John W. Wright. Curator Emeritus. Section of HerpetoIogy. Natural History Museum of Los Angeles County. 900 Exposition Boulevard. Los Angeles, California ...
Biology Of Whiptail Lizards (Genus Cnemidophorus) Editors
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John W. Wright and Laurie J. Vitt
A PUBLICATION OF THE
OKLAHOMA MUSEUM OF NATURAL HISTORY r
AND
THE UNIVERSITY OF OKLAHOMA
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HERPETOLOGISTS' LEAGUE SPECIAL PUBLICATION NO. 3
John W. Wright Curator Emeritus Section of HerpetoIogy Natural History Museum of Los Angeles County 900 Exposition Boulevard Los Angeles, California 90007 USA Laurie J. Vitt
. Assqciate Curator and Associate Professor Oklahoma Museum of NaturaI History and /-
Department of Zoology University of Oklahoma Norman, Oklahoma 73019 Also by Laurie J. Vitt Repteis das Caatingas, P. E. Vanzolini, A. M. M. Ramos-Costa, and L. J. Vitt (Academia Brasileira de Ciencias, Rio de Janeiro, Brazil, 1980) Copyright O 1993 By The Oklahoma Museum of Natural History All rights reserved. This work may not be copied or translated in whole or part without written permission of the publisher (Oklahoma Museum of Natural History, 1335 Asp Avenue, Norman, Oklahoma 73019) except for brief excerpts properly referenced in reviews or scientific publications. Use of part or all of this book in connection with any form of electronic transfer, information storage and retrieval, computer software, or by similar or dissimilar methodology in current use or in the future is forbidden. Permission to photocopy for internal or personal use is granted solely by the OkIahoma Museum of Natural History, 1335Asp Avenue, Norman, Oklahoma 73019 Cite as: Wright, J. W., and L. J. Vitt. 1993. Biology of Whiptail Lizards (Genus Cnemidophoms).Oklahoma Mus. Nat. Hist., Norman, Oklahoma, USA
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Production managed by Laurie J. Vitt Computer expertise provided by Cameron Kacmarcik Printed and bound by Printing Services of the University of Oklahoma ISBN 1-883090-01-6
Chapter 9
Comparative Eggshell Stereology In Two Species Of Whiptail Lizards (Sauria: Teiidae) STANLEY E. TRAUTH and WAYNE R. FAGERBERG
Squamate eggs are grouped into two sharply contrasting categories h ~ e upon d the degree of elasticity of their eggshells; i.e., eggs are either ible-shelled or rigid-shelled (Bellairs, 1970;Packard et d . , 1977, 1982a, b; Sexton et al., 1979).Flexible-shelled eggs, the most common type found in lizards and snakes, owe their pliability to the myriad of tiny fibrils which contribute to the bulk of the eggshell. In addition, this type of eggshell usually lacks a well-developed calcareous component of the surface layer (as 'opposed to the more elaborate crystalline outer layer observed in rigidshelled eggs). Recent investigations on lizard eggshell structure using electron microscopy (Kriesten, 1975; Packard et al., 1982b; Schleich and Kiistle, 1988; Sexton et al., 1979; Trauth and Fagerberg, 1984)have found several layers or zones of fibrils. Zones are distinguished on the basis of fibrillar arrangement (i.e., fibrils may be aligned in a parallel or criss-crossing fashion within a zone or, in other instances, entire zones may run parallel or perpendicular to other zones), fibrillar density (compactness of individual fibrils),'presence or absence of an interfibrillarmatrix, and fibrillar diameter. The presence of intrafibrillar spaces or cavities in eggshells of all squamate species examined thus far suggests that these cavities serve as a means of enhancing the insulative properties of the eggshell, or they possibly act as storage sites. Another prominent morphological feature of flexible-shelled eggs is the undulating nature of the fibrillar zones (observedin both oviductal and oviposited eggs; see Packard et d.,1982b). Regardless of their chemical composition or structural integrity, eggshells perform several vital functions that lead to the successful development and departure of embryos. Among these are: 1) the protection of the embryo from mechanical damage; 2) the protection of the embryo from predation, bacteria, and fungi (as the primary defensive barr); 3) the facilitation of water homeostasis (water exchange with the environment; see Packard et d . , 1982a; Packard and Packard, 1988);4) to act as a source of calcium for skeletogenesis (Packard and Packard, 1988); 5 ) the allowance of gas exchange with the environment, 6) the allowance for growth of the embryo (especially evident in squarnate eggs which, in
Biology of Whiptail Lizards
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most cases, increase in mass andlor volume during incubation [Packard and Packard, 1988; Trauth, 19831); 7) to provide a means of insulation from the environment (Trauth and Fagerberg, 1984);and 8) to allow the embryo to exit the egg at hatching time. Recently, a considerable amount of research has been conducted on the biology of C m t d o p b r u s l a r e d o m , a parthenogenetic whiptail lizard inhabiting the lower Rio Grande Valley of the United States and Mexico (see Walker, 1987). In the present study, we chose to examine the eggshell morphology of C. l a r e d o m for several reasons; in particular, we were interested in comparing eggshells of C. laredo& with those of C. sexlineatus (Trauth and Fagerberg, 1984) mainly because evidence from previous studies (Bickham et al., 1976; McKinney et al., 1973; Wright et al., 1983) indicates that C. laredoensis arose by hybridization between the two biparental species, C. gularis and C . sexlineatus.
Materials and Methods Specimen Preparation Shelled oviductal eggs were obtained from two adult Cnernidopbrus laredoensis (snout-vent lengths, 81 and 75 mm) collected by hand from sandy areas along Chacon Creek in Laredo, Webb Co., Texas, on 16 July 1983. These lizards (informally designated as LAR-A, a clonal hybrid population of C. laredoensis, by Walker, 1986) were kept on ice for 48 hrs. prior to necropsy and eggshell fixation. Eggs of C. gularis were not available for examination in the present study. Eggshell fragments from four eggs were prepared for study by light microscopy (LM) and transmission electron microscopy (TEM). First, eggs were sliced into thirds with a sharp razor blade, and the yolk and extraembryonic membranes were removed. Eggshells were then cut into rectangular pieces and fixed in 3% glutaraldehyde buffered in 0.1 M sodium cacodylate at pH 7.2 for two hrs. Postfixation was carried out in a 2% solution of OsOl (buffered as above) for two hrs. at 4' C. Fragments were transferred through a graded series of ethanol-acetone and embedded in Mollenhauer's Epon-Araldite #2 (Dawes, 1988).Thick sections (1or 2 pm) were sectioned with a glass knife and stained with a metachromatic stain. Silver sections were obtained by sectioning tissue blocks with a diamond knife and stained with uranyl acetate and lead citrate. Specimens for TEM were viewed with a Siemens 102 ,--at an accelerating voltage of 80 kV. Stereological Techniques Stereological methods for determining the % volume (V-) of fibril and/or space within eggshells have been presented elsewhere (Yrauth and Fagerberg, 1984). In brief, the procedures involve the random selection of
Eggshell Stereology
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areas of micrographs representing each eggshell mne (refer to the systematic sampling scheme of Weibel, 1979). Micrographs were taken at an initial magnification of ca. 12,500X and were enlarged to ca. 42,000 X. The V- values were calculated by first counting data points using test grids subrimposed over micrographs (Aherne and Dunnill, 1982; Fagerberg, 1980; Steer, 1981; Underwood, 1970)and then determining ratio estimates from the relationship:
(Mayhewand Cruz-Orive, 1974)where V- is the mean volume %, P is the total number of grid points falling on the fixrils (or intrafibrillar space), and p is the total number of points falling within the boundary of the eggshell zone (or fibril). In order to standardize stereological variables for estimating proportional volumes, we propose the following terminology: Fibrillar V-: the % of the total eggshell volume occupied by v fibrils. Interfibrillar V-: the % of the total eggshell volume occupied by space betweenYibrils. Intrafibrillar V-: the % of whole fibrillar volume occupied by v cavities. Solid Fibrillar V-: the % of whole fibrillar volume occupied by solid fibrillar magx (ie., the inverse of intrafibrillar V-). Mean Fibrillar Diameter: an average diameter of fibriys derived from cross-sectional measurements of each zone. Standard Volume of Eggshell: an idealized volume of eggshell based upon a 100 clm2surface area of eggshell multiplied by the mean eggshell thickness. Standard Volume of Zone: an idealized volume of an eggshell zone determined by multiplying the standard eggshell area (100 ~ mtimes ~ )the mean height of that zone. Mean Height of Eggshell Zone: the average height of each eggshell zone based upon measurements taken from cross sections of eggshell observed with the light microscope. Standard Actual volume of Fibrils: the total volume of fibrils in each zone in real terms (ie., clrn3,mm3, etc.) determined by mul-
Biology of Whiptail Lizards
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tiplying V- (fibrillar) value by the standard volume for each eggshell mXe. 10) Standard Actual Volume of Space: the volume of space between the fibrils in real terms based upon V- (space) value multiplied by standard volume for each eggshell Lne. 11) Actual Volume Solid Portion of Fibril: the actual volume of the fibril excluding the cavities as determined by multiplying Vv (solid fibril) value by standard actual volume of fibrils. 12) Solid Fibril to Interfibrillar Space Ratio: a ratio of solid fibrillar volume to space volume betwken fibrils as determined by dividin the actual volume of solid portion of fibrils by the actual vo ume of interfibrillar space for each zone.
f
Our descriptive terminology for eggshells has been modified from the work of others (see Packard et al., 198213; Sexton et al.,1979), and we follow our previous usage (Trauth and Fagerberg, 1984). The designation "space" is intended to represent non-fibrillar areas of eggshells and does not necessarily imply that these areas are devoid of materials. Statistical methods for ratio estimates follow Weibel (1979); otherwise, all procedures follow Sokal and Rohlf (1981); means f two standard errors are given.
Results Eggshell Morphology Eggshells of oviductd eggs in Cnmdophorus laredoensis consist of three layers or zones of fibrils (Fig. 1) and are basically similar to the eggshell of C. sexlineatus (Trauth and Fagerberg, 1984). Each fibrillar zone, designated Z,, Z2, and & (innermost to outermost, respectively),contains individual, elongated cylindrical fibrils surrounded by either a looselyadhering matrix of unknown composition or by open areas of interfibrillar space. Upon immersion into a dilute solution of hydrochloric acid, the eggshell appears to effervesce which suggests that the interfibrillar matrix (at least in the outermost zone) is comprised of a calcareous substance (see Packard et al., 1982b).With the exception of fibrils in Z,, which lie mostly parallel to one another, the majority of eggshell fibrils are laid down in a criss-crossing manner; moreover, the entire eggshell has an undulating network of fibrils (Figs. 1, 2B). The undulations correspond to smooth -- crests and shallow furrows on the external surface of the eggshell. Fibrils in Z, are scattered along the inner limiting boundary (ILB); the ILB is adherent to constituents of the egg proper (i.e., a yo& mass as seen in Fig. 1 or extraembryonic membranes which would not appear until later in embryonic development). The ILB varies in height, ranging from 2.34 pm
Eggshell Stereology (as in Fig. 1)to as much as 11.78 pm ( z = 4.98 f 1.68 pm; N = 20). This variation did not appear to be an artifact of preparation. Following staining with Paragon stain, the entire eggshell was highly acidophilic which in&cates a strong counterstain reaction (as compared to the deep basophilia observed in yolk bodies found in Fig. 1). Stereology We determined the % voIume (V-) of inter- and intrafibrillarspace for each eggshell zone for ~nemrdopb& Zuredomrir (Table 1). There was a significant change in interfibrillar space between & and Z3 (P < .02) and between fibrillar volume and interfibrillar space of & (PC.001).In Z, and Z,the reIative amount of fibriI compared to space did not differ significantly (P < .001) between each zone. Fibrils in Z, were the most dense, Ireas fibrils in Z2were the least dense. Average fibrillar diameter difiered significantly (P c .001)when each zone was compared to one another, and fibrils in Z, were approximately 1.3 times as large as those in Z,and approximately 2.7 times as large as those in Z,. TABLE 1. Eggshell characteristics in C n e m i d o p b laredo& and in Cnemtdophorus (dab f m Trauth and Fagerberg, 1984).Numerals are mean values; Vv values are percentages,.Note: V- values for the eggshell fibrils are reciprocals (i.e., 100 - V - ) of interfib* space V-. V ! values for the solid portion of the fibrils are reciprocals oY the intrafibrillar space \r-; these values are in parentheses. Ranges are given with fibrillar v diameter (in parentheses).
dineatus
Eggshell Zone
Interfibrillar Space ( vv)
Intrdbrillar Space
(vg)
Fibrillar Diameter (pm)
3' (97) 16' (84) 8' (92)
1 . 1 2(0.79-1.72) ~ 0 . 8 4 (0.48-1 ~ .SO) 0.41 (0.20-0.75)
loe (90) 2Ae (76) 20" (80)
0.83~ (0.76-0.90) 0.80' (0.73-0.82) 0.42' (0.38-0.46)
C.laredo&
23
57 (43) 53C(47) mcd(361d
21
48 (52)
z1
%
C.stdineatus
%
41gd ( ~ 9 ) ~ 51g (49)
z3 r Mmn-Whitney ' U'
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b. ANOVA, P c.001 c t-test., P c.02 d t-test, P
