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

Blow flies (Diptera: Calliphoridae) are generally early colonisers of fresh cadavers, enabling the estimation of a minimum post-mortem interval (minPMI) based on an accurate aging of the oldest immature stages associated with a cadaver. In blow flies, the pupal stage and the subsequent development of the adult take place inside a protective case, the puparium, formed from the hardened and darkened cuticle of the third instar larva. Because the puparium is an opaque structure that shows virtually no external changes, qualitative analyses of the internal tissues can be very informative for determining reliable age-specific morphological markers. Those analyses can be performed using either non-invasive but expensive and not widely accessible techniques, or traditional histological methods, which are invasive as they require the serial sectioning of the sample. Histological methods are often readily available for forensic researchers and practitioners; however, the histological study of blow fly intra-puparial stages has traditionally been hampered by the poor paraffin infiltration of tissues due to the abundance of fat bodies, resulting in usually fragmented sections and the subsequent loss of relevant information. We present here an effective method for the preparation of histological sections of blow fly intra-puparial stages, maximising the paraffin infiltration while enabling the production of clean and entire sections that allow for the use of reliable age-specific morphological markers, thus improving the accuracy of minPMI estimations when access to more costly techniques is not feasible.

Methods for the optimal preservation of blow fly intra-puparial forms for morphological analysis in forensic casework.

Accurate minimum post-mortem interval (minPMI) estimations often rely on a precise age determination of insect developmental stages, which is significantly influenced by environmental temperature. An optimal preservation of the entomological samples collected at crime scenes is pivotal for a reliable aging of immature insect samples. For blow flies (Diptera: Calliphoridae), the most widely used insect indicators in forensic investigations, an appropriate preservation of tissues is particularly important in the case of puparial samples because aging methods for intra-puparial forms usually depend on morphological analyses; however, although informative soft tissues and structures could be discoloured and/or distorted if they are not properly fixed, there is a lack of studies to assess different methods for the optimal preservation of intra-puparial forms collected in forensic investigations. The present study compares three preservation methods for intra-puparial forms of the blow fly Calliphora vicina Robineau-Desvoidy, 1830: (i) direct immersion into 80% ethanol, (ii) puncturing of the puparium and hot water killing (HWK) prior to preservation in 80% ethanol, and (iii) HWK without puncturing before preservation in 80% ethanol. External and internal morphological analyses of intra-puparial forms of different ages were conducted to assess the quality of preservation. The results indicate that direct immersion in ethanol led to poor preservation, affecting both external and internal tissues. Both methods with HWK resulted in a better preservation, but puncturing resulted, in some cases, in physical damage of the specimens. HWK without puncturing emerged as the optimal preservation method, consistently yielding high preservation scores for both external and internal morphological analyses. These findings have practical implications for forensic practitioners and emphasise the need for updating some published guidelines and protocols in forensic entomology.

Larval anatomy of the digestive and excretory systems of the pharyngeal bot fly, Pharyngomyia picta (Diptera: Oestridae).

Oestrid flies (Diptera: Oestridae) are obligate parasites of mammals during their larval stage and show anatomical adaptations for the infestation of host tissues. Unlike the species that parasitize domestic mammals, those oestrid species that infest wild mammal hosts remain poorly known. With the use of x-ray micro-computed tomography, we describe for the first time the anatomy of the digestive and excretory systems of the second and third larval instars of Pharyngomyia picta (Meigen), a parasite of cervids that, like other species within the subfamily Oestrinae, causes nasopharyngeal myiasis. Both larval instars of P. picta show a pair of remarkably large salivary glands arranged in a characteristic 'glandular band', a convoluted and thickly uniform midgut and a greatly enlarged distal region of the anterior pair of Malpighian tubules. These anatomical features also have been described in other species within the subfamily Oestrinae, whereas they differ from the observations in other oestrid subfamilies. We discuss the potential functional significance of the anatomy of the digestive and excretory systems of Oestrinae larvae as specific adaptations to parasitize the nasopharyngeal cavities of mammal hosts.

Morphological and molecular characterization of puparia of Piophilidae species of forensic relevance.

Piophilidae are a small family of Diptera with a worldwide distribution and which are historically associated with human activities. In addition to their economic importance, piophilid larvae can also be of medical and legal relevance. Within a medicolegal context, piophilids are frequently associated with cadavers in advanced stages of decomposition, thus being potentially useful forensic indicators and they have been reported also from archaeo-funerary contexts. An accurate species identification is therefore an essential prerequisite to ensure the reliable analysis of insect material in medical, forensic and archaeological investigations. Identification of the adult piophilid flies is possible because of the availability of identification keys, in contrast immature insects, especially puparia, have been poorly investigated and described. In this paper, puparia of 11 species of forensic interest (Piophila casei, Piophila megastigmata, Parapiophila atrifrons, Parapiophila flavipes, Parapiophila vulgaris, Protopiophila litigata, Liopiophila varipes, Prochyliza nigrimana, Prochyliza xanthosoma and Stearibia nigriceps in subtribe Piophilina and Centrophlebomyia furcata in subtribe Thyreophorina) are described and a molecular analysis, based on the COI sequencing, is presented to show the potential of the molecular approach in their identification.

Micro-CT visualization of a promastigote secretory gel (PSG) and parasite plug in the digestive tract of the sand fly Lutzomyia longipalpis infected with Leishmania mexicana.

Leishmaniasis is a debilitating disease of the tropics, subtropics and southern Europe caused by Leishmania parasites that are transmitted during blood feeding by phlebotomine sand flies (Diptera: Psychodidae). Using non-invasive micro-computed tomography, we were able to visualize the impact of the laboratory model infection of Lutzomyia longipalpis with Leishmania mexicana and its response to a second blood meal. For the first time we were able to show in 3D the plug of promastigote secretory gel (PSG) and parasites in the distended midgut of whole infected sand flies and measure its volume in relation to that of the midgut. We were also able to measure the degree of opening of the stomodeal valve and demonstrate the extension of the PSG and parasites into the pharynx. Although our pilot study could only examine a few flies, it supports the hypothesis that a second, non-infected, blood meal enhances parasite transmission as we showed that the thoracic PSG-parasite plug in infected flies after a second blood meal was, on average, more than twice the volume of the plug in infected flies that did not have a second blood meal.

Looking Back to Move Forward: How Review Articles Could Boost Forensic Entomology.

The Locard's exchange principle (1930) holds that the perpetrator of a crime leaves traces behind that can later be sampled and used as forensic evidence [...].

First report of the forensically important fly, Stearibia nigriceps (Diptera: Piophilidae) in South Korea: Confirmation of specimens from human corpses based on cytochrome c oxidase subunit I barcodes.

Piophilidae are a relatively small family of Diptera that is frequently associated with cadavers at advanced stages of decomposition and are, therefore, considered potentially useful forensic indicators. However, their use in forensic investigations is typically hampered by a deficiency in reliable identification tools. This is particularly evident in countries such as South Korea, where forensic entomology is still in its infancy and the diversity of forensically relevant insect taxa remains largely undocumented. In the present study, we used cytochrome c oxidase subunit I (COI) barcodes to identify samples of piophilid larvae collected during medicolegal investigations performed in South Korea. A total of 174 COI sequences were obtained and have been made publicly available, thus augmenting the reference barcode library for forensically important Piophilidae species. Of the 174 sequenced samples, 172 were identified as Stearibia nigriceps (Meigen), whereas the two remaining samples may represent a previously unsequenced piophilid species. Stearibia nigriceps is recorded from South Korea for the first time, and our results suggest that it might be a particularly relevant forensic indicator in certain case types and scenarios in that country. The findings of this study highlight the utility of COI barcodes for achieving accurate identification of entomological samples, even by non-specialist forensic practitioners. They also contribute to the further development and consolidation of forensic entomology in South Korea and eastern Asia.

Wing morphometrics for the identification of Nearctic and Palaearctic Piophilidae (Diptera) of forensic relevance.

The Piophilidae (Diptera) are a family comprising about 80 species, several of them of high economic and forensic relevance. An unequivocal species identification is crucial for designing effective control measures or to provide reliable estimations of the minimum post mortem interval. However, the identification may sometimes not be possible, either because the diagnostic morphological characters are not easily observable or very fragile, or because of a poor DNA quality and/or unavailability of reference molecular signatures. In the recent years, the use of wing morphometric techniques has emerged as a powerful tool for the identification of different families of forensically important Diptera. The present study applies, for the first time, this technique to the identification of 11 Piophilidae species of forensic relevance in the Nearctic and Palaearctic regions, as well as to the discrimination of sexes, distinct geographical populations and, in the case of the dimorphic species Prochyliza nigrimana (Meigen), seasonal morphs. Wing shape variation was analysed using 14 landmarks located at wing vein junctions and a cross-variation analysis was used to test the reliability of identifications. The present results demonstrate that wing shape can be used to identify most species with relatively high success, whereas cross-validation analyses indicated that discrimination between sexes, populations or morphs was less effective, even if significant differences were observed in every comparison. We conclude that wing morphometrics can be a powerful identification tool that might be used in combination with other methods in order to achieve accurate and reliable species identifications-independently of the sex, geographic origin or colour variation of the samples-even with those piophilid species that have been object of frequent misidentifications.

Estimating crime scene temperatures from nearby meteorological station data.

The importance of temperature data in minimum postmortem interval (minPMI) estimations in criminal investigations is well known. To maximise the accuracy of minPMI estimations, it is imperative to investigate the different components involved in temperature modelling, such as the duration of temperature data logger placement at the crime scene and choice of nearest weather station to compare the crime scene data to. Currently, there is no standardised practice on how long to leave the temperature data logger at the crime scene and the effects of varying logger duration are little known. The choice of the nearest weather station is usually made based on availability and accessibility of data from weather stations in the crime scene vicinity. However, there are no guidelines on what to look for to maximise the comparability of weather station and crime scene temperatures. Linear regression analysis of scene data with data from weather stations with varying time intervals, distances, altitudes and microclimates showed the greatest goodness of fit (R2), i.e. the highest compatibility between datasets, after 4-10 days. However, there was no significant improvement in estimation of crime scene temperatures beyond a 5-day regression period. The smaller the distance between scene and weather station and the higher the similarity in environment, such as altitude and geographical area, resulted in greater compatibility between datasets. Overall, the study demonstrated the complexity of choosing the most comparable weather station to the crime scene, especially because of a high variation in seasonal temperature and numerous influencing factors such as geographical location, urban 'heat island effect' and microclimates. Despite subtle differences, for both urban and rural areas an optimal data fit was generally reached after about five consecutive days within a radius of up to 30 km of the 'crime scene'. With increasing distance and differing altitudes, a lower overall data fit was observed, and a diminishing increase in R2 values was reached after 4-10 consecutive days. These results demonstrate the need for caution regarding distances and climate differences when using weather station data for retrospective regression analyses for estimating temperatures at crime scenes. However, the estimates of scene temperatures from regression analysis were better than simply using the temperatures from the nearest weather station. This study provides recommendations for data logging duration of operation, and a baseline for further research into producing standard guidelines for increasing the accuracy of minPMI estimations and, ultimately, greater robustness of forensic entomology evidence in court.

Visualization of insect metamorphosis.

Metamorphosis and, in particular, holometaboly, the development of organisms through a series of discrete stages (egg, larva, pupa, adult) that hardly resemble one another but are finely adapted to specific roles in the life cycle of the organism, has fascinated and mystified humans throughout history. However, it can be difficult to visualize the dramatic changes that occur during holometaboly without destructive sampling, traditionally through histology. However, advances in imaging technologies developed mainly for medical sciences have been applied to studies of insect metamorphosis over the past couple of decades. These include micro-computed tomography, magnetic resonance imaging and optical coherence tomography. A major advantage of these techniques is that they are rapid and non-destructive, enabling virtual dissection of an organism in any plane by anyone who has access to the image files and the necessary software. They can also be applied in some cases to visualize metamorphosis in vivo, including the periods of most rapid and dramatic morphological change. This review focusses on visualizing the intra-puparial holometabolous metamorphosis of cyclorraphous flies (Diptera), including the primary model organism for all genetic investigations, Drosophila melanogaster, and the blow flies of medical, veterinary and forensic importance, but also discusses similar studies on other insect orders. This article is part of the theme issue 'The evolution of complete metamorphosis'.

Avoidance of carnivore carcasses by vertebrate scavengers enables colonization by a diverse community of carrion insects.

Carrion resources sustain a complex and diverse community of both vertebrate and invertebrate scavengers, either obligate or facultative. However, although carrion ecology has received increasing scientific attention in recent years, our understanding of carrion partitioning in natural conditions is severely limited as most studies are restricted either to the vertebrate or the insect scavenger communities. Moreover, carnivore carcasses have been traditionally neglected as study model. Here, we provide the first data on the partitioning between vertebrate and invertebrate scavengers of medium-sized carnivore carcasses, red fox (Vulpes vulpes (Linnaeus)), in two mountainous Mediterranean areas of south-eastern Spain. Carcasses were visited by several mammalian and avian scavengers, but only one carcass was partially consumed by golden eagle Aquila chrysaetos (Linnaeus). These results provide additional support to the carnivore carrion-avoidance hypothesis, which suggests that mammalian carnivores avoid the consumption of carnivore carcasses to prevent disease transmission risk. In turn, the absence of vertebrate scavengers at carnivore carcasses enabled a diverse and well-structured successional community of insects to colonise the carcasses. The observed richness and abundance of the most frequent families was more influenced by the decomposition time than by the study area. Overall, our study encourages further research on carrion resource partitioning in natural conditions.

Long-term insect successional patterns on pig carcasses in central Spain.

Baseline data on the insect successional patterns on carcasses can be a valuable estimation tool in the investigations of suspicious deaths, particularly when the post-mortem interval is longer than months or years. However, although carrion insect succession is a recurrent topic in forensic science research, the duration of the published studies is typically shorter than 1 year, with only one published study from central Europe documenting successional patterns beyond the first year of decomposition. We provide here the first data on the long-term insect successional patterns in southern Europe, using pig carcasses exposed in the four seasons of the year and documenting the carrion entomofauna during the second and third year of decomposition. Our results confirmed previous observations from central Europe that several Coleoptera species are able to recolonize cadavers during the second and third year after death, with their larvae residing on the remains for long periods. The season of cadaver exposure appears to be a main factor determining the composition of the insect fauna that exploits the remains during subsequent years. Our results suggest that it might be possible to estimate the year and season of death analyzing the composition of the insect fauna occurring on a cadaver. The present data highlight the need for further studies on the long-term insect succession on cadavers in different seasons and geographical areas, in order to fully understand its patterns and ensure its proper application in PMI estimations.

Larval identification key to necrophagous Coleoptera of medico-legal importance in the western Palaearctic.

Several necrophagous Coleoptera species are frequently collected on cadavers, may occasionally act as intermediate or paratenic hosts of parasites, as vectors of pathogens or as allergens, and can also represent major pests of preserved animal products. However, despite their medical, veterinary and economic importance, there is a lack of reliable species identification tools for the larval stages (usually the only entomological evidence associated with medicolegal investigations), thus severely limiting their potential application as forensic indicators. Here, we provide an identification key to the larvae of the necrophagous Coleoptera species which have been recorded on carrion in the western Palaearctic region, based on easily observable morphological characters. In total, we provide diagnostic characters for the reliable identification of 23 necrophagous Coleoptera species within four different families (Cleridae, Dermestidae, Nitidulidae and Silphidae). In addition to the aforementioned families, we provide diagnostic characters for the identification of the larvae of families Histeridae, Staphylinidae and Trogidae, which can also be collected on cadavers. It is expected that the present key will facilitate the identification of larval material of necrophagous Coleoptera collected either in carrion succession studies or during medicolegal investigations, in order to further advance in the potential use of this insects as forensic tools.

3D virtual histology at the host/parasite interface: visualisation of the master manipulator, Dicrocoelium dendriticum, in the brain of its ant host.

Some parasites are able to manipulate the behaviour of their hosts to their own advantage. One of the most well-established textbook examples of host manipulation is that of the trematode Dicrocoelium dendriticum on ants, its second intermediate host. Infected ants harbour encysted metacercariae in the gaster and a non-encysted metacercaria in the suboesophageal ganglion (SOG); however, the mechanisms that D. dendriticum uses to manipulate the ant behaviour remain unknown, partly because of a lack of a proper and direct visualisation of the physical interface between the parasite and the ant brain tissue. Here we provide new insights into the potential mechanisms that this iconic manipulator uses to alter its host's behaviour by characterising the interface between D. dendriticum and the ant tissues with the use of non-invasive micro-CT scanning. For the first time, we show that there is a physical contact between the parasite and the ant brain tissue at the anteriormost part of the SOG, including in a case of multiple brain infection where only the parasite lodged in the most anterior part of the SOG was in contact with the ant brain tissue. We demonstrate the potential of micro-CT to further understand other parasite/host systems in parasitological research.

Annual variation in decomposition and insect succession at a periurban area of central Iberian Peninsula.

The variation in decomposition and insect succession among the four seasons of one year was studied for the first time in a periurban area of central Spain. During the winter trial, the carcasses showed corification, a cadaveric preservation phenomenon which apparently leaded to a significant delay in decomposition processes. The composition of the insect fauna breeding on carcasses changed significantly between trials. Active decay was mainly driven by Calliphoridae (Diptera) larvae in every season except in winter trial, when larvae of Thanatophilus species (Coleoptera: Silphidae) were the main consumers of soft tissues. Advanced decay was characterized by the occurrence of Dermestidae, Silphidae, Cleridae, Nitidulidae (Coleoptera) and Piophilidae (Diptera) larvae. Differences in the species composition in comparison with other regions of the Iberian Peninsula were also observed. The current paper provides baseline and preliminary information on the insect succession on carrion in central Spain, as well as a starting point for further research on forensic entomology in this region.

Early colonisation of urban indoor carcasses by blow flies (Diptera: Calliphoridae): An experimental study from central Spain.

Due to their ubiquity and synanthropy, blow flies (Diptera: Calliphoridae) are generally the first colonisers of cadavers and, therefore, frequently used to estimate a minimum post-mortem interval (minPMI). Whereas in outdoor situations blow flies are expected to locate and colonise exposed cadavers within hours or even minutes after death, it is usually assumed that the colonisation of a cadaver indoors might be delayed for an uncertain period of time. This uncertainty severely limits the informativity of minPMI estimates based on entomological evidence. Moreover, these limitations are emphasised by the lack of experimental data on insect colonisation of indoor carrion and by the fact that most of the forensic cases involving entomological evidence have been reported to occur indoors. In this study we investigate the early colonisation of pig carcasses placed indoors in a building located in the centre of an urban environment in central Spain. Three carcasses were placed in three equal rooms with a window half opened during five experimental trials: summer 2013, autumn 2013, winter 2014, spring 2014 and summer 2014. The species composition and their contribution to the carrion colonisation differed among seasons. Calliphora vicina Robineau-Desvoidy was the sole coloniser of carcasses in winter and colonised the carcasses within the first 24-48h in every season, although Lucilia sericata (Meigen) was the first coloniser of most summer carcasses. On the other hand, Calliphora vomitoria (L.) and Chrysomya albiceps (Wiedemann) colonised the carcasses significantly later in spring and in spring and summer, respectively, with a delay of several days. In autumn, however, there were no significant differences in the colonisation times by C. vicina, L. sericata and Ch. albiceps. C. vicina and L. sericata showed a clear preference for ovipositing in the natural orifices of the carcasses, whereas Ch. albiceps oviposited more frequently on the trunk and legs.

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.

Effect of Temperature on the Survival and Development of Three Forensically Relevant Dermestes Species (Coleoptera: Dermestidae).

Most Dermestes species (Coleoptera: Dermestidae) are scavengers during both larval and adult stages, with a preference for dry organic matter. Because of this, Dermestes beetles are potentially useful indicators in forensic investigations concerning skeletonized and mummified human remains. However, there is a paucity of reference developmental data on most forensically relevant Dermestes species. This study analyses the effect of five constant temperatures (15, 20, 25, 30, and 35 °C) on the survival and developmental rates of three of the forensically most relevant dermestids: Dermestes frischii Kugelan, Dermestes maculatus De Geer, and Dermestes undulatus Brahm. Pig skin was used as rearing substrate, to use a substrate as similar as possible to that exploited in nature. Overall, the temperature had a significant effect on the survival and the duration of development, with optimal values at intermediate temperatures. Both D. frischii and D. maculatus showed similar developmental rates and the shortest developmental times at 30 °C, whereas D. undulatus developed faster at lower temperatures. At 15 °C, both D. frischii and D. undulatus did not oviposit, whereas no D. maculatus individuals survived beyond the pupal stage. An inconsistent number of larval instars per individual were observed across different constant temperatures in the three species. The present study aims to provide baseline developmental data for further advances in the potential use of Dermestes beetles as forensic tools in long postmortem interval cases.

La mayoría de las especies del género Dermestes (Coleoptera: Dermestidae) son carroñeras durante la fase larvaria y la fase adulta, con preferencia por la materia orgánica seca. Debido a esto, los derméstidos son indicadores potencialmente útiles en las investigaciones forenses que impliquen restos humanos momificados y/o esqueléticos. Lamentablemente, los datos de referencia sobre el desarrollo de la mayoría de las especies de Dermestes de relevancia forense son escasos. Este estudio analiza el efecto de cinco temperaturas constantes (15, 20, 25, 30 y 35 °C) sobre la supervivencia y la tasa de desarrollo de tres de los derméstidos de mayor relevancia forense: Dermestes frischii Kugelan, Dermestes maculatus De Geer y Dermestes undulatus Brahm. Como sustrato de cría se utilizó piel de cerdo, con el objetivo de utilizar un sustrato lo más parecido posible al explotado en condiciones naturales. En general, la temperatura tuvo un efecto significativo sobre la supervivencia y la duración del desarrollo, con valores óptimos bajo las temperaturas intermedias. D. frischii y D. maculatus mostraron tasas de desarrollo similares y tiempos de desarrollo más cortos a 30 °C, mientras que D. undulatus se desarrolló más rápido a temperaturas inferiores. A 15 °C, ni D. frischii ni D. undulatus pusieron huevos, mientras que ningún individuo de D. maculatus sobrevivió más allá de la fase pupa. Se observó un número inconsistente de estadios larvarios por individuo en las diferentes temperaturas constantes y en las tres especies. Este estudio tiene como objetivo proporcionar datos de referencia sobre el desarrollo para futuros avances en el uso de los derméstidos como herramientas forenses en casos de intervalos post mortem largos.

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.