An effective method for preparing histological sections of blow fly intra-puparial stages for minimum PMI estimations

The method presented here resulted in complete and fully informative sagittal sections for the four selected IPP percentages (Fig. 1). Although a few cracks (Fig. 1a and b) and wrinkles (Fig. 1e and f) were observed in some sections, they were usually minimal and did not hinder the observation of age-specific morphological markers [4, 12]. Whereas wrinkles appeared at muscle tissue in 100% IPP specimens, cracks were mainly present in the internal tissues at 10% and 20% IPP, corresponding to the cryptocephalic and phanerocephalic pupal stages [3], respectively. These are the periods of most drastic changes, including the eversion of head and legs at 20% IPP [13]. Consequently, the specimens typically show a profusion of fat bodies (Fig. 1a and b) and extensive histolysis and histogenesis [5] that make the appropriate paraffin infiltration of tissues difficult. Nevertheless, the histological sections obtained unequivocally showed the morphological landmarks of these development intervals [4]. At 10% IPP, the partially extruded cephalopharyngeal skeleton [4], the crescent-shaped outer proliferation centre of the brain hemispheres [15] and the large, characteristic gas bubble within the internal tissues of the abdominal region [4, 13] were all observed (Fig. 1a). At 20% IPP, the adult midgut showed a closed-sack shape in sagittal view, occupying the central body region and containing the apoptotic larval midgut or “yellow body”, whereas the thoracic ganglion was located at the most anterior part of the thorax (Fig. 1b). Both characters are diagnostic markers for that development interval [4]. Moreover, the separation of the pupal cuticle from the adult epidermal cells was observed only in some areas (Fig. 1b), indicating that the pupal-adult apolysis was ongoing but not complete [5]. Determining if an apolysis event is complete is crucial for an accurate delimitation of the prepupal, pupal and pharate adult stages [3], but this information could be easily lost if the histological sections are not properly prepared and processed.

The pharate adult stage, which begins once the pupal-adult apolysis is complete [3], is a period of less profound morphological changes than the pupal stage [5]. Nonetheless, it is still possible to determine the IPP percentage during this stage on the basis of age-diagnostic morphological characters [4, 12, 14, 15], all of them clearly observable in the present histological sections (Fig. 1c and f). During the earlier development of the pharate adult, the shape of the adult midgut and the position of the crop are particularly age-informative [4, 5]. As the crop develops, it lengthens and extends from the anterior part of the thorax, reaching its final position in the anterior region of the abdomen at 50% IPP [4]. The present histological sections showed this age-diagnostic character unambiguously (Fig. 1c). The pre-helicoidal region of the adult midgut, which shows a “long-necked bottle” shape in sagittal view at 40% IPP [4], starts to widen uniformly along its length and acquires a more tubular shape at 50% IPP, as observed in the present sections (Fig. 1c). Furthermore, at 50% IPP the indirect flight muscle fibres are easily discernible (Fig. 1c and d) yet not fully formed nor attached to the adult cuticle [4]. In the brain, the lamina, one of the neuropils that form the optic lobe, shows a characteristic “horseshoe” shape at 40% and 50% IPP (Figs. 1d and 2a), before unfolding and extending below newly formed retinular cells at 60% IPP [4, 15].

Finally, at 90–100% IPP, the internal organs and tissues are fully developed and the pharate adult is ready to emerge from the puparium [5]. Particularly noteworthy is the development of the head and thoracic muscles (Fig. 1e and f), which only attach to the adult cuticle (Fig. 2b) at the end of the intra-puparial development [4, 5]. At 100% IPP, the histological sections often showed wrinkles in the muscle fibres (Fig. 1e and f), even in the median plane, where, at that IPP percentage, there is typically no tissue between the two sets of dorsal longitudinal indirect flight muscles in some areas of the thorax [4]. Anyhow, those wrinkles did not hamper the observation of the muscle architecture (Fig. 1e and f), as fully formed indirect flight muscles that occupy the majority of the thoracic region is a diagnostic character for the last IPP percentages [4, 11, 12, 14]. During intra-puparial development, the muscle fibres are mostly surrounded by haemocytes and fatty droplets [11, 22], which can be an obstacle for the proper infiltration of paraffin (Fig. 2b). On the other hand, the present sections showed a narrow tube-shaped midgut in the thoracic region at 100% IPP (Fig. 1e), in contrast to the appearance of that organ at 50% IPP (Fig. 1c). As the indirect flight muscles grow and fill most of the thorax during intra-puparial development, the pre-helicoidal region of the midgut becomes narrower, being a clear indicator of the intra-puparial age [4, 5, 12]. Whereas the volume of the pre-helicoidal region of the midgut decreases, the lumen of the rectum and rectal pouch increases and at 90–100% IPP is typically filled with meconium [4, 5], which was clearly observable in the present histological sections (Fig. 1e and f).

Brown et al. [10] and Davies et al. [11] recommended a physical fixation of blow fly intra-puparial stages for histological analysis using near boiling water, instead of using chemical fixatives traditionally used in insect histology, like formaldehyde, Kahle’s, Bouin’s or Carnoy’s solutions [8, 17, 20, 21]. Hot water fixation has clear advantages in forensic casework: it is a readily available method and the accepted standard for the killing and fixation of blow fly larvae to enable subsequent analyses [1]. Previous studies using micro-CT-based virtual histology have shown that it is certainly an effective method for the fixation of the internal tissues of intra-puparial stages of cyclorrhaphous flies [4, 5, 12‐15, 23], and the present study corroborates that it is also a valid fixation method for preparing traditional histological sections. On the other hand, Brown et al. [10] and Davies et al. [11] recommended piercing with a pin throughout the insect to enable the penetration of the hot water [10] and facilitate the paraffin infiltration of the internal tissues [11]. In addition, Davies et al. [11] recommended the bisection of the intra-puparial form, once extracted from the puparium, to further enhance the paraffin infiltration. We disagree with those recommendations, as piercing and bisection damage the internal tissues and may result in a significant loss of information. Puncturing the puparium (but not the insect inside) after hot water fixation may be useful to enhance the penetration of contrast solutions for micro-CT scanning [4, 5, 12‐14, 23], but, for traditional histological analysis, it is preferable to dissect the puparium and extract the insect inside, which can then be taken through the dehydration, clearing and embedding processes with no need for piercing or bisection. Extracting the entire insect from the puparium also has the advantage of enabling an external morphological analysis [8, 9] prior to the internal histological analysis, thereby increasing the accuracy of the age estimation by combining both methods [8, 12]. Special caution should be taken with recently pupariated (i.e., not fully darkened) specimens, because during the first 5–7.5% IPP some parts of the insect body are still attached to the puparial wall (i.e., the larval-pupal apolysis is not complete) [3, 5]; hence, the extraction might be particularly challenging. However, from a forensic perspective this is not an issue, as the stage of insect development will be evident, hence the age and _minPMI when temperature is taken into account.

To avoid the piercing and bisection of the intra-puparial specimens, we strongly recommend the use of butanol and a vacuum, as it greatly enhances the proper paraffin infiltration of tissues in arthropod samples [17, 20, 24‐27]. Butanol is a solvent of paraffin and its advantages over other dehydrating agents like absolute ethanol or xylol have long been highlighted, as it prevents the hardening and shrinking of tissues [17, 24, 25, 27]. It has proven to be particularly useful for the serial sectioning of arthropods with a weakly chitinized cuticle [25, 27], which is the case of both the pupal and pharate adult cuticles [5]. Regarding the use of a vacuum oven, it facilitates the paraffin infiltration of tissues by providing negative atmospheric pressure conditions, thus accelerating the complete removal of solvents and air bubbles [26, 27]. Most modern tissue processors are equipped with a vacuum pump connection [27], so access to this facility should be readily available. Although the procedure described here is more time-consuming than other standard procedures [11], it enables the preparation of sections of the entire specimen (Fig. 1), minimising the risk of damaging informative tissues and structures. In order to optimise time and effort, we recommend sectioning the samples in the sagittal plane, as it enables the visualisation of most age-diagnostic morphological markers, not only for C. vicina [4, 5, 12, 15], but also for other forensically relevant Calliphoridae species and other families of cyclorrhaphous flies [4, 14, 23]. Regarding the staining method, the trichrome stain [20, 21] used here has shown to be very effective, but other widely used staining techniques, like haematoxylin-eosin [8, 11], may also provide good results. In conclusion, the method presented here should be easily applicable in most forensic institutions with access to histology facilities, providing an alternative for improving the accuracy of _minPMI estimations based on intra-puparial samples when access to more expensive techniques is not feasible.