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.

Eclosion muscles secrete ecdysteroids to initiate asymmetric intestinal stem cell division in Drosophila.

During organ development, tissue stem cells first expand via symmetric divisions and then switch to asymmetric divisions to minimize the time to obtain a mature tissue. In the Drosophila midgut, intestinal stem cells switch their divisions from symmetric to asymmetric at midpupal development to produce enteroendocrine cells. However, the signals that initiate this switch are unknown. Here, we identify the signal as ecdysteroids. In the presence of ecdysone, EcR and Usp promote the expression of E93 to suppress Br expression, resulting in asymmetric divisions. Surprisingly, the primary source of pupal ecdysone is not from the prothoracic gland but from dorsal internal oblique muscles (DIOMs), a group of transient skeletal muscles that are required for eclosion. Genetic analysis shows that DIOMs secrete ecdysteroids during mTOR-mediated muscle remodeling. Our findings identify sequential endocrine and mechanical roles for skeletal muscle, which ensure the timely asymmetric divisions of intestinal stem cells.

An examination of the intrapuparial development of Chrysomya albiceps (Wiedemann, 1819) (Calliphoridae: Diptera) at three different temperatures.

The determination of intrapuparial development periods and development times of insects with holometabolous metamorphosis is necessary both in terms of developmental biology and for minimum Post-mortem interval (PMImin) calculations in forensic entomology. In this study, Chrysomya albiceps (Wiedemann, 1819), which is a cosmopolitan species and one of the most rapidly attracted to carrion, was studied. The focus was the intrapuparial development periods of this species at varying temperatures (20, 25, and 30 °C), knowledge which is used in forensic entomology for the estimation of Post-mortem interval (PMI) and is a type of black box. At the specified temperatures, pupae were collected and puparia were dissected hourly; thus, developmental periods were determined, and minimum and maximum starting times of these periods were calculated. With this research, a total of 20 periods, nine of which are new, were determined. The hourly determination of intrapuparial development periods at three different temperatures is the first in the world for this species.

Within- and Trans-Generational Life History Responses to Diurnal Temperature Amplitudes of the Pupal Stage in the Diamondback Moth.

Diurnal temperature fluctuations in nature can have a significant effect on many ectodermic traits. However, studies on the effects of diurnal temperature fluctuations on organisms, especially the effects on specific life stages, are still limited. We examined the immediate effects of the same average temperature (25°C) and different temperature amplitudes (±4, ±6, ±8, ±10, ±12°C) on the development and survival of Plutella xylostella (Lepidoptera: Plutellidae). We also assessed carry-over effects on adult longevity, reproduction, development, and survival of offspring across generations. The effect of moderate temperature amplitudes was similar to that of constant temperature. Wide temperature amplitudes inhibited the development of pupae, reduced total reproduction, lowered intrinsic rates of population growth, and slowed the development and survival of eggs on the first day, but the proportion of females ovipositing on the first three days increased. Insects coped with the adverse effects of wide temperature amplitudes by laying eggs as soon as possible. Our results confirmed that a logistic model based on daily average temperature cannot predict development rates under wide temperature amplitudes. These findings highlight the effect of environmental temperature fluctuations at the pupal stage on the development and oviposition patterns of P. xylostella and should be fully considered when predicting field occurrence.

The morphology of the preimaginal stages of Cleopomiarusmicros (Germar, 1821) (Curculionidae, Coleoptera) and notes on its biology.

As yet little is known of the bionomics of weevils of the genus Cleopomiarus Pierce, 1919; current knowledge is limited to data on the morphology and biology of the preimaginal stages of certain species. This paper includes original information on the life cycle of Cleopomiarusmicros (Germar, 1821). It presents the morphology of the egg, last larva (L3) and pupa. Data on the host plant (Jasionemontana L.) and breeding plant (Campanulapatula L.) and on the oviposition and phenology of the species are updated. The anatomy of the third-stage larva of C.micros shares certain traits with other species of the tribe Mecinini Gistel, 1848. Comparison of the morphology of preimaginal stages of C.micros with those previously described for other species of the genera Cleopomiarus and Miarus Schönherr, 1826 - previously considered the same genus - reveals species differences in larval body length, colour of the body and epicranium, and chaetotaxy of head and body.

First description of the larva of Dinaraea Thomson, 1858, with comments on chaetotaxy, pupa, and life history based on two saproxylic species from Europe (Staphylinidae, Aleocharinae, Athetini).

The paper describes the morphological ultrastructure of the previously unknown early (L1) and late larval instars (L2-3) of Dinaraea, including chaetotaxy, pupal cocoon, prepupa, and pupa, based on the saproxylic species D. aequata Erichson and D. linearis Gravenhorst. Diagnostic larval characters for the genus Dinaraea are given for the first time. Morphological differences between mature larvae of these two species relate to the colouration and degree of flattening of the body, details of antennal structure, anterior margin of the labrum, mandibles, and mala. The differences are relatively small, probably because of the similar ecological preferences of both species. As in the case of other aleocharine larvae, L1 in Dinaraea differs from L2-3 in the lack of some setae on the dorsal surface of the head and thorax, and on the abdominal tergites and sternites; the presence of a subapical seta on the urogomphi; egg bursters on some thoracic and abdominal tergites; a darker antennal segment III; and the relatively longer urogomphi and their apical setae. The differences established in the features of the chaetotaxy of L1 and L2-3 between Athetini (Dinaraea), Oxypodini (Thiasophila) and Homalotini (Gyrophaena) correspond with the molecular marker-based relationships of these taxa.

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.

Looking into the puparium: Micro-CT visualization of the internal morphological changes during metamorphosis of the blow fly, Calliphora vicina, with the first quantitative analysis of organ development in cyclorrhaphous dipterans.

Metamorphosis of cyclorrhaphous flies takes place inside a barrel-like puparium, formed by the shrinking, hardening and darkening of the third-instar larval cuticle. The opacity of this structure hampers the visualization of the morphological changes occurring inside and therefore a full understanding of the metamorphosis process. Here, we use micro-computed tomography (micro-CT) to describe the internal morphological changes that occur during metamorphosis of the blow fly, Calliphora vicina Robineau-Desvoidy 1830 (Diptera: Calliphoridae) at a greater temporal resolution than anything hitherto published. The morphological changes were documented at 10% intervals of the total intra-puparial period, and down to 2.5% intervals during the first 20% interval, when the most dramatic morphological changes occur. Moreover, the development of an internal gas bubble, which plays an essential role during early metamorphosis, was further investigated with X-ray images and micro-CT virtual sections. The origin of this gas bubble has been largely unknown, but micro-CT virtual sections show that it is connected to one of the main tracheal trunks. Micro-CT virtual sections also provided enough resolution for determining the completion of the larval-pupal and pupal-adult apolyses, thus enabling an accurate timing of the different intra-puparial life stages. The prepupal, pupal, and pharate adult stages last for 7.5%, 22.5%, and 70% of the total intra-puparial development, respectively. Furthermore, we provide for the first time quantitative data on the development of two organ systems of the blow fly: the alimentary canal and the indirect flight muscles. There is a significant and negative correlation between the volume of the indirect flight muscles and the pre-helicoidal region of the midgut during metamorphosis. The latter occupies a large portion of the thorax during the pupal stage but narrows progressively as the indirect flight muscles increase in volume during the development of the pharate adult.

Functional activity of allatotropin and allatostatin in the pupal stage of a holometablous insect, Tribolium castaneum (Coleoptera, Tenebrionidae).

Allatotropin (AT) and allatostatin (AS) neuropeptides are known to regulate the biosynthesis of juvenile hormones (JH) in insects. Furthermore, they possess myoregulatory and other activities in a wide range of insect species. The genome of Tribolium castaneum encodes two AS and one AT precursors. Here we cloned the cDNAs of the precursors, followed their expression patterns during the pupal stage, and established their putative roles in adult development and oviposition of the females using RNA interference (RNAi). Cloning of the cDNA and gene structure analyses of the Tc-AT gene confirmed that the gene is expressed in three mRNA isoforms. Real-time PCR data demonstrate that the Tc-AT isoforms and the AS genes, Tc-AS C and Tc-AS B, are expressed in discerning developmental and tissue-specific patterns. Single injections of dsRNAi (targeted against the Tc-AT, Tc-AS C, and Tc-AS B, respectively), into young pupae resulted in abnormal adult phenotypes, whereby about half of the animals (P1 phenotype) looked relatively normal, but the females laid low numbers of eggs. The other halves (P2) exhibited strong developmental defects with abnormal duration of the pupal stage, abnormal head and body sizes, short elytra, and incomplete sclerotization. Moreover, these females deposited no eggs and died within one week after emergence. Individual silencing of the Tc-AT mRNA isoforms showed that Tc-AT3 had the most disruptive influence on adult development and fecundity of the females. Our findings clearly indicate a significant role of AT and AS neuropeptides in the pupa. The distinct mechanisms of action, however, remain to be determined.

Emergence and pupal mortality factors of Anastrepha obliqua (Macq. ) (Diptera: Tephritidae) along the fruiting season of the host Spondias dulcis L. / Emergência e fatores de mortalidade pupal de Anastrepha obliqua (Macq. ) (Diptera: Tephritidae) no período de frutificação do hospedeiro Spondias dulcis L.

A emergência e os fatores de mortalidade pupal de Anastrepha obliqua (Macq.) (Diptera: Tephritidae) durante a frutificação da planta hospedeira Spondias dulcis L. (Anacardiaceae) foram estudados no campo e no laboratório em Sertãozinho, SP. Nas duas condições experimentais, após os períodos de emergência, foram registrados os números de pupários fechados e abertos. Os números de moscas e parasitóides emergidos dos pupários foram registrados. Os pupários fechados foram analisados e, conforme o estado da pupa dentro do pupário, as mesmas foram classificadas em vivas (dormentes) e mortas. Os fatores de mortalidade considerados foram: dessecação, doenças e predação. Foramanalisados 1204 pupários, sendo que de 53% emergiram adultos e 47% permaneceram fechados; do total de pupários fechados, 25,3% eram pupas em estado de dormência e 21,7% eram pupas mortas por predadores, doenças e dessecação. Das pupas em estado de dormência, 17,8% eram moscas e somente 0,2% completaram o estágio pupal; 7,5% continham parasitóides, sendo que 4,7% deles emergiram. Oparasitismo inicial foi de 8,6% e após a emergência das pupas em dormência aumentou para 15,5%. A ação predatória em condições naturais foi acentuada, especialmente quando o tempo de exposição foi prolongado. Em condições de laboratório, a dessecação foi o principal fator de mortalidade pupal. O parasitismo também contribuiu significativamente para a mortalidade pupal enquanto que, as doenças provocadas por patógenos (fungos e bactérias), parecem ter sido menos significativas. Predadores e parasitóides atuaram efetivamente no controle populacional dessa mosca-das-frutas. Entretanto, os fatores que regulam o estado de dormência, ainda devem ser determinados...

The emergence and pupal mortality factors of Anastrepha obliqua (Macq.) (Diptera: Tephritidae) along the fruiting season of the host plant, Spondias dulcis L. (Anarcadiaceae), were studied in Sertãozinho, SP, Brazil, under field and laboratory conditions. Eclosed and uneclosed puparia were recorded in two experimental conditions. The number of emerged flies and parasitoids weredetermined in the eclosed puparia. The uneclosed puparia were inspected and classified into living (dormant) and dead. The following pupal mortality factors were considered: disease, desiccation, predation and parasitism. Out of the total of 1,204 puparia analyzed, 53% emerged and 47% remained uneclosed. Out of the uneclosed puparia, 25.3% contained dormant pupae and 21.7% showed no signs of emergence. Among the dormant pupae, 17.8% were flies, 0.2% of which emerged; 7.5% were parasitoids, 4.7% of them emerged. The initial parasitism was 8.6%, increasing to 15.3% after theemergence of dormant pupae. Predatory activity (natural condition), especially when time of exposure was long, and desiccation (laboratory condition) were the predominant causes of pupal mortality. Variation in mortality caused by parasites and pathogens (bacteria and fungi) seems to play a minor role. Control by predators on fruit fly population is significant. However, the factors, which regulate induction, maintenance and termination of dormancy, are still to be determined. An estimate of the dormancy and of the biotic and abiotic pupal mortality factors are essential to understand the adaptivestrategies of A.obliqua and its parasitoids and to develop effective methods of control in tropical regions.