MYRICK ET AL.: DENTAL LAYERS IN HAWAIIAN SPINNER DOLPHINS 



calcification removes TCL labels (Nielsen 1972). We 

 prepared the spinner teeth using both methods. 



Untreated, mid-longitudinal thin sections, 100 [xm 

 thick, were prepared by hand grinding and polishing 

 teeth using 240 and 600 grit AI2O3 on a glass plate. 

 Other teeth were decalcified in RDO 7 for 6-8 h, 

 rinsed, and cut with a microtome in longitudinal 

 plane to produce 30 /xm thick sections that were 

 stained in Mayer's haematoxylin for 15-30 min. 

 Untreated and D/S preparations were mounted on 

 slides in Permount or glycerin gel and covered with 

 coverslips. 



To determine the pattern components of GLGs, the 

 D/S and untreated thin sections were examined in 

 plain transmitted light 39X and 150X with a Zeiss 

 photomicroscope. TCL labels were viewed at the 

 same magnifications with UV reflected light using a 

 Zeiss fluorescent vertical illuminator with a filter- 

 reflector No. 44-75-05 combination attached to the 

 same instrument. 



Retrospective Calibration of 

 Dentinal GLGs 



Dates and durations of treatment, date of birth (for 

 one specimen) or capture, and dates of death (for four 

 carcasses) were taken from clinical records main- 

 tained for each dolphin during its captive life at Sea 

 Life Park (for summaries see Myrick et al. in press). 

 Data for each specimen were transcribed onto a cali- 

 bration chart as the chronological series of event blocks, 

 the relative width of a given block corresponding to 

 the length of a given period of treatment. 



In each thin section showing distinct fluorescent 

 labels under UV light, label thicknesses and 

 interlabel distances were measured. Label- 

 measurement data for each dolphin were entered on 

 its chart as a series of blocks below the event blocks, 

 with spacing and thickness scaled to the correspond- 

 ing measurements. The treatment and label blocks 

 were compared for spacing and thickness to identify 

 the date each label was introduced. Connecting lines 

 were drawn from the beginning and the end of each 

 matched pair of blocks (Fig. 1C). 



A UV photograph of each thin section was used to 

 identify and letter key labels that enclosed 6- or 12- 

 mo segments of dentine. Labels and structural 

 landmarks in the UV photograph were traced with a 

 china marker on an overlay of transparent plastic. 

 Using the landmarks, the tracing was lined up on the 

 corresponding plain-light photograph onto which the 



1 A commercial rapid decalcifying agent available through Dupage 

 Kinetic Laboratories, Inc., Plainfield, 111. 



labels were reproduced to delineate layering pat- 

 terns within the time segments. Each marked 

 photograph was then inspected for repeating layer 

 components to define GLGs and their subunits in the 

 untreated thin section. GLGs defined in the labeled 

 dentine of each thin section were used as a basis for 

 identifying similar GLGs in the unlabeled regions of 

 the dentine and permitted a complete series of GLG- 

 thickness measurements and an estimate of dentinal 

 age in years to be made for each animal. 



Dentinal GLGs in dolphin teeth are most easily dis- 

 cerned in the region of the "shoulder", i.e., along a 

 transect from near the base of the neonatal line (the 

 first layer of the postnatal dentine), downward and 

 inward at about a 30°-40° angle to the margin of the 

 pulp cavity (for examples see Perrin and Myrick 

 1980: fig. 2; Hui 1978: figs. 1, 2, 3). For consistency, 

 measurements of GLG and label thickness, taken 

 perpendicular to the long axis of the teeth of the 

 Hawaiian spinner dolphins, were made along tran- 

 sects at a similar position and angle (Figs. 1A, B). 

 However, a GLG or label may vary in thickness in 

 localized regions of the dentine and may not be the 

 same on both sides of a tooth because of tooth asym- 

 metry. For these reasons, measurements were made 

 on the most symmetrical side of a tooth and in regions 

 where GLGs and labels were clearest and least vari- 

 able in thickness; departing slightly from a uniform 

 angle of transect. GLGs in the dentine of the corres- 

 ponding D/S thin sections were defined and counted 

 with the aid of GLG-thickness measurements 

 obtained from the untreated section. 



Retrospective Calibration of 

 Cemental GLGs 



Because fewer labels were observed in the cemen- 

 tum than in the dentine of the same untreated thin 

 section, it was assumed that those visible represent- 

 ed condensed forms of only the brightest, thickest, or 

 closely spaced groups of dentinal labels. This has 

 been verified in bottlenose dolphins (Myrick 1980b) 

 and recently in the present sample of Hawaiian spin- 

 ners by observations that bright dentinal labels at the 

 tooth base are continuous with cemental labels. 

 Hence, cemental labels were lettered to correspond 

 to the brightest dentinal labels, and the cemental 

 layers between labels were calibrated using the time 

 segments represented between the dentinal labels. 



The annual GLG pattern was defined as precisely as 

 possible using the calibrated segments of the tissue, 

 and the cemental GLG definition was tested by com- 

 paring the dentinal GLG count with the cemental 

 GLG count in untreated thin sections. In D/S thin 



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