We previously reported that two of three patients with severe Usutu virus (USUV) disease carried circulating autoantibodies (auto-Abs) neutralizing type I IFNs (AAN-I-IFN). Here, we studied four additional patients, bringing the total number of European patients with life-threatening USUV infection to seven (Austria, n = 1; France, n = 1; Hungary, n = 4; Italy, n = 1). Three of these patients (all male, aged 43, 77, and 78 years, one French and two Hungarian) had blood auto-Abs neutralizing at least 10 ng/ml IFN-α2 and IFN-ω and at least 1 ng/ml IFN-β. They suffered from meningitis (n = 1), meningoencephalitis (n = 1), or myocarditis (n = 1). The other four patients were similar demographically and clinically but had no detectable AAN-I-IFN. No AAN-I-IFN were found in the 63 individuals with silent USUV infection tested (P = 6.4 × 10-4, Fisher's exact test). These AAN-I-IFN increased the risk of life-threatening USUV disease by 98.8-fold (95% CI: 4.12-2,369; P = 0.0046, Firth's bias-corrected logistic regression) and, thus, account for almost half the cases of life-threatening USUV infection in Europe.

Blowflies of the genus Lucilia (Insecta: Diptera: Calliphoridae) are important in forensic science, medical research, and agriculture, particularly due to their role as facultative ectoparasites of domesticated sheep. The Australian sheep blowfly, L. cuprina dorsalis, is a primary causative agent of cutaneous myiasis, leading to substantial economic losses to the wool industry. In contrast, the closely related subspecies L. cuprina cuprina does not induce myiasis in sheep, but functions as a necrophagous subspecies in urban regions of Australia. Differences in the physiology, behavior, and host interactions of these subspecies might be attributed to their bacterial community structures as demonstrated in other blowfly species. However, our understanding of the bacterial communities associated with these flies is limited. This study aimed to characterize the microbiomes of two geographically distinct subspecies of L. cuprina-L. c. dorsalis (rural) and L. c. cuprina (urban) using 16S rRNA gene sequencing. The findings indicate that, while both subspecies harbor broadly similar microbial compositions, their respective urban and rural environments influence the prevalence of specific bacterial genera. The bacterial communities of these flies were predominantly composed of members of the phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. The genus Ignatzschineria was highly abundant in L. c. dorsalis, whereas Pseudomonas was more prevalent in L. c. cuprina, highlighting distinct microbial compositions between the two subspecies. These findings provide fundamental insights into the microbial composition of Lucilia species, contributing to a broader understanding of their ecological and biological roles, with potential applications in forensic entomology, medical research, and agricultural pest management. KEY POINTS: • The microbial communities of Lucilia cuprina dorsalis and Lucilia cuprina cuprina have a broadly similar composition, but the abundance of particular bacterial genera is notably different. • Proteobacteria is the most abundant phylum in both subspecies. • The genus Ignatzschineria is notably abundant in Lucilia cuprina dorsalis, while Pseudomonas is predominant in Lucilia cuprina cuprina, emphasizing differences between the microbial community members between these subspecies.

The current study details the first species list of flesh flies (Diptera: Sarcophagidae), collected using carrion baited traps at three locations within Athens, Greece. The research aims to understand the local flesh fly fauna and discuss their potential use in forensic applications. Employing traps baited with beef liver, fish, and chicken stomach, the present study assessed the efficacy of these different bait types in attracting Sarcophagidae. The study elucidates species diversity, richness, species assemblage, abundance, and bait preference. A total of 1125 Sarcophaga spp. specimens were captured, of which 241 were males, representing 12 species. The predominant species were Sarcophaga africa (Wiedemann) and Sarcophaga melanura Meigen. Overall, among the identified males, beef liver attracted the highest number and greatest species diversity of flesh flies, followed by fish and finally chicken stomach. Identification of this insect group contributes to the discipline of Forensic Entomology in Greece by providing a comprehensive database of local flesh fly species associated with carrion that may provide information about the circumstances concerning the time since death (TSD) in crime scene investigations.

Synthesiomyia nudiseta (van der Wulp, 1883) is widely distributed across pantropical and subtropical biogeographic regions. Despite its recognized forensic importance, comprehensive developmental data for it remains scarce. This study observed the development of S. nudiseta from egg to adult at six constant temperatures in the range of 20-35 °C, and found that the times required were 732.7 ± 7, 587.3 ± 4.7, 449 ± 21.3, 393.7 ± 5.7, 361.3 ± 8.3 h, respectively. It cannot complete development at 35 °C. We established three development models to estimate the age of the immature insect: isomegalen diagram, isomorphen diagram, and thermal summation model. A regression analysis was performed on the relationship between body length and total development time from hatching to pupariation. The thermal summation constant during the development of S. nudiseta was 7870.1 degree-hours, and the developmental threshold temperature was 9.28 °C. In addition, we observed development of S. nudiseta under four fluctuating temperature regimes; developmental duration exhibited a negative correlation with increasing mean temperature; however, inclusion of 35 °C within the fluctuating temperature cycle prevented successful adult eclosion, with the vast majority of pupae succumbing to mortality before reaching the adult stage. The results of this experiment provide a basis for the use of S. nudiseta in the estimation of PMImin.

Rising atmospheric CO2 levels may alter behavioral dynamics that mediate species interactions, a largely overlooked dimension of ecosystem functioning. Aphids offer a tractable model for examining these modifications, as their primary protection from natural enemies relies on the alarm pheromone (E)-β-farnesene (Eβf). Facultative aphid endosymbionts like Candidatus Hamiltonella defensa complicate this dynamic by modifying parasitoid resistance and associated behaviors, yet their interaction with elevated CO2 remains largely unexplored. Using a single clone of the pea aphid Acyrthosiphon pisum (Harris), we investigated how CO2 levels and H. defensa D3 status, independently and interactively, altered Vicia faba L. plant size, aphid fecundity, and aphid dispersal responses to Eβf. Here we show that dry shoot weights of bean plants grown under elevated CO2 increased, compared to those grown under ambient conditions. Irrespective of CO2 level, however, aphids harboring H. defensa produced significantly more offspring, and dispersed at significantly higher rates in response to Eβf than symbiont-free individuals. Under the standardized Eβf assay, the difference in dispersal between H. defensa-harboring aphids and those that were symbiont-free was large under ambient CO2 (59% vs. 9%) and smaller under elevated CO2 (23% vs. 12%). Thus, elevated CO2 reduced symbiont-associated differences in dispersal under standard assay conditions in this aphid clone with implications for tri-trophic interactions, biological control efficacy, and symbiont prevalence under future atmospheric conditions.

Real-time quantitative polymerase chain reaction (RT-qPCR) is widely used for gene expression quantification, and stably expressed reference genes are essential for accurate data normalization. However, validated reference genes are not yet available for the hide beetle, Dermestes maculatus De Geer, 1774 (Coleoptera: Dermestidae), an economically important stored-product pest and a forensically relevant necrophagous beetle. In this study, 8 candidate reference genes (RPL4, RPL13A, RPL27, RPS3, RPS18, RPS28b, GAPDH, and EF1α) were systematically evaluated across 8 developmental stages, 6 tissue types, and all samples. All primer pairs showed good amplification efficiency and specificity. Expression stability was assessed using the comparative ΔCt method, BestKeeper, NormFinder, and geNorm, and the rankings were integrated using RefFinder. The resulting rankings showed that RPS28b was the most stable gene across developmental stages, whereas RPL4 ranked first across tissues and all samples. Pairwise variation analysis using geNorm showed that 2 reference genes were sufficient for normalization in all 3 datasets, supporting RPL4 and RPS28b as the optimal reference-gene combination. GAPDH was consistently the least stable gene in all datasets. Validation of PMP7 expression supported RPL4 and RPS28b as suitable reference genes, whereas GAPDH altered the apparent expression pattern. This study provides the first validated RT-qPCR normalization framework for D. maculatus and supports future studies of developmental regulation, RNA interference, functional genomics, pest management, and forensic entomology.

Phoretic associations with insects are widespread in the Mesostigmata. These temporary symbiotic associations have been important in driving the evolutionary development of this taxon. The phoretic mite genus Poecilochirus, which is considered obligately associated with burying beetles, is of great value in forensic entomology. However, due to the morphological conservatism and the difficulty of obtaining adult specimens, the taxonomy of this genus has long been based on deutonymphal characters, and the true species diversity is far from resolved. Here we used next-generation sequencing to determine, for the first time, the complete mitochondrial genomes of P. austroasiaticus, P. subterraneus, and two distinct lineages of P. carabi (BJ1 and BJ2). All the sequenced mitogenomes contain 37 genes with a gene order identical to that of the ancestral arthropod arrangement and they all show pronounced AT bias and negative GC skew. All 13 protein-coding genes are under strong purifying selection (Ka/Ks < 1). Sliding window analysis identifies nad2 and nad5 as nucleotide diversity hotspots, which could serve as high-resolution markers for future species delimitation. Phylogenetic reconstruction tentatively recovered Poecilochirus as paraphyletic with Parasitus nested within it (ML = 44%, BI = 0.982), while Parasitinae and Pergamasinae are not reciprocally monophyletic. The cox1 genetic distance between P. carabi BJ1 and BJ2 reached 0.409, with both mPTP and ASAP analyses of the mitogenomic dataset partitioning them into separate candidate molecular operational taxonomic units. Bayesian divergence time estimation revealed that the two lineages diverged in the Eocene about 52.4 million years ago (Ma), before the split between the morphologically recognized species P. subterraneus and P. davydovae (39.0 Ma). Our results give strong molecular evidence for cryptic speciation within the P. carabi complex and provide a solid genomic foundation for the future systematic and taxonomic research in the Parasitidae.

Forensic entomology plays an important role in estimating the postmortem interval (PMI). However, during cold seasons, necrophagous insects may enter diapause, resulting in developmental arrest. Currently, effective methods for estimating diapause duration are limited, which may compromise the accuracy of PMI estimation based on insect evidence. Here we show that multi-dimensional data fusion of gene expression and infrared spectral features could improve the accuracy of diapause duration estimation. Using the forensically important flesh fly Sarcophaga peregrina as a model, we performed transcriptome sequencing across the diapause period and identified 14 core differentially expressed genes (DEGs) with pronounced temporal dynamics. After qPCR validation, 11 DEGs were retained. ATR-FTIR analysis revealed stage-dependent shifts in macromolecular absorption peak features. Source-level integration of the two data types, followed by partial least-squares dimensionality reduction and support vector regression, produced a model (test R[2] = 0.922) that substantially outperformed either dataset alone. These findings suggest a potential molecular-spectroscopic strategy that may help address the long-standing challenge of PMI estimation in cold-season forensic cases, and offer a preliminary framework for exploring multi-dimensional data fusion in other complex biological processes.

In this work, spectral data was used to identify the species and weathering time of blow fly puparia, which helps with the estimation of the minimum post-mortem interval (PMImin) in forensic science. After a preliminary model selection stage, the two best-performing modeling approaches were compared: a classical Logistic Regression (LR) model and a more complex Convolutional Neural Network (CNN). By evaluating both models on these two tasks, we aim to assess the performance trade-offs between competitive traditional machine learning approaches and modern deep learning techniques. Moreover, beyond comparative performance results, an interpretability analysis was conducted to investigate which regions of the spectrum were most informative for weathering time estimation and species classification. These insights may guide future research in forensic entomology using IR spectroscopy by highlighting spectral regions associated with key biological changes related to ageing and species differentiation.

Post-mortem interval (PMI) estimation remains a complex yet crucial task in medico-legal death investigations, as accurate and reliable estimates could establish timelines and contextualize physical evidence. Taphonomic studies often use pig carcasses as human analogues, and while they perform well in some entomology experiments, comparative studies have shown interspecies differences in biomolecular degradation during decomposition. Further, few studies use fluorimetry, leaving the degradation of fluorophores understudied in both species. Herein, we integrated PMI analysis into DNA extraction protocols by probing typically discarded cell lysates, enabling multiple analyses from a single sample. Our dataset included human and pig remains: 50 biopsy punches collected from 14 human donors at the Research in Experimental and Social Thanatology (REST) facility in Québec, Canada and 21 biopsy punches from two pigs placed in Oshawa, Canada, with PMIs ranging from 0-1185 days (9.9-11 281.7 accumulated degree days; ADD). An excitation-emission matrix was acquired for each lysate and deconvoluted using parallel factor analysis (PARAFAC) to identify individual fluorophores. PARAFAC models revealed similar fluorophores and time-dependent trends in both species. Tryptophan fluorescence decreased over time, while fluorescence between 400-500 nm increased as PMI progressed, attributed to an accumulation of fluorescent oxidation products and advanced glycation end-products. Although partial least squares discriminant analysis demonstrated fluorescence differences between species, principal component analysis did not yield species-specific groupings and displayed common PMI/ADD-related trends for both species. Our findings suggest that interspecies differences are not primarily driven by PMI/ADD, and that pig carcasses are a useful human analogue in taphonomic studies using fluorimetry.

Medicolegal forensic entomology allows investigators to estimate the minimum postmortem interval (mPMI) through the predictable attraction of blow flies (Diptera: Calliphoridae) to decomposing remains. Because homicide victims are frequently concealed within plastic bags, the behavioral responses of flies to these physical barriers requires formal investigation. Furthermore, the rising prevalence of scented trash bags introduces an unstudied olfactory variable to carrion colonization. To assess these potential physical and olfactory barriers, raw chicken legs were either left unbagged (control) or completely enclosed within white plastic bags (unscented or lavender-scented low-density polyethylene (LDPE)) and put inside plastic bottle traps to monitor fly attraction over a one-week period. Here we show that Lucilia coeruleiviridis was the overwhelmingly dominant species captured, followed in decreasing abundance by Phormia regina, Sarcophaga spp., Musca spp., and Cochliomyia macellaria. Bagging treatment had no significant effect on trap captures, and the absence of a species-by-treatment interaction indicates similar behavioral responses across all observed taxa. Consequently, thin LDPE scented trash bags do not disrupt blow fly attraction to remains, providing reassurance to medicolegal investigators that these commercial products are unlikely to bias mPMI estimates.

Diptera and Coleoptera (Arthropoda: Insecta) are of great importance in forensic entomology for providing valuable information that contribute to investigations of the cause of death, cases of neglect and mistreatment or for estimating post-mortem interval (PMI), among others. Considering that the period of insect activity (PIA) reliably and accurately represents the PMI and the increasing use of entomological evidence in forensic cases, we report a case in which Hemilucilia segmentaria (Fabricius) (Diptera, Calliphoridae) was used as entomological evidence to estimate the PMI in a suspected death of a woman whose body was found in a wild environment in southeastern Brazil. Additionally, we report the breeding of Sarcophaga (Lyopygia) ruficornis (Fabricius) (Diptera, Sarcophagidae) in the corpse, highlighting the need of data development of this species for accurately estimation of PMI, and postmortem lesions caused by the previous fly species and by the carrion beetle Oxelytrum discicolle (Brullé) (Coleoptera, Staphylinidae, Silphinae), emphasizing the importance of the knowledge about the biology and behaviour of necrophagous species in order to avoid misinterpretation of evidences.