Intra-puparial Period Research Articles

Development and age estimation of the intrapuparial period of Phormia regina (Diptera: Calliphoridae) for postmortem interval estimation.

Phormia regina (Meigen, 1826; Diptera: Calliphoridae) is a Holarctic species that rapidly colonizes carcasses and has been used as an indicator for determining the minimum postmortem interval. However, studies using morphological methods to estimate the intrapuparial age of P. regina are lacking. In this study, morphological changes within the puparium were observed under a stereomicroscope at 7 constant temperatures ranging from 16 °C to 34 °C. The intrapuparial period was categorized into 12 substages. Morphological indicators, including compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen, were recorded in detail. The observed morphological changes were divided into 6-10 substages, and the duration of each substage was also recorded in detail. The results of this study provide primary data for using the intrapuparial morphology of P. regina when pupae are collected at a crime scene and estimating the minimum postmortem interval.

Age estimation of Phormia regina pupae based on ATR-FTIR and chemometrics

Accurate postmortem interval estimation is vital in the investigation of homicides, suicides, and accidental deaths. It is key in narrowing suspect lists, improving crime-solving efficiency, and offering solace to bereaved families. The intra-puparial period, comprising about half of a fly’s developmental cycle, presents challenges for morphological age estimation. External changes are limited to color shifts and the appearance of respiratory horns on the puparium only within several hours after pupariation, while detailed internal development analysis often requires invasive methods like removing the puparium, which can be damaging. Additionally, these techniques usually depend on a forensic entomologist’s expertise, which lead to subjective biases. This study employed attenuated total reflection-fourier transform infrared spectroscopy, a rapid, non-destructive method for analyzing proteins, chitosan, and chitin in puparia. Data showed a consistent reduction in the concentration of the amide I band within the puparium during the intra-puparial development at five constant temperatures (19 °C, 22 °C, 25 °C, 28 °C and 31 °C). This trend in the spectral data effectively distinguishes pupae at various stages of intra-puparial development, facilitating precise age estimation, which is critical for the estimation of the minimum postmortem interval (PMImin). Finally, this work combined the total reflection-fourier transform infrared spectroscopy with chemometric analysis and successfully developed a partial least squares discriminant analysis model and a random forest model, with accuracies of 88 % and 81 %, respectively. These models enable the non-invasive age estimation of P. regina in its intra-puparial period, a stage traditionally difficult to assess morphologically, thus laying the groundwork for PMImin estimation using fly pupae.

A methodological approach to age estimation of the intra-puparial period of the forensically relevant blow fly Calliphora vicina via Fourier transform infrared spectroscopy.

Estimating the age of immature blow flies is of great importance for forensic entomology. However, no gold-standard technique for an accurate determination of the intra-puparial age has yet been established. Fourier transform infrared (FTIR) spectroscopy is a method to (bio-)chemically characterise material based on the absorbance of electromagnetic energy by functional groups of molecules. In recent years, it also has become a powerful tool in forensic and life sciences, as it is a fast and cost-effective way to characterise all kinds of material and biological traces. This study is the first to collect developmental reference data on the changes in absorption spectra during the intra-puparial period of the forensically important blow fly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). Calliphora vicina was reared at constant 20°C and 25°C and specimens were killed every other day throughout their intra-puparial development. In order to investigate which part yields the highest detectable differences in absorption spectra throughout the intra-puparial development, each specimen was divided into two different subsamples: the pupal body and the former cuticle of the third instar, that is, the puparium. Absorption spectra were collected with a FTIR spectrometer coupled to an attenuated total reflection (ATR) unit. Classification accuracies of different wavenumber regions with two machine learning models, i.e., random forests (RF) and support vector machines (SVMs), were tested. The best age predictions for both temperature settings and machine learning models were obtained by using the full spectral range from 3700 to 600 cm-1. While SVMs resulted in better accuracies for C. vicina reared at 20°C, RFs performed almost as good as SVMs for data obtained from 25°C. In terms of sample type, the pupal body gave smoother spectra and usually better classification accuracies than the puparia. This study shows that FTIR spectroscopy is a promising technique in forensic entomology to support the estimation of the minimum post-mortem interval (PMImin), by estimating the age of a given insect specimen.

Intrapuparial development and age estimation of Sarcophaga peregrina (Diptera: Sarcophagidae) for postmortem interval estimation

Sarcophaga peregrina Robineau-Desvoidy, 1830 (Diptera: Sarcophagidae) is a widely distributed species in the Palearctic, Oriental and Australian regions. Numerous studies and case reports have confirmed the value of this species in forensic investigations, but methods for estimating the age of the intrapuparial period of this species are lacking. In this study, S. peregrina pupae were sampled at 16 °C, 19 °C, 22 °C, 25 °C, 28 °C, 31 °C, and 34 °C. Morphological changes within the puparium were observed under a stereomicroscope. The results of the study showed that S. peregrina could complete its intrapuparial development at each of the seven tested constant temperatures. As the temperature increased, the average minimum duration of the intrapuparial period of S. peregrina decreased from 706.7 ± 28.1 h at 16 °C to 170.7 ± 4.6 h at 34 °C. The intrapuparial morphological changes of S. peregrina can be divided into 12 sub-stages. While some structures such as the compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen can be used individually to estimate intrapuparial age, we observed all these structures with local magnification photographs and classified the developmental process of these structures into 6–11 stages. The time range for the appearance of each sub-stage or structure was recorded in detail. The results of this study provide important basic data for estimating the minimum postmortem interval using the intrapuparial morphology of S. peregrina.

Development of Forensically Important Sarcophaga peregrina (Diptera: Sarcophagidae) and Intra-Puparial Age Estimation Utilizing Multiple Methods at Constant and Fluctuating Temperatures.

Sarcophaga peregrina (Robineau-Desvoidy, 1830) has the potential to estimate the minimum postmortem interval (PMImin). Development data and intra-puparial age estimation are significant for PMImin estimation. Previous research has focused on constant temperatures, although fluctuating temperatures are a more real scenario at a crime scene. The current study examined the growth patterns of S. peregrina under constant (25.75 °C) and fluctuating temperatures (18-36 °C; 22-30 °C). Furthermore, differentially expressed genes, attenuated total reflectance Fourier-transform infrared spectroscopy, and cuticular hydrocarbons of S. peregrina during the intra-puparial period were used to estimate age. The results indicated that S. peregrina at fluctuating temperatures took longer to develop and had a lower pupariation rate, eclosion rate, and pupal weight than the group at constant temperatures did. Moreover, we found that six DEG expression profiles and ATR-FTIR technology, CHCs detection methods, and chemometrics can potentially estimate the intra-puparial age of S. peregrina at both constant and fluctuating temperatures. The findings of the study support the use of S. peregrina for PMImin estimation and encourage the use of entomological evidence in forensic practice.

Estimating the intra-puparial period of Hydrotaea spinigera (Stein,1910) (Diptera: Muscidae) with morphological and gene expression changes

Hydrotaea spinigera (Stein, 1910) (Diptera: Muscidae) is a forensically important sarcosaprophagous species widely distributed throughout the Oriental and Australasian regions. At the advanced decomposition stage or the skeletonize stage, the immature stages of H. spinigera, especially the pupae, can still be found in large quantities and could be used as important indicators to estimate the minimum postmortem interval (PMImin). However, there have been no studies on the intra-puparial period of this species. Herein, we studied morphological and differential gene expression changes during the intra-puparial development of H. spinigera, aiming to accurately estimate the intra-puparial age of H. spinigera. The intra-puparial morphological changes of H. spinigera were observed at seven constant temperatures ranging from 16 °C to 34 °C and divided into 12 sub-stages. Structures that could be used to estimate the intra-puparial age, such as compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen, were observed in detail, and the developmental process of each structure was divided into 5 to 10 stages. The time range of each sub-stage, or when a structure appeared, was recorded. For the gene expression section, the most suitable reference genes were screened by geNorm, NormFinder, BestKeeper, and ΔCt methods. Based on the selected reference genes, real-time quantitative PCR (RT qPCR) was used to detect the expression changes of the white and hsp90 genes with developmental time at 19 °C, 25 °C, and 31 °C. Results showed that the trend of hsp90 gene expression under different temperatures was not consistent, while white genes exhibited regular changes during development, and could thus be used for age estimation of H. spinigera. This study provides an important basis for forensic entomology to use morphological and differential gene expression for estimating the age of H. spinigera during the intra-puparial period. Moreover, the combination of the two methods can produce a more accurate minimum postmortem interval (PMImin) estimate compared to when each method is used separately.

Multimethod combination for age estimation of Sarcophaga peregrina (Diptera: Sarcophagidae) with implications for estimation of the postmortem interval.

Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a forensically important flesh fly with potential value for estimating the minimum postmortem interval (PMImin). Here, the developmental patterns of S. peregrina were investigated at 5 constant temperatures (15-35°C). Morphological changes at different developmental stages of the pupa were observed at 4 constant temperatures (15-30°C) by removing the puparium and staining the pupa with hematoxylin and eosin. Furthermore, differentially expressed genes (DEGs) were analyzed at 25°C in the intrapuparial period to estimate the age of S. peregrina during the intrapuparial stage. S. peregrina completed development from larviposition to adult eclosion at 15°C, 20°C, 25°C, and 30°C; the developmental durations were 1090.3 ± 30.6h, 566.6 ± 21.9h, 404.6 ± 13.01h, and 280.3 ± 4.5h, respectively, while the development could not be completed at 35°C. The intrapuparial period of S. peregrina was divided into 12 sub-stages on the basis of the overall external morphological changes; 6 sub-stages on the basis of individual morphological structures such as the compound eyes, antennae, thorax, legs, wings, and abdomen; and 10 sub-stages on the basis of internal morphological changes detected using histological analysis. The period of each sub-stage or structure that appeared was determined. Moreover, we found that 6 genes (NDUFS2, CPAMD8, NDUFV2, Hsp27, Hsp23, and TPP) with differential expression can be used for the precise age estimation of S. peregrina during the intrapuparial period. This study provided basic developmental data for the use of S. peregrina in PMImin estimation, and we successfully estimated PMImin in a real forensic case by using a multimethod combination.

An X-ray micro-computer tomography study of the Malpighian tubules of the Blue Bottle Blow Fly (Calliphora vomitoria) Diptera: Calliphoridae

Malpighian tubules are the insect equivalent of mammalian kidneys and normally drain into the gut at the junction between the mid and hind gut. The Malpighian tubules of the fruit fly Drosophila melanogaster are increasingly being used as a model for studying human renal tract development, histology, nephrolithiasis and urolithiasis. In the present study we report when using X-ray micro-computer tomography techniques, the larval, intrapuparial and adult stages of the larger Calliphora vomitoria can contain large amounts of calcium-rich concretions which are tightly packed in the lumen of both anterior Malpighian tubules. We show that it is feasible to utilise these calcium-rich concretions as a form of marking agent to delineate the various developmental stages of the Malpighian tubules including the crucial phase when the Malpighian tubules reconnect with the hind gut. In the majority of cases during the intrapuparial period the ureters of the Malpighian tubules did not start to re-canalise and thus reconnect with the developing hind gut until the 7th day of the 10-11 day. Just prior to ecdysis, virtually all the radio-opaque concretions in the Malpighian tubules had emptied into the hind gut and had then been completely excreted by the time the imago emerged from its puparium. In contrast, we show that in flies developing from larvae previously stained by ingesting Rhodamine B, a known substrate for both the Multi Xenobiotic Resistance and Multi Drug Resistant membrane transport systems, the efficiency with which these calcium-rich concretions are excreted by the imago as it emerges from its intrapuparial period can be significantly impaired. Therefore, it might be useful to include C. vomitoria as a model when studying renal tract development and urolithiasis using X-ray micro-computer tomography.

Overwintering behaviour of the skipper fly (Diptera: Piophilidae) of forensic importance in Québec, Canada

AbstractLaboratory experiments and field observations from August 2019 to April 2020 demonstrated that skipper flies (Diptera: Piophilidae) overwinter as larvae and likely present an obligatory winter diapause in Québec, Canada. Diapause was confirmed by the very few emergences of adults in the laboratory following collection from carrion at the end of summer and in fall, the migration of larvae deep in the soil, and the persistence of larvae inside carrion during the winter until the following spring when they became active again and initiated pupation (i.e., intra-puparial period). To our knowledge, our observations represent the first report of the overwintering of skipper fly larvae inside an animal carcass in North America. In addition, this winter diapause should be considered in forensic entomology when using the presence of skipper fly larvae on a body to estimate the time elapsed since death.

Evaluation of Reference Genes and Age Estimation of Forensically Useful Aldrichina grahami (Diptera: Calliphoridae) During Intrapuparial Period.

The minimum postmortem interval (PMImin) could be evaluated from the developmental stage of forensically important insects colonize a corpse, such as blow flies (Diptera: Calliphoridae). Unlike larvae, the developmental stage of which is well established according to their morphology, estimating the age of pupae is proven to be challenging. Recently, several studies reported the regulation of special genes during the development of blow fly pupae. However, gene regulation in Aldrichina grahami during the intrapuparial period remains to be studied. Therefore, we set out to investigate the mRNA levels of heat shock protein 23 (Hsp23), heat shock protein 24 (Hsp24), and 1_16 during the metamorphosis of A. grahami pupae. First, we examined seven candidate reference genes (ribosomal protein 49 (RP49), 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), beta-tubulin at 56D (β-tubulin), Ribosomal protein L23 (RPL23), glutathione S-transferase (GST1), and Actin. Three widely used algorithms (NormFinder, BestKeeper, and geNorm) were applied to evaluate the mRNA levels of reference gene candidates in puparium at three stable temperatures (15, 22, and 27°C). Next, mRNA expression of Hsp23, Hsp24, and 1_16 during A. grahami metamorphosis was examined. We demonstrated that mRNA expression levels of Hsp23, Hsp24, and 1_16 showed time-specific regulation. In summary, our study identified three gene markers for the intrapuparial period of A. grahami and might provide a potential application in PMImin estimation.

Intrapuparial Development of Hemilucilia semidiaphana (Diptera: Calliphoridae) and Its Use in Forensic Entomology.

In forensic entomology, the minimum postmortem interval (PMImin) estimative is usually based on the oldest immature recovered from a local of death. The time spent by fly immatures in the intrapuparial period comprises more than 50% of their complete life cycles. An accurate estimate of the duration of this period will improve PMImin estimates. The blow fly Hemilucilia semidiaphana (Rondani 1850) was found in six criminal cases in the city of Curitiba. Even though there is data on the morphology of the larval instars and developmental rate of H. semidiaphana, the intrapuparial period has not been investigated. Here, we provide a detailed description of the intrapuparial morphological changes of H. semidiaphana, which might be useful to estimate minimum PMI. Samples of H. semidiaphana in the intrapuparial period were obtained from immatures reared on an artificial diet in incubators adjusted to 25°C or 20°C temperature regimes. Blow fly puparia of H. semidiaphana were fixed at intervals of 3 and 6 h until emergence of the adult. The external morphological traits of sampled immatures were analyzed using light microscopy. Our analysis of the intrapuparial period of H. semidiaphana provided 21 traits from which nine were age informative. These nine characteristics divide the developmental time (144 h at 25°C and 192 h at 20°C) into smaller sections. The developmental data provided, together with the time line allows a practical way to make interspecific comparisons as well as to estimate the age of H. semidiaphana based on the intrapuparial development.

Gene expression during the intra-puparial stage of Chrysomya megacephala: Implications for postmortem interval estimation

Abstract Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) is one of the most predominant calliphorid species which arrives and colonizes a cadaver first in its native range of the Australian and Oriental regions, and the intra-puparial stage of this species accounts for about half of its immature stage. Therefore, establishing a reliable aging method of the intra-puparial stage of C. megacephala is very important to accurately estimate the minimum postmortem interval (PMImin). In this study, actin was used as an internal reference gene to study the expression of three genes, ecdysone receptor gene ecr, white-eye gene white, and heat shock protein gene hsp70, at different time points during intra-puparial development of C. megacephala. Quantification through real-time PCR revealed that these genes can be used to age the intra-puparial period of C. megacephala as they exhibit regular changes and gene expression level was temperature-dependent. The overall gene expression profile of ecr showed a downward trend throughout the intra-puparial stage. White expression increased steadily until it peaked when intra-puparial development reached 45%, and the expression began to decrease when the intra-puparial development reached 55%. hsp70 was first down-regulated at 0–15% of intra-puparial development, and then slightly up-regulated at 15–40%, and finally down-regulated again until the end of intra-puparial development. This study provides molecular indicators of age during the intra-puparial stage of C. megacephala, and combining gene expression with the morphological methods can lead to more accurate estimation for PMImin.

Estimating the age of Lucilia illustris during the intrapuparial period using two approaches: Morphological changes and differential gene expression

Lucilia illustris (Meigen, 1826) (Diptera: Calliphoridae) is a cosmopolitan species of fly that has forensic and medical significance. However, there is no relevant study regarding the determination of the age of this species during the intrapuparial period. In this study, we investigated the changes in both morphology and differential gene expression during intrapuparial development, with an aim to estimate the age of L. illustris during the intrapuparial stage. The overall intrapuparial morphological changes of L. illustris were divided into 12 substages. Structures such as the compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen, each capable of indicating age during the intrapuparial stage, were observed in detail, and the developmental progression of each of these structures was divided into six to eight stages. We recorded the time range over which each substage or structure appeared. The differential expression of the three genes 15_2, actin, and tbp previously identified for predicting the timing of intrapuparial development was measured during L. illustris metamorphosis. The expression of these genes was quantified by real-time PCR, and the results revealed that these genes can be used to estimate the age of L. illustris during the intrapuparial period, as they exhibit regular changes and temperature dependence. This study provides an important basis for estimating the minimum postmortem interval (PMImin) in forensic entomology according to changes in intrapuparial development and differential gene expression. Furthermore, combination of the two approaches can generate a more precise PMImin than either approach alone.

Initial investigations of spectral measurements to estimate the time within stages of Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae)

Current applications of forensic entomology to post-mortem interval estimations involve ageing the insects colonizing the remains based on minimum time to reach the oldest stage of development. Immature species of blow fly develop at a predictable rate to each stage of development in their lifecycle. Unfortunately, the minimum time to reach a stage of development can be a rather unrefined estimate of tenure on the body in the sometimes lengthy time frame of the later stages. In a successful attempt to narrow this time frame, daily spectral measurements of the immature stages of Protophormia terraenovae (Robineau-Desvoidy) raised at a mean temperature of 24.6°C were collected and the functional data analysis was completed. Functional regressions and coefficient functions were examined for model prediction and generalization. P. terraenovae is a Holarctic species as well as an early colonizer of human remains and is therefore, an excellent indicator species in North American death investigations.Spectral measurements can be used successfully to estimate the day of development in the third instar including post feeding stage. In the intra-puparial period, however, only the last day of development could be distinguished from the earlier days of the intra-puparial period. Distinguishing day within second instar is also possible for P. terraenovae raised at a mean temperature of 24.6°C and, although not fully within the pointwise 95% confidence interval, it still accurately predicts the day.The results of this proof of concept research are promising and show a potential method for narrowing the original death estimates and offering a better overall estimate of age of P. terraenovae larvae and, therefore; estimated time since death.

Age estimation during the blow fly intra-puparial period: a qualitative and quantitative approach using micro-computed tomography

Minimum post-mortem interval (minPMI) estimates often rely on the use of developmental data from blow flies (Diptera: Calliphoridae), which are generally the first colonisers of cadavers and, therefore, exemplar forensic indicators. Developmental data of the intra-puparial period are of particular importance, as it can account for more than half of the developmental duration of the blow fly life cycle. During this period, the insect undergoes metamorphosis inside the opaque, barrel-shaped puparium, formed by the hardening and darkening of the third instar larval cuticle, which shows virtually no external changes until adult emergence. Regrettably, estimates based on the intra-puparial period are severely limited due to the lack of reliable, non-destructive ageing methods and are frequently based solely on qualitative developmental markers. In this study, we use non-destructive micro-computed tomography (micro-CT) for (i) performing qualitative and quantitative analyses of the morphological changes taking place during the intra-puparial period of two forensically relevant blow fly species, Calliphora vicina and Lucilia sericata, and (ii) developing a novel and reliable method for estimating insect age in forensic practice. We show that micro-CT provides age-diagnostic qualitative characters for most 10% time intervals of the total intra-puparial period, which can be used over a range of temperatures and with a resolution comparable to more invasive and time-consuming traditional imaging techniques. Moreover, micro-CT can be used to yield a quantitative measure of the development of selected organ systems to be used in combination with qualitative markers. Our results confirm micro-CT as an emerging, powerful tool in medico-legal investigations.

Spectral Signatures of Immature Lucilia sericata (Meigen) (Diptera: Calliphoridae).

Hyperspectral remote sensing is an innovative technology with applications in many sciences and is a non-destructive method that may offer more precise aging within development stages. Hyperspectral reflectance measurements from the anterior, midsection, and posterior of Lucilia sericata (Meigen) larvae and pupae were conducted daily from samples of the developing insects beginning at second instar. Only midsection measurements were conducted on second instar larvae due to their size, to ensure that the measurement was not of reflective surroundings. Once measured, all insects were washed with deionized water, blotted with filter paper, and re-measured. Daily age prediction during the post-feeding stage was not impacted by the unwashed insect measurements and was best predicted based on posterior measurements. The second and third instar larvae, which move about their food source, had different contributing coefficients to the functional regression model for the hyperspectral measurements of the washed compared with unwashed specimens. Although washing did not affect the daily prediction within these stages, it is still encouraged in order to decrease the effect of food source on spectral reflectance. Days within the intra-puparial period were best predicted based on anterior measurements and were not well distinguished from one another in the first few days based on midsection and posterior measurements.

The 'dance' of life: visualizing metamorphosis during pupation in the blow fly Calliphora vicina by X-ray video imaging and micro-computed tomography.

The dramatic metamorphosis from larva to adult of insect orders such as Diptera cannot usually be witnessed because it occurs within an opaque structure. For the cyclorrhaphous dipterans, such as blow flies, this structure is the puparium, formed from the larval cuticle. Here, we reveal metamorphosis within the puparium of a blow fly at higher temporal resolution than previously possible with two-dimensional time-lapse videos created using the X-ray within a micro-computed tomography scanner, imaging development at 1 min and 2 min intervals. Our studies confirm that the most profound morphological changes occur during just 0.5% of the intrapuparial period (approx. equivalent to 1.25 h at 24°C) and demonstrate the significant potential of this technique to complement other methods for the study of developmental changes, such as hormone control and gene expression. We hope this will stimulate a renewed interest among students and researchers in the study of morphology and its astonishing transformation engendered by metamorphosis.

Isolation of the Male-Specific Transformer Exon as a Method for Immature Specimen Sex Identification in Chrysomya megacephala (Diptera: Calliphoridae).

Being able to efficiently differentiate between male and female individuals in the immature forms of insects allows for investigations into sexually dimorphic patterns of growth rates and gene expression. For species lacking sex-specific morphological characteristics during these periods, alternative methods must be devised. Commonly, isolation of sex determination genes reveals sex-specific band patterns and allows for markers that can be used in insect control. For blow flies, a family that includes flies of medical and forensic importance, sex has previously been identified in some members using the male-specific exon in the transformer gene. This gene is relatively conserved between members of the genera Cochliomyia and Lucilia (Diptera: Calliphoridae), and we isolated a portion of this gene in an additional forensically and medically important blow fly genus using the widespread Chrysomya megacephala (F.). We found a relatively high level of conservation between exons 1 and 2 of transformer and were able to amplify a region containing the male-specific exon in C. megacephala. A sex-specific molecular diagnostic test based on the presence of sexually dimorphic PCR product bands showed the expected genotype for adults and intrapuparial period specimens of known sex. The same result could be obtained from single third-instar larval specimens, opening up the possibility to not only determine if development rates are sex dependent, but also to investigate the development of sexually dimorphic traits of interest in C. megacephala.

Intra-puparial development in Oestrus ovis (Diptera: Oestridae).

The phenology of intrapuparial development in Oestrus ovis L. is described, based on 302 specimens collected from the head cavities of goats and reared in the laboratory at a photoperiod of 12:12 (L:D) h and 32 and 16 degrees C. Dissection and histology of puparia at pupariation and at 3, 6, 9, 12, 15, 18, 21, 24, 30, 36, 42, 48, 66, and 72 h after pupariation and every day of the intra-puparial period showed that pupariation was achieved in approximately 12 h in heavily pigmented larvae (range, 2-46 h in postfeeding period). Larval-pupal apolysis began immediately after pupariation and was completed by 18-36 h after pupariation (prepupal period). The cryptocephalic pupa was found from this time to the 5th d, when head eversion occurred. Pupal-adult apolysis was initiated before head eversion and completed by day 7. The pharate adult presented progressive coloration in compound eyes (transparent, white, yellow, orange, red, brown, silver) while integumental pigmentation and sclerotization were in progress. Adult emergence occurred at 22 and 23 d in males and females, respectively. Changes in the weekly puparial weight of specimens reared under both field and laboratory conditions was described. It was concluded that although the intra-puparial development of O. ovis displayed some unique characteristics, it was essentially similar to other cyclorraphous flies. The actual pupal period of O. ovis lasted from the 2-7 d post-pupariation, whereas approximately two-thirds of the intra-puparial period was used for the maturation of the pharate adult.