Embryonic morphological landmarks
Previous studies on the egg development of forensically important blow flies suggested that the only external age-diagnostic markers in non-dechorionated eggs become visible in the very last hours of the embryonic development, when distinct mouthparts and spine bands can be visualised as the first instar larva is almost ready to hatch [
10,
12]. Particularly, Sanit et al. [
12] only observed clear spine bands from the 80 to 90 % period of total embryonic development in the blow fly
Hypopygiopsis tumrasvini Kurahashi, with no obvious features at earlier stages. This severely limited the temporal resolution of an egg aging method based on the identification of morphological landmarks. However, visualisation of the diagnostic characters can be solved with dechorionation of eggs, as it reveals the morphology of the embryo, making it possible to identify the morphological changes occurring during egg development as already applied to other Diptera species [
16,
17,
19,
20]. Dechorionation of eggs is a very simple method, not requiring special equipment or particular expertise. Although staining methods can enhance the contrast between cell layers and yolk, thus enhancing the visualisation of some internal morphological characters [
18], a simple and fast mounting of the embryos using Hoyer’s medium or a solution of sodium chloride [
16] is suitable for visualising and identifying the age-specific morphological landmarks determined here (Table
1). It must be noted that the variation in the extent of the germ band during embryonic development (Figs.
2i,
3b) could be an additional informative marker (see Campos-Ortega and Hartenstein [
17] for further information on this structure). Nevertheless, it has not been included in the current table of diagnostic landmarks (Table
1) because it is not always easily discernible, becoming only prominent during shortening (Fig.
3b), probably due to an increasing cell density [
17]. Before shortening, the maximum extent of the germ band can be identified by a dorsal indentation (Fig.
2i), but it must be emphasised that this is a variable feature and frequently obscured by the folds of the serous membrane [
17].
Overall, the morphological characters described and their chronology are consistent with those described for other Diptera species [
15‐
20,
32], including the previous study on
C. vicina embryogenesis [
26]. Nevertheless, it must be highlighted that the 5–7 divergent dorsal folds observed during gastrulation at 30 % of embryonic development of
C. vicina (Fig.
2f, g) are more numerous than the three folds observed in
D. melanogaster [
17], being more similar to the up to six folds observed in the more closely related blow fly
Lucilia sericata (Meigen) [
32]. Mellethin et al. [
32] suggested that such higher number of dorsal folds formed during gastrulation might be due to the different dimensions of the body axes, with a much greater length-to-thickness ratio in blow fly eggs than in
Drosophila eggs.
Regarding the timing of the developmental intervals, it must be noted that previous descriptions of the embryonic stages included only approximations of the duration of each stage and highlighted the difficulty of delimiting the embryonic stages [
17]. Indeed, when applying the current age-specific morphological landmarks (Table
1), it should be taken into account that embryonic development is a continuous process and that the morphological events observed at the end of a development interval continue during part of the following interval. In any case, the time intervals described here are consistent with the times suggested for
C. vicina embryogenesis by Starre-van der Molen [
26]. Interestingly though, when compared to the times suggested for
D. melanogaster at 25 °C [
17], the first stages of embryonic development appear to progress slightly faster in
C. vicina (Table
1). Mellenthin et al. [
32] also suggested shorter stages in the first part of the embryonic development of
L. sericata. Different duration of the embryonic stages has been observed in closely related species of the genus
Drosophila [
20], so the development intervals determined in the current study for
C. vicina might not be valid for other Calliphoridae species.
It is worth mentioning the difference observed at the 20 % development interval between the two temperature set-ups (Table
1), suggesting a proportionately slightly shorter development during the first stages of embryogenesis at lower temperatures. Lower tolerance of young embryos to cold has been observed in
L. sericata [
33] and
Drosophila suzukii (Matsumura) [
20], so shortening of earlier stages at lower temperatures might be an adaptive strategy of cold-tolerant species like
C. vicina. However, our current data do not allow us to draw further conclusions on this issue and, to the best of our knowledge, there are no studies on the duration of the embryonic stages at different temperatures in other blow fly species. It is also beyond the scope of the current study to discuss the difference in the ADH requirements between the two experimental temperatures (Fig.
1). However, our result concurs with other studies of
C. vicina that report higher values of ADH at lower temperatures [
3,
27,
34]. This variation requires further investigation, including use of more experimental temperatures than the two used in the present study.
Preservation of egg samples
Direct placement of living entomological samples into ethanol has been shown to cause marked decomposition of tissues following death of the samples, frequently resulting in shrinkage and discolouration [
4,
28,
29]; this is also clearly the case in blow fly eggs as indicated by the current results (Fig.
4). Moreover, as ethanol does not instantly kill the specimens, it might lead to erroneous PMI
min estimations, particularly in the last hours of egg development, when first instar larvae may hatch in the preservative (Fig.
4). For these reasons, direct placement of live eggs into ethanol should be avoided when collecting egg samples at the forensic scene.
As mentioned, HWK fixation prior to preservation in 80 % ethanol is already recommended for larval and pupal samples collected at forensic scenes [
27,
29]. This fixation and preservation method also allows for the visualisation of most age-diagnostic morphological landmarks in egg samples (Fig.
5; Table
1); hence, it is also recommended for eggs. Dechorionation may not be strictly needed in some cases as the chorion usually becomes semi-transparent after several days of storage and the fixed embryo can be visualised inside (Figs.
5h, l). However, to achieve the best possible resolution, it is preferable in most cases to remove the chorion.
Finally, as a guideline for forensic practice, the egg samples collected at a forensic scene should ideally be divided into two batches: one should be placed on moistened tissue paper in vials, stored in a cool bag with a temperature data logger and transferred to an expert for rearing as soon as possible, and the other one should be HWK fixed (as soon as possible after collection, recording the time) prior to storage in 80 % ethanol for a subsequent morphological analysis at the laboratory. This recommendation of fixation and storage method unifies the protocol for collecting entomological evidence as already recommended for both larval and pupal samples [
27,
29].