A Study of the Pupal Development of Five Forensically Important Flies (Diptera: Brachycera)

Holometabolous insects undergo complete metamorphosis, and hence, they have different phases of development (egg, larva, pupa, and adult), which occupy distinct ecological niches. The pupae of several fly species are surrounded by the puparium, which is a rigid structure, usually formed by the integument of the last larval instar. The puparium presents unique characteristics distinct from those of the larval and adult phases. During intrapuparial development, it is possible to distinguish at least four fundamental and continuous steps, namely: 1) larval–pupal apolysis, 2) cryptocephalic pupa, 3) phanerocephalic pupa, and 4) pharate adult. The objective of this work was to describe the external morphology of the distinct phase of development for five species that were collected, identified, and raised in the laboratory; intrapuparial development was studied by fixing immature specimens at regular intervals; the morphological analyses were performed with the aid of both light and scanning electron microscopy. Under the conditions established (27 ± 1.0 or 23 ± 1.0°C, 60 ± 10% relative humidity, 12 h of photoperiod), the minimum time for intrapuparial development was: 252 h for Megaselia scalaris (Loew 1966) (Phoridae), 192 h for Piophila casei (Linnaeus 1758) (Piophilidae), Fannia pusio (Wiedemann 1830) (Fanniidae), and Musca domestica (Linnaeus 1758) (Muscidae), and 96 h for Chrysomya megacephala (Fabricius 1794) (Calliphoridae). Intrapuparial development has defined steps, and distinct species responded differently to the same environmental conditions. In addition, it is possible to establish a sequential rule without ignoring the specific characteristics of each taxon.

The optimal post-eclosion interval while estimating the post-mortem interval based on an empty puparium

The puparium is the hardened exoskeleton of the last larval instar of a fly, inside which a prepupa, a pupa and a pharate adult fly successively develop. Empty puparia are frequently collected at death scenes, especially in cases with a long post mortem interval (PMI). Although we are not able to estimate the interval between the eclosion of an adult fly and the collection of an empty puparium (i.e. the post-eclosion interval (PEI)), empty puparia may still provide valuable evidence about the minimum PMI. However, because of the unknown PEI, it is impossible to determine the time when the fly emerged, and thus when the retrospective calculation of the minimum PMI should start. In this study, the estimation of PMI (or minimum PMI) for empty puparia of Protophormia terraenovae Rob.-Desv. (Calliphoridae) and Stearibia nigriceps Meig. (Piophilidae) was simulated, to gain insight into the changes in estimates, when different PEIs and different temperature conditions were assumed. The simulations showed that the PEI (in a range of 0–90 days) had no effect on the PMI (or minimum PMI) when the puparium was collected in winter or early spring (December–April). In late spring, summer, or autumn (May–November) the PMI (or minimum PMI) increased with the PEI. The increase in PMI was large in the summer months, and surprisingly small in the autumn months, frequently smaller than the PEI used in the estimation. The shortest PMI was always obtained with a PEI of 0, indicating that the true minimum PMI is always estimated using a PEI of 0. When the puparium was collected during spring, simulations indicated that oviposition had occurred in the previous year, while in summer the previous-year oviposition has been indicated by the simulations only when longer PEIs had been assumed. These findings should guide estimation of the PMI (or minimum PMI) based on an empty puparium.

'Awareness' in metamorphosing pupae (Lepidoptera: Pieridae)

Neurobiological studies of model insect species have established that the nervous system retains some larval innervations, remodels others, and develops other new adult innervations during metamorphosis. Using a simple behavioral response – the ability to ‘kick’ by pupae of the pierid, Catopsilia pomona (Fabricius, 1775) - it was possible to assay for the retention of environmental awareness during ontogenetic reorganization. All pupae kicked 24h after ecdysis, 48% of pupae kicked at the pharate adult stage, and 28% of pupae kicked every day of their development (6.52 d ± 0.10). Both the mode and temporal expression of the response indicate that this retained awareness has larval origins. Variability in the response supports the inference that this response is mediated, to some extent, by prior experience rather than purely ‘reflex’. This is consistent with a Darwinian explanation of the behavior (and retained environmental awareness itself) as serving a protective function in pupae.

First Insights into the Intrapuparial Development of Bactrocera dorsalis (Hendel): Application in Predicting Emergence Time for Tephritid Fly Control

Intrapuparial development is a special pattern of metamorphosis in cyclorrhaphous flies, in which the pupa forms in an opaque, barrel-like puparium. This has been well studied in forensic insects for age estimations. In this study, the intrapuparial development of a quarantine agricultural pest, Bactrocera dorsalis (Hendel), was studied under a constant temperature of 27 ± 1 °C and 70 ± 5% relative humidity. Results showed that intrapuparial development could be divided into five stages: Larval-pupal apolysis, cryptocephalic pupa, phanerocephalic pupa, pharate adult, and emergent adult. It lays a morphology-based foundation for molecular mechanism studies and enhances the understanding of the physiological basis for changes in intrapuparial development. More importantly, the chronology of intrapuparial development can be used to predict the emergence time of tephritid flies, indicating when to spray insecticides to control these phytophagous agricultural pests. This may be an effective approach to reduce the use of insecticides and slow down the evolution of insecticidal resistance.

Intra-puparial development of Lucilia eximia (Diptera, Calliphoridae)

ABSTRACT There are few studies about the intra-puparial development in Diptera, nonetheless its importance has been increased because several dipteran species are of forensic interest. Studies on the life cycles of flies often disregard the changes that occur inside the pupae. The objectives of this research were to analyze the intra-puparial development of Lucilia eximia, and to describe chronological and morphological changes that occur during this stage. Around 1.600 specimens were laboratory-reared. The pre-pupae were identified by the reduction of their size and change in coloration, and 10 individuals were sampled every three hours (n=1.000) until adult emergence. The specimens were fixed in 96% alcohol, subsequently immersed in Canoy solution for 24 hours and in formic acid (5%) for 48 hours, to facilitate dissection and analysis of the morphological changes of the individuals. Four stages of the intra-pupal development of L. eximia were observed: 1) Larva-pupa apolysis, which lasted 23 ± 1.08 h; 2) Cryptocephalic pupa, 5 ± 0.53 h; 3) Phanerocephalic pupa, 92 ± 1.94 h; and 4) Pharate adult: Transparents eyes, 125 ± 2.15 h; Yellow eyes, 23 ± 0.89 h; Pink eyes, 14 ± 0.73 h; and Red eyes, 20 ± 0.60 h. The pharate adult is completely formed after 296 hours and the emergence of the adult occurred after 302 ± 3.81 h. In addition, there were included the stage of pre-pupa, pupariation and the beginning of the adult stage, Imago and adult emergence. Each stage is described and compared with those described for Chrysomya albiceps (Calliphoridae).

New chironomid pupal types from Norway, one with a male pharate adult: Pseudosmittia paraspinispinata n.sp.

Three new pupal types from Norway are described, one with an associated male: Pseudosmittia parspinispinata n.sp.