Table 1.
Sample sizes for each age determination technique by sample group.
Fig 1.
Film holder for dental radiography.
A plate holder was manufactured from two appropriately sized translucent plexiglass sheets affixed on the end of a ½ inch PVC pipe. A high-resolution standard dental #4 size digital phosphor plate was encased in a sealed plastic sleeve, inserted between the plexiglass sheets and positioned in the dolphin’s mouth for dental radiographs.
Fig 2.
Periapical dental X-ray procedure on wild bottlenose dolphins.
During health assessments, water-proofed, portable X-ray equipment was used to collect lateral views of the maxillary or mandibular teeth, both while dolphins were held in the water (A) or evaluated on a processing vessel (B). The lead handler positioned the film holder, while the two other handlers closest to the head helped minimize side-to-side head movements. The individuals appearing in Fig 2 have given written informed consent (as outlined in PLOS consent form) to publish this picture.
Fig 3.
Periapical dental X-ray radiograph from a wild bottlenose dolphin.
Three maxillary teeth per individual (A) were selected to calculate the tooth area (B) and pulp area (C) using similar methods to those described by White et al. [55]. Briefly, the lasso and histogram tools in Adobe Photoshop were used to count the number of pixels in each area, then the pulp:tooth area ratio was used to estimate the age of the individual.
Fig 4.
Each radiograph typically captured five to seven teeth, allowing analysts to identify and exclude non-vital (dead) teeth (blue arrows) from the pulp and tooth area calculations, as these teeth no longer accumulate dentin layers as the dolphins age.
Fig 5.
Fit and prediction error of the linear-log regression analysis.
(A) Plot of the training dataset used in the linear-log regression analysis to evaluate how well pulp:tooth area ratio can predict age. The points represent individual dolphins with both periapical dental radiographs and life history-based age estimates from SB and BB (n = 40). Due to the low number of samples for pulp:tooth area ratios between 0.04 and 0.17, as well as the wide range of life history-based ages for pulp:tooth area ratios less than 0.04, samples (grey points) with pulp:tooth area ratios < 0.1 (vertical grey line) were excluded. The remaining samples (black points, n = 26) were used to fit a linear-log model of pulp:tooth area ratio against predicted ages (black line, R2 = 0.85, confidence limits are given as dashed lines). Due to the low sample size, site or sex were not added as covariates, but there did not appear to be obvious differences between males (triangles) and females (circles), nor between the sites, above the 0.1 threshold. (B) Residual errors for the linear-log model including SB and BB dolphins (n = 26). The increasing variation in residuals with increasing age predictions indicates that the model performs better with larger pulp:tooth area ratios (younger animals).
Table 2.
Sample sizes for each age determination technique by sample group for pulp:tooth area ratio ≥ 0.1.
Fig 6.
Predicted ages of wild dolphins using periapical dental radiography.
Using the linear-log model, the pulp:tooth area ratio was used to predict ages for 91 living dolphins (from health assessments in SB, BB, and MS) and 10 dead, stranded dolphins (from BB). Age distributions should not be interpreted as representative for the populations in question, as the study design for each health assessment, sample availability and model criterion (pulp:tooth area ratio ≥ 0.1) biased the data set.
Fig 7.
Plot of the linear-log model based on periapical dental radiographs compared to samples with tooth GLG and life history age estimates.
Orange points represent the training data set with life history-based age estimates; blue points represent samples without life history information, but with both tooth GLG-based age estimates and periapical dental radiographs. The data set includes both wild, living dolphins (from BB [circles], MS [triangles], and SB [squares]) and dead, stranded dolphins (crosses). The model (black solid line with prediction limits given as dotted lines) consistently predicts lower ages than the GLG-based age estimates (RMSPE = 1.65 years, compared to RMSE = 0.75 for the life history-based age estimates). Data are only shown for samples with a pulp:tooth area ratio ≥ 0.1 (vertical grey line).
Fig 8.
Dental radiography-based age estimates compared to total body length.
Data from females are plotted in purple and males are plotted in green. McFee et al. [31] identified three phases of growth in dolphins from Mississippi waters based on GLG age estimates (grey lines represent median fitted values with 95% CI). Although our data set is limited to dolphins generally between the ages of 1–10 years old, the general trend in the periapical age estimates versus length show similar rapid growth of dolphins in this age range.
Fig 9.
Periapical radiographs of dolphin YN9.
Even in cases where the pulp:tooth area ratio is below 0.1 and excluded from age estimation by the model; dental radiographs can help confirm other age estimation techniques. Here, dolphin YN9’s pulp area is very small (A), indicating an older animal. However, radiography shows that the single tooth extracted for GLG-based age estimation is in fact non-vital (B), and therefore likely results in a younger age estimation.