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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.

Background The pupal stage in holometabolous insects is a critical transition between larval and adult forms, during which feeding ceases and survival depends on stored energy reserves. Mosquito pupae exhibit active diving behaviour in response to threats, which is energetically costly due to their positive buoyancy. Whether pupae are able to adjust diving behaviour according to environmental conditions, balancing predator avoidance and energy expenditure, remains poorly understood. Here, we investigated how water salinity affects the diving behaviour of Aedes mariae pupae, a species inhabiting Mediterranean rock pools characterised by highly variable salinity conditions. Methods Pupae were maintained and tested in two salinity conditions: low (50%) and high (150%). Diving behaviour was recorded following an automated mechanical stimulus, and we measured: (i) time spent underwater, (ii) pupal activity (i.e. the number of abdominal movements during the immersion and the ratio of movements to time spent underwater) and (iii) the proportion of time spent by a pupa at different depths along the height of the water column (space use). Results We found that pupae in high-salinity conditions spent 20.6% less time underwater than those in low salinity. They also performed fewer abdominal movements during dives but showed no significant differences in movements per unit time. Analysis of space use showed that pupae in high salinity spent more time in the upper part of the water column and less time in the middle and lower parts. Conclusions Ae. mariae pupae modify their diving behaviour in response to different salinity conditions, adopting energy-efficient responses to external stimuli that promote survival in variable habitats. These findings highlight the importance of pupal behavioural flexibility for overall fitness and underscore the need to investigate pupal behavioural plasticity, which remains largely unexplored. Graphical abstract

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.

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 (L 3 ) 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.

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.

Parasitoid populations having optimum male: female ratios are generally released in the field during the cocoon stage for an effective bio-control program. Due to the distinctive habit of forming a cocoon within the shrunken larval body of the host, cocoons of both sexes of the icheneumonid wasp, Hyposoter ebeninus, displayed morphological similarities. Therefore, male and female cocoons cannot be differentiated based on normal morphological parameters. In this report, we tested several combinations of morphological parameters and size indices and developed a simple technique for separation of sexes at the cocoon stage of this wasp. Among ten possible parameters tested, the weight of the cocoon was found to be the most appropriate and reliable parameter for sex separation with a 100% success rate. Since this technique involves only one parameter, it is simple, robust and requires minimum technical skill. This technique would be very useful for research workers, commercial biological control laboratories and other related agencies working on H. ebeninus.

Survivorship of larvae of the small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae), was measured after they were raised on one of six diets. The effects of container shape (wide and shallow vs. narrow and deep), soil depth (0, 0.5, 1.0, 2.0, 4.0, and 8.0 cm), and temperature (28°, 32°, or 35° C) on pupation success was measured. Diet influenced larval survivorship, but did not have a strong effect on larval weight. The larvae fed only bee brood survived the shortest period of time. The larvae that were denied pupation substrate, fed only honey and pollen, and no other food or water after 20 days, had a median survivorship of 47.6 days, with a maximum of 61 days, while those fed only brood had a median survivorship of 18.2 days. Pupation substrate was essential for successful pupation, and the depth of the substrate, not its top surface area, was the crucial factor. Pupation success in narrow and deep containers was 95.6% on average, but only 12.5% in wide and shallow containers, using the same soil volume. In narrow and deep containers, most or all larvae kept in 4–8 cm of soil pupated at all temperatures, few larvae kept at 2 cm soil depth pupated, one out of 240 kept at 1.0 cm pupated, and no larvae kept at soil depths of 0 or 0.5 cm pupated.

Endemicity of onchocerciasis (river blindness) in humans is linked to the location of Simulium spp. (black fly). The distribution of immature stages of Simulium in Sholur, Pykara, Gudalur, Coonoor and Kotagiri streams of the Nilgiris hills of Tamil Nadu was investigated during the months of May and July 2012. Out of these five streams, only Sholur was infested with larval and pupal stages of Simulium spp. Out of six plants collected from various water bodies, larval and pupal stages were found on the leaves and stems of an aquatic plant Nasturtium officinale and on the roots and leaves of Pennisetum glandulosum. The stages of Simulium were observed only during the summer month of May.

A species of Notodontidae, Nystalea ebalea was discovered feeding on leaflets of the invasive weed Schinus terebinthifolia Raddi (Sapindales: Anacardiaceae) in south Florida. The larvae of this species generally have 5 instars and require 20–22 days to reach the pupal stage. Discovery of wild populations of this Neotropical species in the weed's invasive range precluded its consideration as a classical biological control agent.

Although forensic entomology may seem to be a narrow speciality, it is applicable in a wide variety of cases. Forensic entomologists identify insects associated with dead bodies and estimate time since death. They also play an important role in other areas where insects and crime intersect, such as drugs, poisons, and location of stolen goods.