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

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.

The quality of developmental reference data in forensic entomology: Detrimental effects of multiple, in vivo measurements in Creophilus maxillosus L. (Coleoptera: Staphylinidae).

Although in vivo measurements of larval insects are often performed during developmental studies of necrophilous beetles, their impact on development has not been studied. During measurements insects are taken out of the incubator for a few minutes at room temperature, which may affect the development and eventually the quality of the developmental reference data. Additionally, while being measured larvae are under stress which may have an effect on their development. We conducted an experiment using predatory beetle species Creophilus maxillosus L. (Coleoptera: Staphylinidae) which often occurs and breeds on large vertebrate carcasses. We tested the hypothesis that multiple, in vivo measurements affect the development of C. maxillosus by increasing its duration and changing adult insect size at emergence. As a consequence, we predicted that the multiple insect measurement protocol will affect the accuracy of age estimates using the resultant reference developmental data. Development of C. maxillosus was studied at 7 constant temperatures. All individuals were inspected for developmental landmarks; half of them were also repeatedly measured and weighed. Measured beetles developed longer than non-measured beetles (e.g. 1.59 days longer at 22.5 °C) and at emergence were distinctly smaller (e.g. 1.5 mm shorter and 22 mg lighter at 22.5 °C). The accuracy of age estimates was greater while using the model for non-measured beetles. These results support the claim that multiple in vivo measurements of insects reduce the quality of resultant developmental data. The measurements were particularly detrimental for the adult insect size. Consequently, particular attention should be paid to isomegalen diagrams which are based on insect length. Our findings indicate that these diagrams, when based on multiple, in vivo measurements of larval beetles, will systematically overestimate their age.