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Insect pupae sampled at a death scene may be used to estimate the post-mortem interval. The pupal age is however difficult to estimate, as there are no good quantitative markers for the age of a pupa. We present a novel method for pupal age estimation based on the quantification of contrast in intensity between the eyes of a pupa and the middle grey photography card as a standard background. The intensity is measured on a standardized scale from 0 (perfect black) to 255 (perfect white) using computer graphical software and pictures of the eye and the background taken with a stereomicroscope. Eye-background contrast is calculated by subtracting the average intensity of the eye from the average intensity of the background. The method was developed and validated using pupae of  Necrodes littoralis  (Linnaeus, 1758) (Coleoptera: Silphidae), one of the most abundant beetle species on human cadavers in Central Europe. To develop the model, pupae were reared in 17, 20 and 23 °C, with a total of 120 specimens. The method was validated by three raters, using in total 182 pupae reared in 15, 17, 20, 23 and 25 °C. We found a gradual increase in eye-background contrast with pupal age. Changes followed generalized logistic function, with almost perfect fit of the model. Using our method pupal age was estimated with the average error of 8.1 accumulated degree-days (ADD). The largest error was 27.8 ADD and 95% of age estimates had errors smaller than 20 ADD. While using the method, different raters attained similar accuracy. In conclusion, we have demonstrated that eye-background contrast is a good quantitative marker for the age of  N. littoralis  pupae. Contrast measurements gave accurate estimates for pupal age. Our method is thus proven to be a candidate for a reliable approach to age insect pupae in forensic entomology.

Eye-background contrast as a quantitative marker for pupal age in a forensically important carrion beetle Necrodes littoralis L. (Silphidae)