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The symbiotic microbiota profoundly affect many aspects of host physiology; however, the molecular mechanisms underlying host-microbe cross-talk are largely unknown. Here, we show that the pyrroloquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) activity of a commensal bacterium, Acetobacter pomorum, modulates insulin/insulin-like growth factor signaling (IIS) in Drosophila to regulate host homeostatic programs controlling developmental rate, body size, energy metabolism, and intestinal stem cell activity. Germ-free animals monoassociated with PQQ-ADH mutant bacteria displayed severe deregulation of developmental and metabolic homeostasis. Importantly, these defects were reversed by enhancing host IIS or by supplementing the diet with acetic acid, the metabolic product of PQQ-ADH.

We provide recommendations for sampling and identification of introduced larval parasitoids of spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae). These parasitoids are either under consideration for importation (aka classical) biological control introductions, or their adventive (presumed to have been accidentally introduced) populations have recently been discovered in North America and Europe. Within the context of the ecology of D. suzukii and its parasitoids, we discuss advantages and disadvantages of estimating larval parasitism levels using different methods, including naturally collected fruit samples and sentinel baits. For most situations, we recommend repeated sampling of naturally occurring fruit rather than using sentinel baits to monitor seasonal dynamics of host plant–Drosophila–parasitoid associations. We describe how to separate Drosophilidae puparia from host fruit material in order to accurately estimate parasitism levels and establish host–parasitoid associations. We provide instructions for identification of emerging parasitoids and include a key to the common families of parasitoids of D. suzukii. We anticipate that the guidelines for methodology and interpretation of results that we provide here will form the basis for a large, multi-research team sampling effort in the coming years to characterize the biological control and nontarget impacts of accidentally and intentionally introduced larval parasitoids of D. suzukii in several regions of the world.

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.

The weathering time of empty puparia could be important in predicting the minimum postmortem interval (PMImin). As corpse decomposition progresses to the skeletal stage, empty puparia often remain the sole evidence of fly activity at the scene. In this study, we used empty puparia of Sarcophaga peregrina (Diptera: Sarcophagidae) collected at ten different time points between January 2019 and February 2023 as our samples. Initially, we used the scanning electron microscope (SEM) to observe the surface of the empty puparia, but it was challenging to identify significant markers to estimate weathering time. We then utilized attenuated total internal reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to detect the puparia spectrogram. Absorption peaks were observed at 1064 cm−1, 1236 cm−1, 1381 cm−1, 1538 cm−1, 1636 cm−1, 2852 cm−1, 2920 cm−1. Three machine learning models were used to regress the spectral data after dimensionality reduction using principal component analysis (PCA). Among them, eXtreme Gradient Boosting regression (XGBR) showed the best performance in the wavenumber range of 1800–600 cm−1, with a mean absolute error (MAE) of 1.20. This study highlights the value of refining these techniques for forensic applications involving entomological specimens and underscores the considerable potential of combining FTIR and machine learning in forensic practice. •Morphology observed by SEM is helpless in estimating puparium weathering time.•Most absorption peaks in the waveband region of 1800–600 cm−1.•XGBR perform well with the spectral data to estimate weathering time.

Forensic entomology uses the age of insects, such as blow flies, to determine a minimum post-mortem interval (PMImin). Recent research has focused on using the analysis of specific cuticular hydrocarbons (CHCs) in adult insects and their empty puparia to estimate their age, as it has been shown that their profile changes are consistent with age. The current work is based on the weathering of five CHCs from empty puparia of Calliphora vicina that were stored in soil (field/outdoor) and non-soil (room/indoor conditions) based pupariation media for a total of six months. The experiment was conducted in a controlled environment chamber at a constant temperature of 25 ± 2 °C under constant darkness. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the cuticular hydrocarbons after they were extracted in n-Hexane. n-Pentacosane, n-Hexacosane, n-Heptacosane, n-Octacosane, and n-Nonacosane were the five CHCs investigated. Results showed that CHCs weathered more quickly in the soil than in the non-soil environment. It was also found that the abundance of Heptacosane increased in the samples during the fifth month when stored in a non-soil medium, while the abundances of all five CHCs were not detected after eight weeks onwards in soil pupation medium. [Display omitted] •Blow flies used to determine PMImin; research focuses on CHCs in insects and puparia.•The study investigated weathering of CHCs in Calliphora vicina puparia stored in soil and non-soil environments.•Gas chromatography-mass spectrometry was used to analyze the cuticular hydrocarbons after extraction in hexane.•CHCs weathered faster in soil; Heptacosane increased in non-soil medium after fifth month.•The relative abundance of all five CHCs were not detected after eight weeks onwards in soil pupation medium.

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.

Drosophila suzukii is native to East Asia and an invasive pest of fruit crops widely established in the Americas and Europe. The lack of effective indigenous parasitoids of D. suzukii in the invaded regions prompted surveys for co-evolved parasitoids in Yunnan Province, China, from 2013 to 2016. From banana-baited traps (2013–2015), 458 parasitoids of drosophilids were reared, comprised of Braconidae (49.56%), Figitidae (37.55%), Diapriidae (7.42%), and Pteromalidae (5.46%). Larval parasitoids included seven braconid species, all Asobara and primarily Asobara mesocauda , and five figitid species, primarily Leptopilina japonica japonica . Pupal parasitoids were the diapriid Trichopria drosophilae and the pteromalid Pachycrepoideus vindemiae . Collections from wild fruits (2016) provided more interesting results. From the puparia of drosophilids collected, comprised of D. suzukii and Drosophila pulchrella , emerged 1354 parasitoids. The larval parasitoids Ganaspis brasiliensis and L. j. japonica were the prevalent species, reaching a fairly high percentage parasitism of fly puparia collected from berries of Rubus foliosus (22.35%), R. niveus (18.81%), Fragaria moupinensis (19.75%), and Sambucus adnata (63.46%). Ganaspis brasiliensis was the dominant species and was collected only from D. suzukii and D. pulchrella -infested fruits and never from banana-baited traps. Molecular analysis showed two G. brasiliensis lineages, which are discussed with respect to previous Japanese collections. Quarantine tests showed that G. brasiliensis developed from D. suzukii and two closely related hosts ( Drosophila melanogaster and Drosophila simulans ) but did not develop from seven non-target drosophilid species. Our results suggest that G. brasiliensis is a promising classical biocontrol agent for release in invaded regions.

There is a consensus that forensic methods must be valid. The high quality of the method may be fully demonstrated only through validation. Unfortunately, there are very few experimental or casework validations of entomological methods for estimating post-mortem interval (PMI). Here, we present the first part of the results from the field validation of minimum PMI (PMImin) estimates based on insect development. From eight pig carcasses (24–46.4 kg) exposed in a forest habitat of Western Poland, one every one or two months, we collected insect evidence with standard entomological techniques. Using weather station temperatures and the thermal summation method, PMImin was estimated based on insect life stages that were reared under controlled laboratory conditions. Through rearing an insect until the next developmental landmark (or until eclosion), its age at the time of collection (and eventually PMImin) may be estimated by subtracting thermal accumulation in the laboratory from the corresponding thermal constant. We hypothesized that rearing insect evidence significantly improves the accuracy of PMImin compared to the estimation based on non-reared insect evidence. The results clearly supported this hypothesis. However, the accuracy significantly increased only in the case of these insects that developed normally during rearing. When their development in the laboratory was prolonged, PMImin was significantly less accurate. For the normal development sample the accuracy improved in all species and life stages. The largest accuracy gains from rearing were recorded for Calliphora vomitoria (Diptera: Calliphoridae) and Stearibia nigriceps (Diptera: Piophilidae). Moreover, when puparia or third instar larvae were reared, gains were larger than in the case of earlier life stages. In conclusion, this study demonstrated that the method of rearing insect evidence to improve the accuracy of PMImin is valid. However, it needs to be used with caution, since substantial part of the evidence may die or slow down their development in the laboratory, which violates assumptions of the method. ●Further rearing of insects sampled on a death scene increases accuracy of PMImin.●The rearing provides accuracy gains across species and life stages.●The rearing of third instar larvae and puparia yields the largest gains.●The rearing is risky due to high mortality and prolonged development.

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.

Calliphora grahami (Aldrich, 1930) (Diptera: calliphoridae) is a forensically important blow fly that is widely distributed across Asia, North America, Russia, and Mexico. Calliphora grahami is frequently found on corpses during the spring, autumn, and winter seasons. It is among the early colonizers of cadavers during the cold season, and sometimes, the only necrophagous blow fly on cadavers. Therefore, this species is of forensic significance, although very few studies have explored the application of its intrapuparial age for PMI estimation. This study aimed to examine the intrapuparial development of C. grahami and establish a method for estimating its intrapuparial age. Herein, the C. grahami puparia were studied under six different temperatures (13, 16, 19, 22, 25, and 28°C) for the intrapuparial age estimation, and a total of 5776 puparia were sampled. The morphological changes were divided into 11 stages based on the 1) development of legs and wings, 2) differentiation of the head, thorax, and abdomen, 3) growth and color of the bristles, and 4) color changes of the compound eyes. The corresponding time of each stage was determined. Moreover, the observation and classification of individual morphological features, including compound eyes, antennae, mouthparts, thorax, abdomen, legs, and wings was used to improve the precision of intrapuparial age estimation. The findings of this study provide important information on the use of C. grahami intrapuparia to estimate the minimum postmortem interval (PMImin).