Estimating the age of Calliphora vicina eggs (Diptera: Calliphoridae): determination of embryonic morphological landmarks and preservation of egg samples.

Blow fly eggs may sometimes be the only entomological evidence recovered in a forensic case, especially in cooler weather when hatching might take several days: hence, a method for estimating their age is greatly needed. However, developmental data on blow fly eggs are mainly limited to records of the time to larval hatching. The current paper describes the morphological changes occurring during embryogenesis of the blow fly Calliphora vicina Robineau-Desvoidy and their timing in relation to temperature, in order to determine those characters which can be used for simple egg age estimation using light microscopy. At 7.3 and 25 °C, 15 easily visualised morphological landmarks were determined in C. vicina living embryos, allowing for their age estimation with a resolution of 10-20% of total egg developmental time. The observed age intervals were compared to the embryonic stages described for the fruit fly Drosophila melanogaster Meigen, which are used as reference data in multiple developmental studies. Moreover, current guidelines for preservation of egg samples, which recommend the placement of living eggs directly into 80% ethanol, were tested against the hot water killing (HWK) method prior to preservation in 80% ethanol, recommended for larval and pupal specimens. Direct placement of eggs into 80% ethanol caused marked decomposition of samples, and no morphological landmarks were discernible. On the other hand, HWK fixation prior to preservation in 80% ethanol enabled visualisation of 11 of the 15 age-specific morphological landmarks that were discernible in living embryos. Therefore, HWK fixation prior to preservation in 80% ethanol is recommended for egg samples, thus unifying the protocols for collecting entomological evidence.