meso­ are mUch coiled and form the epididymis in conjunction with persistent 442 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIANS mesonephric tubules. The posterior Wolffian ducts form the ducti defe­ rens, which open into the cloaca near the base of a groove that runs along the upper surface of the single penis (Moens, 1912; Reese, 1924; Neal and Rand, 1936; Bellairs and Attridge, 1975). The development of the croco­ dilian clitero-penis has been described by Rathke (1866), Voeltzkow (1892, 1899), Moens (1912), and Reese (1924). It grows out from the ventral ab­ dominal wall, anterior to and distinct from the cloaca. As the cloacal lips develop, the clitero-penis withdraws between them and differentiates into its adult component parts (Figs. 34A-H). Anal musk glands and abdominal pores arise by epithelial invagination (Reese, 1921, 1924). Although sex is fully determined and the gonads are differentiated at the time of hatching in all crocodilian species investigated (Ferguson and Joanen, 1982, 1983), the degree of differentiation of the external genitalia is variable. Sexing by cloacal examination is impossible in Alligator mississip­ piensis (Joanen and McNease, 1978) or in Cavialis gangeticus (Singh, per­ sonal communication) less than 0.6 m in length. However, male and fe­ male Crocodylus porosus, C. johnsoni, and C. niloticus can be distinguished at hatching (Webb et aI., 1983c; Hutton, personal communication). These differences may be the result of the shorter incubation times of some species, for example, A. mississippiensis (see Table III) as suggested by Webb et a1. (1983c), but this would not explain the etiology in C. gangeticus and possibly other crocodilians. Striking differences in the degree of differ­ entiation of the external genitalia and in the size of the latter are present from Stage 7 and marked after Stage 14 (Figs. 34A-H) between A. missis­ sippiensis, C. johnsoni, and C. porosus. Thus, development of the external genitalia may be different from the onset in alligators and crocodiles. The influence of incubation temperatures on the development of the external genitalia is unknown, as is the influence of the degree of differentiation of the external genitalia at hatching (and their subsequent rate of develop­ ment) on the age or size at which sexual maturity is attained in various crocodilians Cfable I). Perhaps the onset of gametogenesis is only one of the factors that determine sexual maturation. In Alligator mississippiensis, both laboratory and field experiments have shown that incubation temperature determines the sex of the hatchling I and hence of the adult (Ferguson and Joanen, 1982, 1983), and this is Fig. 34. Crocodylus niloticus. Drawings of the development of the external genitalia (after confirmed by preliminary data for Crocodylus johnsoni (Webb et aI., 1983e; Voeltzkow, 1899, 1901). (A, B) Stage 10. Ventral and lateral views. (C, D) Stage 13. Ventral and Webb and Smith, 1984), C. porosus (Webb, personal communication) and C. lateral views. (E, F) Stage 19. Ventral and lateral views. (G, H) Stage 25. Ventral and lateral niloticus (Hutton, personal communication). Temperature dependent sex views. (I) Higher power view of the cross-section of the umbilical stalk seen in E. A, Allantois; determination is Widespread among turtles and lizards (Bull, 1980). That G, gut loops; GA, genital anlage; GB, genital bulb (glans in male); GF, genital fold; GS, shaft of genital primordium; L, limb buds; 0, omphalo-mesenteric artery; U, umbilicus; UA, umbilical heteromorphic sex chromosomes appear to be absent from all living artery; UV, umbilical vein. crocodilians studied to date (21 out of the 29 species) (Cohen and Gans, 1970) makes it likely that all crocodilian species have a temperature depen­ dent mechanism. Female alligators are HY pOSitive, similar to snakes and turtles (V. Lance, personal communication). In Alligator mississippiensis, incubation of eggs at 340C produces only 444 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS ORGANOGENESIS 445 males, whereas incubation at 30°C or below produces only females (Fergu­ is Stage 22 (Bull and Vogt, 1981). Double shift experiments in which a pulse son and Joanen, 1982, 1983). At 32°C, the sex ratio varies from 87% fe­ of one temperature occurs on a background of the other revealed that the males: 13% males to 100% males, whereas the sex ratio is approximately time required to produce males (25°C) is less than to produce females 50% females:50% males at 31°C (Ferguson and Joanen, 1982, 1983, unpub­ (31°C) (Bull and Vogt, 1981). Therefore in Alligator mississippiensis, C. lished observations). Similarly, in Crocodylus niloticus (Hutton, personal porosus, and emydid turtles, cooler temperatures act earlier and more rap­ communication) incubation at 34°C produces males, and at 30°C, it pro­ idly in determining sex (females in crocodilians, males in turtles). duces females. In C. porosus (Webb and Smith, 1984; Webb, personal com­ The situation is more complex in biomodal species with two temperature munication) incubation at 32°C produces 100% males and at 30°C 100% thresholds. No data are available for Crocodylus johns toni but the TSPs of females; 34°C incubation causes a high mortality of C. porosus eggs. Despite Chelydra serpentina are between Yntema Stages 16 and 20 for shifts from 26° this apparent uniformity amongst species, there is at least one exception. to 30°C, Stages 12 and 15 for shifts from 30° to 26°C, Stages 13 and 17 for In C. johnsoni, incubation at 34°C produces 100% females, at 31°-33°C shifts from 20° to 26°C, and Stages 12 and 18 for shifts from 26° to 20°C males (in various ratios), and at 26°-30°C 100% females (Webb and Smith, (Yntema, 1979). Thus, it appears that less incubation at 30°C (to Yntema 1984). This bimodal pattern of production of females at both high and low Stage 16) is required to determine femaleness than is required at 26°C (to temperatures and males at intermediate temperatures is similar to the situ­ Yntema Stage 19) to determine maleness. Embryos of C. serpentina, in­ ation in Chelydra serpentina (Yntema, 1976, 1979), which itself contrasts to cubated at the male producing temperatures of 22° or 24°C must be ex­ the normal turtle pattern in which high temperatures produce females and posed to 30°C for at least four hours per day in order to ensure female low temperatures produce males (Bull and Vogt, 1979, 1981; Bull, 1980). In development (Wilhoft et al., 1983). all experiments differential embryonic mortality has been eliminated as a To date the data on temperature sensitive periods for sex determination factor producing the skewed sex ratios, which have also been recorded in (in both crocodilians and turtles) have remained puzzling and no one has natural nests in different environments (Ferguson and Joanen, 1982, 1983; satisfactorily explained them or assimilated them into any model of the Webb and Smith, 1984) and in adult populations (see Table II). mechanism for temperature dependent sex determination. Part of the rea­ In an attempt to define the temperature sensitive period (TSP) for sex son for this may have been the tendency to assume that the TSP equates determination, experiments have been performed involving the shift of with the time of embryonic sex determination (which would therefore have eggs from an all male to an all female producing temperature and vice to occur over at least 2 to 3 widely different stages of development). This versa at various times of development. If eggs of Alligator mississippiensis assumption may be erroneous, and the concepts and nomenclature of TSPs are shifted from 30° to 34°C, the TSP is between days 14 and 21 (Stages 13­ and even temperature-dependent sex determination itself may be mislead­ 16), but if eggs are shifted from 34° to 30°C, the TSP is between days 28 and ing, and the term gonadal size-dependent sex determination may reflect 35 (Stages 20-21); (Ferguson and Joanen, 1983). Thus, the temperature of more accurately the underlying biological processes. A new theory regard­ egg incubation prior to the TSP (run-in temperature) does influence sex ing these phenomena has been proposed (Ferguson, submitted). determination but not irreversibly. The relationship is opposite to what one might expect in that the higher run-in temperature (which accelerates P. Endocrine Glands macroscopic external development) delays the TSP and embryos remain sexually labile for much longer. The development of the reptilian pituitary gland is reviewed by Pearson In Crocodylus porosus (Webb, personal communication) shifts from 30° to (Chapter 9, this volume); that of crocodilians is discussed in Section VII.I. 32°C give a TSP between 20 and 25 days (Stages 15-18), whereas shifts The development of the thyroid gland resembles the basic vertebrate from 32° to 30°C give a TSP between 40 and 45 days (Stage 23). The TSP of pattern. With the approximation of the mandibular arches, a deep narrow C. johnsoni is poorly documented, but preliminary data for shifts from 31° groove is formed in the anterior floor of the pharynx; it is uncertain to 30°C put it between 20 and 30 days (Stages 15-20) (Webb and Smith, Whether this location implies a first pouch origin. The epithelium of the 1984). The embryonic stages during the TSPs both for switches of high to caudal part of this groove thickens ventrally and invaginates to form the low or low to high temperature are remarkably similar in these three anlage of the thyroid gland. The gland becomes cut off from the pharynx species. Moreover, the stages are similar to the equivalent ones for Chelydra and migrates caudally in the form of solid cords, which break up into (Yntema, 1968); the TSP for shifts of the eggs of emydid turtles from 25°C groups of cells that form the primary follicles. These then become encap­ (male producing temperature) to 31°C (female producing temperature) oc­ sulated to form the adult gland (Reese, 1908, 191Oa, c, 1915a, 1931b; Lynn, curs at Stage 16 (Yntema, 1968), whereas the TSP in shifts from 31° to 25°C 1960. 1970; Gabe and Saint Girons, 1970a). qq& REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCODILIANS ORGANOGENEBIS qq7 The parathyroids, like those of reptiles in general, are derived from branchial pouches three and four (Gabe and Saint Girons, 1970b). Van (1939), Kniisel (1944), Devillers (1954c), De Beer (1959), Guibe (1970g, k), Bemmelen (1886, 1888) described a single pair of parathyroids derived from Pasteels (1970), Walker (1972) and Honig (1984). Rathke (1866), Voeltzkow pouch three in late embryonic and adult specimens of Alligator mississip­ (1899), Reese (1908, 1915a), Wettstein (1937, 1954), Deraniyagala (1939), piensis and Crocodylus porosus. Hammar (1937) observed two pairs of and Ramaswami (1946) mention some features of tail development. parathyroid glands in young embryos of C. porosus, but noted that the pair In Alligator mississippiensis, the limb buds first appear about Stage 6 from pouch four disappeared in adults, a finding supported by the work of (Figs. 23 and 35). The posterior buds develop faster than the anterior, at Clarke (1970) on Caiman crocodilus. The glands derived from pouch four are least during the early stages of their embryology, contrary to Reese (1915a). small, and it is probable that they fuse with those from pouch three during The chronology of limb development is outlined in Section VI and is dia­ late development (Clarke, 1970). The parathyroids are not associated with gramed in Fig. 23. The limb buds develop distinct apical ectodermal ridges the thyroid glands but are located in or near the thymus, in dose proximity (Figs. 23 and 35A-H). Grafting experiments within alligator embryos and to their embryonic relationship with the third and fourth aortic arches. between alligator and chicken embryos (Honig, 1984) have revealed that In Alligator mississippiensis, the primitive adrenal cortex arises from a the apical ectodermal ridge and zones of polarizing activity (ZPA) regulate thickened mass of coelomic epithelium lying on the ventromedial surface differentiation as has been shown in other tetrapods (see reviews in Fallon of the mesonephros and extending laterally from the dorsal mesentery to and Caplan, 1983). These experimental digit duplications resemble those the genital primordium (Forbes, 1940a; Gabe, 1970a; Martoja, 1970). seen as natural abnormalities in the wild (Fig. 36L). Normally, there are Shortly after its appearance, buds arise and extend rapidly as cortical cords five digits on the forelimb, but only four on the hindlimb (Fig. 23). Claws into the mesenchyme between the mesonephros and aorta. Adrenal med­ differentiate on the tips of three digits of both limbs, but are absent from ullary tissue appears as aggregations of basophilic cells in the mesenchyme the two outer digits of the forelimb and the one outer digit of the hindlimb dorsal to the cortical cords and medial to the mesonephros. It migrates (Fig. 23). The ventral concaVity of the embryonic claw is filled by the ventrally into the cortical mass (Lawton, 1937; Forbes, 1940a). neonychial pad, a soft rounded cushion formed by a thickening of the epidermis superficial to the sole of the claw (Kerr, 1919). These structures G. Thymus and Immune System prevent the embryo from tearing its membranes as it moves about during late development; they detach shortly after hatching, leaving behind the The observations available on the thymus are mainly topographic descrip­ functional claws, the morphogenesis of which has not been reported. tions of its size and position in late embryos and young adults, in which it Explanted limb buds that are grown either in vitro or as grafts on the occupies much of the total length of the neck (Rathke, 1866; Van Bemme­ chick chorioallantoic membrane show good mesenchymal differentiation, len, 1888; Hammar, 1909; Reese, 1908, 191Oc, 1915a; Pischinger, 1937; particularly in the cartilages (Honig and Ferguson, unpublished data). Wettstein, 1954; Bockman, 1970). Histological descriptions of the early thy­ The development of skeletal elements in the limb has been described by mus report nothing about its development (Dustin, 1914; Taguchi, 1920; Kiikenthal (1893), Voeltzkow (1899), and Steiner (1934). There is a similar­ Wettstein, 1954; Bockman, 1970). The crocodilian thymus, like that in other ity between avian and crocodilian embryos as regards the lateral deflexion reptiles, derives from the branchial pouches (Reese, 191Oc), but precisely of the wrist-hand axis from that of the forearm and in the reduction of the which ones is unknown (Bockman, 1970), although the second has been two outer digits. The tarsus of the avian embryo has a distinctly crocodilian suggested (Reese, 191Oc). Study of the development of the structure and appearance, with a dorsolateral process of the astragalus contacting the function of the crocodilian thymus, which is known to be involved with lower end of the fibula above the calcaneum (Walker, 1972). In the embryo, adaptive immunity (Bockman, 1970), would be of great interest as a com­ the iliac, pubic, and ischiadic cartilages are in continuity and the ace­ parison with the avian immune response, which involves the unique Bursa tabulum is closed. The piercing of the acetabulum and separation of the of Fabricius. Bursa equivalents in crocodilians remain to be investigated. pubis take place late in development (Goodrich, 1930). Little is known about tail development (Figs. 18-22) apart from com­ R. Limbs and Tail ~ents on its changing relative length. Deraniyagala (1939) described a highly vascularized terminal hook (or kink) on the tail of embryos of Aspects of the development of crocodilian limbs have been described by Crocodylus porosus, which arises by fusion of the last three rings of caudal Rathke (1866), Kiikenthal (1893), Zuckerkandl (1895a, b), Voeltzkow scales. This hook is clearly evident in Alligator mississippiensis, C. porosus, (1899), Goeldi (1900), Braus (1906), Reese (1908, 1915a), Kerr (1919), Tornier and C. johnstoni (Figs. 18-22). Ramaswami (1946) reported that the kink (1928), Goodrich (1930), Steiner (1934), Wettstein (1937), Deraniyagala Contains only connective tissue cells and concluded that it was a transient feature of unknown function, but Deraniyagala (1939) hypothesized that Fig. 36. Alligator mississippiensis. Details of the caruncle. (A) Dorsal view of the snout of a hatchling illustrating the position of the caruncle. (B) Scanning electron micrograph of hatch­ ling, note the smooth pedicle and bifid points of the caruncle, that are used to slit the eggshell membranes. (C) Stage 24. Coronal histological section of the caruncle. Note the vascular loose fibrous central core and the enlarged strateum corneum. (D) Stage 28. Coronal histological section. Note the denser avascular fibrous core and the greatly enlarged strateum corneum.

(day 9). Lateral SEM view of the forelimb bud. Note that it lacks an apical ectodermal ridge Fig. 35. Alligator mississippiensis. Early limb development (compare with Fig. 23.). (A) Stage 7 and is less well developed than the hindlimb bud. (E) Edge on SEM view of the hindlimb bud (day 7). Scanning electron micrograph (SEM) of the hindlimb bud (H). Note also the devel­ depicted in C. Note the apical ectodermal ridge (arrowed). (F) Edge on SEM view of the oping tail (T) and allantois (A). (B) Stage 11 (day 12). Histological section of the apical ectoder­ forelimb bud depicted in D. The apical ectodermal ridge is not apparent. (G) Stage 10 (day 10). mal ridge (AER) on the forelimb bud. Photograph courtesy of Dr. J. Fallon. (C) Stage 9 (day 9). SEM of a hindlimb bud. Note the apical ectodermal ridge (arrowed). (H) Stage 10 (day 10). Lateral SEM view of the hindlimb bud. Note the apical ectodermal ridge (AER). (D) Stage 9 SEM of a forelimb bud. Note the apical ectodermal ridge (arrowed). DEVELOPMENTAL ABNORMALITIES 450 REPRODUCTIVE BIOLOGY AND EMBRYOLDGY OF CROCODILIANS 4/51 its hypertrophy could have produced the terminal caudal fin of some ex­ unrelated to the dentition. In this regard, it is very different from the true tinct crocodiles. "egg teeth" of the Squamata, which are formed as part of the premaxillary dentition (De Beer, 1949; Fioroni, 1962; Edmund, 1969; Guibe, 1970m). S. Integument and Its Glands Some minor confusion may result when the crocodilian caruncle is called the "egg tooth" by field workers (e.g., McIlhenny, 1935; Deraniyagala, Information on reptilian integumentary embryology is reviewed by Mader­ 1936, 1939; Pooley, 1962, 1969a; Neill, 1971; Singh, 1975). son (Chapter 7, this volume). The most detailed description for croco­ In Crocodylus niloticus, C. porosus, C. johnsoni, and Alligator mississippien­ dilians is by Voeltzkow (1899) who also reports the development of the sis, the caruncle first appears at Stage 15 as a pair of epithelial outgrowths integumentary glands and bony scutes (osteoderms). The latter have been on the tip of the snout (Sluiter, 1893; Voeltzkow, 1899; Fig. 26A). By Stage extensively described by Schmidt (1914), and much of their development 20, these have approximated, and the tissue between them has grown out occurs posthatching. According to Deraniyagala (1939), embryos of to form a single consolidated mass (Figs. 26B and C). The ensemble forms a Crocodylus porosus form scales about 26 days after egg laying; the process is lightly pigmented horny outgrowth terminating in two points (Fig. 260). It largely complete by 45 days when pigmentation apparently begins. The has been suggested that the caruncle forms entirely by a thickening of the macroscopic appearance and chronology of scale formation are described strateum corneum (Rose, 1892a; Sluiter, 1893; Voeltzkow, 1899; Fioroni, in Section VI. There is substantial variation in the coloration pattern of 1962; Guibe, 1970m) in which calcium salts may be deposited (Fioroni, hatchling alligators, the yellow stripes being variable in both form and 1962). However, coronal sections (Figs. 36C, D) of the caruncle in A. missis­ position. We do not know what determines this pattern and how it sippiensis show a previously overlooked sizable fibrous, dermal core, in changes with growth and development. addition to the hyperplastic stratum corneum. Initially this dermal core is Data on the development of the various types of integumentary glands composed of loosely arranged fibroblasts and numerous blood vessels (Fig. indicate only that these arise as invaginations of ectoderm into the dermis 36C). (Reese, 1921); the surrounding mesenchyme forms gland capsules of con­ As development proceeds the dermal core becomes less vascular and nective tissue and muscle (Reese, 1921). more fibrous; at hatching it is almost completely fibrous (Fig. 360). Post­ hatching, this fibrosis continues until the caruncle breaks off, a process T. Caruncle facilitated by epithelial ingrowths at the base and the diminished blood supply in the dermal core. The epithelial ingrowths then establish a normal The small caruncle facilitates slitting of the extraembryonic and shell mem­ epidermis. In parallel with the changes of the connective tissue, the branes during hatching. It has been described for most crocodilians keratinocytes of the stratum corneum of the developing caruncle become (Voeltzkow, 1891, 1892, 1893, 1899; Pooley, 1962, 1969a; Deraniyagala, more numerous and densely packed as keratinization proceeds (Figs. 36C, 1936, 1939; McIlhenny, 1935; Joanen, 1969; Singh, 1975). At the time of D). The thickness of the peridermal layer decreases but the distinctive hatching, it is a small hard structure (approximately 1 mm in length and 2 interface between periderm and stratum corneum persists (Figs. 36C, D). mm in diameter) located at the tip of the snout (Figs. 36A, B). It is bifid and The presence of a fibrous core seems logical because an unsupported pile ends in two sharp points; as the embryo "nods" its head in ovo, these rub of dead epidermal cells, 2-mm broad by 1-mm high, would be weak and against, pierce, and slit the enclosing membranes (Figs. 26G, H, I, 36A and prone to breakage, unlike the actual condition at the time of hatching. The B). The chronology of its macroscopic development is described in Section crocodilian caruncle with its fibrous core is not dissimilar to that of mono­ VI and illustrated in Fig. 26. It is initiated (and present histologically) at tremes in which the core contains a nodule of bone (Hill and De Beer, 1949) Stage 15 and becomes evident at Stage 16. It persists for approximately two so that the two are more likely to be homologous than has hitherto been weeks posthatching, then falls off leaving a scar that later heals. Hatchlings assumed (De Beer, 1949). show a 1-2 mm "halo" of smooth and lightly pitted integument around the base of the caruncle (Figs. 36A and B). The structure and development of the caruncle were first described in VIII. DEVELOPMENTAL ABNORMALITIES Crocodylus porosus (Sluiter, 1893), and this work formed the basis for subse­ quent descriptions and illustrations (Voeltzkow, 1899; De Beer, 1949; Case reports garnered from the literature are listed in Table VIII. In most Fioroni, 1962; Edmund, 1969; Guibe, 1970m). The caruncle of crocodilians, cases, the etiology and pathogenesis of such malformations are unknown. like that of Testudines, Sphenodon and birds, is epidermal and is formed Comparable summaries exist for lizards, snakes, and turtles (A. d'A. Bel­ principally by a hyperplastic stratum corneum so that it is structurally lairs, 1981; Ewert, 1979). ~, ' TABLE VIII TABLE VIII A Systematic Catalogue of Reported Developmental Abnormalities (Continued) in Crocodilians

Type oi Maliormation Species Notes Reference Type oi Maliormation Species Notes Reierence (b) cyclopia A. mississippiensis Teratogen induced Fig. 37c; Ferguson Double yolks A. mississippiensis Often laid first or Ferguson and Joanen and spontaneous (l982b, 1984) O. tetraspis last by young ie­ (1983) Tryon (1980) (c) anophthalmia A. mississippiensis males Spontaneous Ferguson (unpublished C niloticus Blomberg (1979) data) C. palustris C. aculus Neill (1971) Whitaker and Whitaker C. POroSU5 Webb et a1. (1983b) (1976a, b) Twins A. mississippiensis Stage 1 and Stage 25 Reese (1906); Joanen C. niloticus Pooley (1969a) (personal communica­ C. gangeticus Deiective develop­ Singh and Tandan tion) ment oi optic (1978); Subba-Rao and o tetraspis Hatchlings Tryon (1980) placodes and Bustard (1979); Singh C. nilolicus Hutton and Loveridge vesicle and Bustard (1982) (d) exophthalmia (personal communica­ A. mississippiensis Ferguson (1981a. 1982b, tion) 1984) (e) corneal and iris C. porosus Stage 18 Ferguson and Webb C gangelicus Singh and Bustard malformations (unpublished data) (1982) (2) Jaw deiects C. jo/msol1i Stage 20, hatchlings Ferguson and Webb (a) cleit lip and palate (unpublished data) A. mississippiensis Teratogen induced Figs. 37i-i; Ferguson Axial biiurcation and par­ A. mississippiensis Single head with Fig. 37b or spontaneous, (l981a, 1982b, 1984) tial twinning cleft lip and palate see text C. niloticu5 Two vertebral col­ Spontaneous Hutton and Loveridge umns, 2 sets oi (personal communica- tion) limbs and tails (b) reduced upper or A. mississippiensis Surgically or terato­ Ferguson (1981a, 1982b, Stage 21 double­ Joanen (personal com­ lower jaws headed embryo munication) genically induced 1984); Fig. 37e C. nilolicus Double head, two Hutton and Loveridge and spontaneous, tails and iour hind (personal communica­ see text C. porusus legs tion) Hot and cold nests Webb and Messel eNS maliormations (1977); Webb et a1. (l983b) (1) Spina biiida A mississippiensis Often laid by young Fig. 37a; Ferguson C. johnsoni iemales (1981a, 1982b, 1984) Webb and Manolis (1983) (2) Exencephaly and A. mississippiensis Often laid by young Ferguson (unpublished C. niloticus anancephaly females data) Pooley (1969a); Hutton (3) Encephalocele A. mississippiclIsis Often laid by young Ferguson (unpublished and Loveridge (per­ iemales data) sonal communication) C. palustris Kalin (1936a, b) C. nilotirus Hutton and Loveridge O. tetraspis (personal communica­ Tryon (1980) C. gangeticus Basihyal valve tion) Singh and Bustard absent (1982) (4) Microcephaly C. nilolic"s Hu tton and Loveridge (c) laterally skewed A. mississippiensis (personal communica­ lower jaws Ferguson (unpublished data) tion) C. porosus Webb and Messel A. mississippiens;s High incubation Ferguson (unpublished temperatures data) (1977); Webb et a1. (1983b) (5) Hydrocephalus A. mississippiensis Teratogen induced Fig. 37j; Ferguson C. ;011ll50ni (1981a, 1982b, 1984) Webb and Manolis (1983) (6) Inner ear and cere­ A. mississippiensis Low incubation tem­ Joanen (personal com­ C. gangeticus bellar maliormations peratures. see Sec­ munication); Ferguson Singh and Bustard tion II (unpublished data) (3) Dental deiects (1982) C. porGsus Several teeth in one De Jong (1928) Craniofacial nlalformations socket (1) Eye deiects A. mississippiensis Tooth erupting into Ferguson (1982b) (a) microphthalmia A. mississippiensis Teratogen induced Fig. 37d; Ferguson the nose and spontaneous (1982b, 1984) (continued) TABLE VIII TABLE VIII (Continued) (Continued)

Type of Malformation Species Notes Reference Reference Type of Malformation Species Notes (3) Vertebral column Skeletal malformations (a) twisted back A. mississippiensis Eggs incubated up­ Fig. 37k; Ferguson (un­ (1) Limbs (scoliosis, right, i.e., at right published data) Ferguson (observation (a) extra limbs A. mississippiensis Extra fifth hind leg kyphosis or angles to the nest of animal at St Au­ attached to dorsal lordosis) base and spon­ gustine Alligator surface of the taneous back at the pelvic Farm, Florida) C. porosus Desiccation Kar (1979) level High and low incu­ Webb et at. (1983b) A. mississippiensis Extra fifth leg at­ bation tempera­ tached to right tures hindleg C. niloticus Modha (1967); Pooley Eight toes on each Giles (1948) (b) extra digits A. mississippiensis (1969a) of the front limbs G. gangeticus Singh and Bustard and the left hind (1982) foot, seven on the (b) extra vertebrae A. mississippiensis Case (1896) right hind foot, C. porosus Rheinhardt (1874); Baur teratogenical1y in­ Ferguson (1981a, 1982b) (1886, 1889) duced and spon­ C. niloticus Kalin (1936a, b) taneous (c) missing vertebrae C. porosus Webb et at. (1983b) C. porosus Eight toes on each Deraniyagala (1936, of the hind feet 1939) Visceral malformations (1) Herniation of thoracic A. mississippiensis Fig. 37a; Ferguson (un­ C. johnsoni Fig. 371 and/or abdominal published data) (c) absent digits A. mississippifllsis Teratogenically in­ Ferguson (1982b) duced and spon­ contents G. gangetieus Singh and Bustard taneous (1982) C. porosus Webb et al. (1983b) C. niloticus Hutton and Loveridge (personal communica­ C. niloticus Hutton and Loveridge tion) (personal communica­ tion) A. mississippiensis High incubation Ferguson (unpublished (d) syndactyly (2) Hermaphroditism temperatures data) A. mississippiensis Evident only on Forbes (1940a) gonadal histology (2) Tail C. johnson; High incubation Webb and Smith (1984) (a) kinked tail A. mississippiensis High incubation Ferguson (unpublished temperatures and data) temperatures, evi­ eggs incubated dent only on upright, i.e, at gonadal histology right angles to the Color malformations nest base (1) Albinism or partial A. mississippiensis Some genetic cases Ferguson, unpublished; G. gangeticus Singh and Bustard albinism and also related to McIlhenny (1935); (1982) incubation tem­ Allen and Neill (1956); C. novaeguineae High incubation Bustard (1969, 1971) peratures Neill (1971) temperatures C. porosus Kar and Bustard, 1982; C. niloticus Pooley (1969a); Hutton Ferguson and Webb and Loveridge (per­ (unpublished data) sonal communication) C. ;ohnstoni Ferguson and Webb C. palustris Deraniyagala (1939); (unpublished data) Whitaker and C. niloticus Blake and Loveridge Whitaker (1976a, b, (1975) 1977b) C. acutus Neill (1971) C. porosus Kar (1979); Kar and Bus­ C. novaeguineae Photograph of a Whitaker (personal com­ tard (1982b); Webb et pure white albino munication) al. (1983b) wi th red eyes (2) Melanistic (b) reduced or absent A. mississippiensis Ferguson (unpublished C. crocodi/u$ Completely black Allen and Neill (1956) (3) Erythristic tail data) A. mississippiensis Red and yellow Allen and Neill (1956) C. porosus Genetic cause sus­ Webb et al. (1983b) colour pected C. niloticus Modha (1967); Pooley

(1969a) dF;Pi, 456 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS

Embryos produced by young and very old females are frequently abnor­ mal (Section IlD and Table IV). Defects include spina bifida (Fig. 37A), exencephaly, cyclopia (Fig. 37C), microphthalmia (Fig. 370), exophthal­ mia, hydrocephalus (Fig. 37J), reduced upper or lower jaw (Fig. 37E), cleft lip and palate (Figs. 37F-I), scoliosis (Fig. 37K), and herniation of the thoracic or abdominal viscera. During incubation, extremes of temperature, abnormalities in hydric or gaseous environment, and variations in the orientation of eggs (i.e., sitting upright as opposed to lying flat) all cause malformations (Table VIII and Section II). Animals incubated at high temperatures, which hatch pre­ maturely, often have a bump or bulge on their cranial platform (Alliga­ tor mississippiensis, Ferguson, unpublished data; Crocodylus porosus, Webb et al., 1983b; C. johnsoni, Webb, personal communication; C. niloticus, Modha, 1967). This represents the midbrain bulge and is probably caused by enhanced ossification of the skull before the cranial flexure has straight­ ened out. As with gonadal growth, this emphasizes how an alteration in incubation temperature may introduce asynchronies in development. Craniofacial malformations have been induced experimentally in em­ bryos of Alligator mississippiensis by either surgical excision of various re­ gions of the neural crest or by teratogens such as 5-fluoro-2-desoxyuridine (FUDR) or hydrocortisone (Ferguson, 1981a, 1982b, 1984a). Teratogens can be injected into the albumen or the yolk. Injection into the chorioallan­ toic blood vessels is technically more difficult, but preferable because uni­ form, homogeneous deformities result from eggs treated with the same dose of the same drug at the same developmental stage. Table IX sum-

Fig. 37. Alligator mississippiensis. Examples of malformed embryos. (A) View of embryo from egg laid by a young mother. Note the meningicoel covering the thoracic spina bifida (ar­ rowed), the thoraco-abdominal herniation (A) and the kyphotic spine. Such embryos tend to die after 10-20 days incubation. (B) Spontaneous malformation recovered from a 50-day egg laid by a young female. Radiograph illustrating axial bifurcation. There are two vertebral columns, two pairs of upper and lower limbs, two pelves, two shoulder girdles. and two tails, but only one head. The latter has bilateral cleft lip and palate. (C) Lateral view of a 24-day embryo with FUOR induced monorhiny (one stage before cyclopia). Note the single nasal proboscis (arrowed), absent midface and small mandible. The eyes contact each other in the ventral midline. (D) Right lateral view of a developmentally retarded 24-day embryo with M FUOR induced microophthalmia (right side) and anophthalmia (left side). (E) Macroscopic genioglossus (G), hyoglossus (H), intermandibularis (I), palatal submucosa (S), junction be­ view of a hatchling alligator treated with 0.01 mg FUOR on day 10. The otherwise normal tween nasal ciliated columnar and oral stratified squamous epithelia (arrowed) and continu­ embryo has almost no lower jaw. but a normal palate and upper jaw. (F) Lateral view of a 29­ ous oral and nasal cavities. (J) Lateral view of a hatchling with FUOR induced hydrocephalus day embryo with FUOR induced bilateral cleft lip and palate (arrowed). The lower jaw is and bilateral cleft lip and palate. (K) Dorsal view of a hatchling with kyphosis and scoliosis. trapped behind the cleft premaxilla. (G) Intraoral view of a hatchling alligator with FUOR ~is type of spontaneous malformation is frequently seen in eggs incubated at an incorrect induced bilateral cleft lip (large arrows) and palate (small arrows). The wide palatal cleft onentation, e.g., at 45-90° to the nest base. Such embryos are usually unable to hatch spon extends into the nasal cavities anteriorly and into the nasopharyngeal duct posteriorly. (H) taneously and the malformations often become progressively worse after hatching. (L) Transverse section through the head of a 20-day embryo with FUOR induced bilateral cleft lip Crocodylus johnsoni. View of a hatchling with complete duplication of the digits in its right pes. and palate. H. & E. and Alcian Blue. Note the small slender projections representing the (M) Detail of part L. Similar malformations can be produced experimentally by grafts of palatal shelves (P). (I) Transverse section through a hatchling with FUOR induced bilateral SUpernumerary ZPA's (zones of polariZing activity). Unless otherwise indicated the scale cleft lip and palate. Mallory. Note the cleft maxilla, distorted tongue and floor of the mouth, markings are divided into 1 mm intervals. 459 DEVELOPMENTAL ABNORMALITIES

c: marizes the results of experiments using FUDR to induce specific craniofa­ '0 o . ~ ~ CIJ.5 ... E >,"0 ~ .n prosencephalic neural crest cell formation, division and migration. This ;:l ~ bO~CIJ.8Cl ~ ~ ~ Q) o CIJ :-g , >,>,>,>,>, -g Q.S E"5 qJ placodes to form a joined or single eye (Fig. 37C). A deficiency in the '" 6 ::J~'..c: ... , CIJ ClJU £ Cl::O o , '"0 .... >, >, >, co >,>, .n ~ -0 ::J ... 0 <1l <1l C'O .~ such deficiencies is that the small processes fail to unite and the nasal pit aJ ... ..c: co '" ~ qJ ~ 4-> "0 "0 "0 co "0"0 I::i=l-< OJ C 0_ ~ .­ Q) bO co co :>­ co bO ..c: ~ slit (Section VII. B) persists as a cleft lip. Because the right nasal pit slit ><:a~ '0 co C u ~ ~ ~·c ~ CIJ 0 :::l E E E CIJ E E -, , .... o .29 P­ o 0 .n teratogen administration (Table IX) is a reliable method of producing a ~ ~.~ It .\3 8 I:Q >< 8 o o oXi::' 00 specific unilateral cleft lip (Ferguson, 1981a, 1982b, 1984a); this is the first < £ ... ~ !-< aJ 0 c 0 .... ~, g: animal model in which this has been possible. Bilateral cleft lip and palate I ... N aJ Q) , -foOJ • "0 .­ .... (Figs. 37F, G, H, and I) results from deficient medial nasal, lateral nasal, o ... ._E .5.!.._ C'O' ..c: ~ ...... ,::0 >,>, -E .§ .., -. r..IJ ,...... :2 '" co bll== C .-"i 2 . co Q) U Q) < o<1lt_ C C .~ ~ o 0 .9L. __ .s.... 0 cr 1 1 (Ferguson, 1982b, 1984a). Hyaluronic acid normally binds water, which Q~.2!.,~E ::J ::J r-< X .... r-< r-< C 0 :3­ ~ .~ I:: .;, causes the shelves to swell, approximate, and close. In cleft palate, the ~ 0 .n '" I:: .... "01jS .... palatal shelves are too small to touch each other in the midline (Ferguson, aJ ::: ~ 1982b, 1984a). Induced palatal clefts can be experimentally repaired in ovo -­ "0 E J...l u ..... rJ'J - = o o ..c: ~ Q) ..c: '" - Q) '" qJ C .... - ~ +1 bO ... v E E 6 produced so that induced facial clefts repaired shortly after their appear­ 6 ~ ._ ...c ~ a ~"t:: ..9:l 2 ~ .... 'C C:':= .2 0 rE co ..c: P­ E ­ '" ~ ~'""O.- ance neither scar nor show marked wound contraction in contrast to facial § .~ t:: 9 rfJ Z' .& § § ~ ~ 0 Q) Q) m-5 o 0 0 C - ...... P­ ... Q) ~~~ E clefts repaired after hatching. The alligator embryo may prove a useful <.8 "U C b >-Q).t ~ ~ Q)..c: >... 0 ._ &;. Cf} OJ C .t E 6 51 ..c: .... Q) OJ - a o _ model for research into embryonic surgery. ~ u:::E:::E >~ 0 OE-<~"U !­ 0 460 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCOOILIANS

Craniofacial malformations can also be induced by altering the diet of penned breeding animals (Ferguson 1981a, 1982b, 1984a). However, such regimes produce a wide spectrum of malformations and are therefore of less experimental value but important in farming, management, conserva­ tion, and population studies. , IX. SHELL-LESS, SEMI·SHELL-LESS, AND IN VITAO CULTURE TECHNIBUES

In many surgical and teratological investigations, it is useful to be able to follow development throughout incubation by observing, even manipulat­ ing, the embryo through the shell. Numerous investigators have explanted DAY 12 chicken embryos and egg contents into petri dishes and grown them in DAY 20 DAY 28 shell-less culture (for review, see Ferguson, 1984a). The developing al­ ligator is a particularly good animal for shell-less culture because it obtains little calcium from the shell as compared to turtles and birds (Section IILD). Alligator embryos collected within 12 hours of egg laying, before attach­ ment to the shell, may be prepared for shell-less culture (Ferguson, 1981a). An incision made around the longitudinal axis of the shell permits removal of the top one-third; this is followed by sterile excision of the underlying shell membrane. The egg contents are then explanted into a 120 mm sterile, vented petri dish under a laminar flow hood. This dish is covered and placed inside a larger one containing filter paper saturated with sterile water for high humidity. The entire assembly is incubated at 30°C and 100% humidity under sterile conditions. Development proceeds normally in such preparations up to approximately Stage 18, after which malforma­ DAY 30 DAY 35 DAY 40 tions, particularly in the snout, cranium, and limbs, occur. Probably these result from altered tensile forces impinging on the embryo. As the yolk and albumen of alligator eggs are more viscous than those of avian eggs, a technique of semi-shell-less culture has been developed; with slight modifications, this can be used successfully at any period of develop­ ment (Ferguson, 1981a, 1982b, 1984a). Sterile incisions are made around the longitudinal axis of the shell, as described earlier, and the top one-third MM of the shell and shell membrane are removed. The yolk and albumen are viscous enough to remain intact inside the lower two-thirds of the shell so that the natural geometry of the egg contents is maintained (Fig. 38). Main­ tenance of such embryos in sterile incubators at 30°C and 100% humidity results in normal development (Fig. 38), which can be filmed and/or ex­ perimentally manipulated. The modest extrinsic calcium needs of the em­ bryo are met by the intact lower two-thirds of the shell. For optimum DAY 55 results, the technique should be performed within 24 hours of egg laying, Fig. 38. Alligator mississippiensis. Montage illustrating the development of embryos in semi­ before the embryo has attached to the top of the shell membrane or devel­ Shell-less culture. Note the chorioallantoic blood vessels overlying the embryos. oped a chorioallantois; such embryos will develop normally and hatch approximately 65 days later (Fig. 38). There are no statistically significant 1­ ACKNOWLEDGMENTS 4S3 4S2 REPRODUCTIVE BIOLOGY AND EMBRYOLOGY OF CROCODILIANS differences between the weights and crown-rump lengths of these em­ ACKNOWLEOGMENTS bryos and those recovered from normally incubating eggs of the same age. The technique, combined with the application of 0.01 mg. FUDR to a In 1978 the alligator specimens referred to in this chapter were collected Stage 10 embryo (to inhibit lower jaw formation), has permitted the direct during the tenure of a Winston Churchill Travelling Fellowship from the observation of palatal development in normal and teratogen-treated em­ Winston Churchill Memorial Trust, London, in 1980 during the tenure of a bryos in situ. Initially, the treated embryos are developmentally retarded, Sir Thomas and Lady Edith Dixon Research Scholarship from The Queen's but they exhibit rapid compensatory growth and apart from an extremely University of Belfast, and in 1981 during the tenure of a Wellcome Trust small lower jaw, they exhibit no other macroscopically detectable abnor­ Research Visiting Fellowship from the Wellcome Trust, London. I am malities. The treated embryos remain viable and hatch around 65 days but grateful to all these Bodies for their support and confidence. die shortly thereafter because they cannot feed. The semi-sheIl-less culture I would like to thank sincerely Mr. Ted Joanen and the staff of the technique may be equally useful for studying the development of other Rockefeller Wildlife Refuge, Louisiana, for their outstanding assistance in structures. obtaining alligator specimens an,d for their generous hospitality and advice Alligator eggs can be windowed for experimental purposes, preferably during my visits with them. The Department, Belfast, secre­ during the first 12 hours after egg laying, before the embryo attaches to the taries, Miss Anne Richardson and Miss Janice Smith, and the Divisional shell. Candling and removal of the eggshell and shell membrane gives secretaries in Ann Arbor, Ms. S. Konchal and Mrs. K! Vernon, transformed poorer results in analyzing older embryos; many embryos die because they my scribbles into a beautifully typed manuscript. I would also like to thank drop from the inside of the shell membrane into the underlying yolk, Mr. George Bryan for photographic assistance, Dr. K. Lewis and the staff tearing their chorioallantoic blood vessels. Only 5% survive this later win­ of the Q. U. B. Biomedical and Medical Libraries for assistance in procur­ dowing technique (Honig, 1984; Honig and Ferguson, unpublished data). ing copies of many obscure papers, Professors A. d'A. Bellairs, C. Gans, Embryonic rudiments, e.g. mandibular arches, palatal shelves, limb P. F. A. Maderson, and Drs. F. Billett, G. Webb, and B. Westergaard for buds, teeth, genital ridges and lungs have been successfully cultured in critical analysis of the original manuscript, and the Academic Council of vitro (Ferguson et al., 1982, 1983a, b; Ferguson and Honig, 1984). Tissues The Queen's University of Belfast for a grant to offset the cost of translating can be cultured for long time periods in either chemically defined or serum some important German papers cited in this chapter. Dr. G. Webb and the supplemented media, and at the abnormally high temperature of 37°C; Conservation Commission of the Northern Territory, Australia, greatly procedural details have been presented previously (Ferguson et al., 1982, facilitated my study of Crocodylus porosus and C. johnsoni embryos and 1983, 1984; Ferguson and Honig, 1984). provided valuable financial assistance toward my traveling expenses. Many individuals volunteered copies of unpublished data or papers in press and participated in useful discussions: I thank them all, but espe­ x. CONCLUSIONS cially Ted Joanen, Larry McNease, Grahame Webb, Jon Hutton, John Loveridge, Alistair Graham, Bjarne Westergaard, and Larry Honig. The embryology of crocodilians is an interesting and important topic, with Alligator research expenses were generously provided by grants from applications not just in herpetology but also in medicine, dentistry, and the Medical Research Council of Great Britain (Grants No. G979/386/C and general developmental biology. Further research is required and areas in 811361OCB), the Eastern Health and Social Services Board, Northern Ire­ need of attention have been highlighted. There is a need to extend what land (Grant No. E109/74/75), the National Institutes of Health, U.S.A. little knowledge we now possess by systematic studies of development in (Grant No. DE-02848 and DE-03569), and the Louisiana Wildlife and F~sheries various species, particularly in the more atypical such as the gavial and Commission. This work was carried out under my U.S. Federal Tomistoma. One could hardly do better than to close with a quotation from FISh and Wildlife permit No. PRT2-2511 and under my Northern Ireland Parker's (1883) monograph on the development of the crocodilian skull, Wildlife permit No. B/WLl/78, B/WL2/80. which is as relevant today as it was 100 years ago: "As the crocodile is . Finally, it is with deep regret that I record the sudden death of my good known to be one of the most ancient types inhabiting this terraqueous friend Professor J. J. Pritchard, for whose advice and encouragement I will globe, his development is full of interest in relation to those countless always be grateful. His death, shortly after the work for this chapter had Reptilian forms that have succumbed to secular changes of the earth, and commenced was a great shock and loss to me, and doubtless this chapter have left neither son nor nephew in the regions where they once were would have been enriched by his critical editorial pen had he lived to see it materialize. dominant." *". 464 REPROOUCTIVE BIOLOGY ANO EMBRYOLOGY OF CROCODILIANS REFERENCEB 465 REFERENCES Bellairs, A. d'A. (1971). The senses of crocodilians. In Crocodiles. Proceedings of the 1st Meeting of Crocodile Specialists. I. U. CN. 32, 181-191. Acharjyo, L. N., Biswas, S., and Misra, R. (1975). Some notes on gharial [Cavialis gangeticus Bellairs, A. d'A. (1977). The nose and Jacobson's organ in reptiles: a review. Cotswold Herpet. (Gmelin)] in captivity. ]. Bombay na!. His!. Soc. 72(2), 558-560. Symp. Report 3, 27-36. Ackerman, R. A. (1980). Physiological and ecological aspects of gas exchange by sea turtle Bellairs, A. d'A. 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