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The yellow dung fly Scathophaga stercoraria is a widely distributed species in high-altitude regions of the Northern Hemisphere. It plays important roles as a decomposer, predator, and pollinator in the ecosystem. As a staple model organism, S. stercoraria serves as a standard test species for assessing the toxicity of drug residues in livestock dung and has been the focus of numerous studies. The genetic mechanisms underlying the ecological adaptability of S. stercoraria remain poorly understood. To fill the gap, we first assembled a high-quality chromosome-level genome of S. stercoraria, resulting in a final assembly size of 549.64 Mb, with a contig N50 of 4.06 Mb, and 92.53 % of the sequence anchored to six chromosomes. Gene family analysis revealed an expansion of Toll (Toll1), GNBP3, Cyp303a1, Cyp4d14, Cyp6g1, OR67d, and yolk protein genes in the S. stercoraria genome. Transcriptome analysis indicated that most genes in the trypsin and carboxypeptidase gene families are predominantly expressed during the larval stage, whereas the α-Amylase gene family is mainly expressed during the adult stage. Additionally, PGRP-SC is highly expressed during the larval stage, OBPs are primarily expressed during the adult stage, and yolk protein genes exhibit female-biased expression. Our study not only provides a new resource for the dung flies genomic pool, but also identifies the expression patterns of key ecologically adaptative genes and gene families at the developmental stages, which provides new insights into the ecological adaptive evolution of dung flies., Competing Interests: Declaration of competing interest The authors have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The leafminer Liriomyza trifolii causes severe economic damage on ornamental and horticultural crops in China. Rapid cold hardening (RCH) is a phenomenon where cold tolerance in insects can be significantly enhanced after a short-term acclimation to low temperatures. In this study, the regulation of transcription in response to cold hardening was investigated in L. trifolii adults, and fatty acids and cryoprotectant levels were measured. The composition of fatty acids changed after RCH treatment, and glucose and trehalose levels showed significant accumulation after acclimation, thus indicating that changes in fatty acids and cryoprotectants contribute to RCH in L. trifolii. RNA-seq was used to analyze transcriptional regulation after a 4 h hardening period and showed that differentially expressed genes clustered in multiple metabolic pathways, which indicates the importance of transcriptional regulation in RCH. This study expands our knowledge of biochemical and transcriptional changes in L. trifolii during cold hardening and provides a basis for further investigations aimed at understanding thermal adaptation in insects., (© 2024. The Author(s).)

Urbanization is a leading factor effecting global biodiversity, driving rapid evolutionary processes in the local biota. Species that adapt and proliferate in city environments can become pests, with human activities facilitating their dispersal and excessive outbreaks. Here we present the first genome data of Plecia longiforceps, a lovebug pest in Eastern Asia with intensive aggregations recently occurring in the Seoul Metropolitan Area of Korea. PacBio HiFi and ONT Pore-C sequencing data were used to construct a highly continuous assembly with a total size of 707 Mb and 8 major pseudochromosomes, its integrity supported by the N50 length of 98.1 Mb and 96.8% BUSCO completeness. Structural and functional annotation using transcriptome data and ab initio predictions revealed a high proportion (69.3%) of repeat sequences, and synteny analysis with Bibio marci showed high levels of genomic collinearity. The genome will serve as an essential resource for both population genomics and molecular research on lovebug dispersal and outbreaks, and also implement studies on the eco-evolutionary processes of insects in urbanizing habitats., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Forensic entomology plays a crucial role in criminal investigations by providing vital insights into minimum postmortem interval (PMI min ) and corpse relocation by identifying insect species that colonize in decomposing remains. This study aimed to identify and analyze the genetic variation of forensically significant fly species in Thailand, using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I COI gene. A total of 3,220 fly specimens were collected from 18 provinces across six regions of Thailand from October 2017 to September 2022. These specimens were classified by morphological identification into 21 species among three Dipteran families: Calliphoridae, Muscidae, and Sarcophagidae, with Chrysomya megacephala Diptera: Calliphoridae being the most abundant species. DNA barcoding confirmed the morphological identifications with 100 % accuracy, showing low intraspecific K2P distances0.0 to 1.1 %) and significant interspecific K2P distances 2.5 % to 17.2 %. A Neighbour-Joining (NJ) analysis was conducted to assess the molecular identification capabilities of the barcoding region. This analysis successfully recovered nearly all species as distinct monophyletic groups. The species groupings obtained were generally consistent with both morphological and molecular identifications. These findings underscore the effectiveness of DNA barcoding for precise species identification and contribute to a comprehensive database of forensically important flies in Thailand, thus facilitating improved forensic investigations and biodiversity studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tissue-specific gene promoters are desired as they provide the specificity needed for control of gene expression in transgenic animals. Here we describe a relatively rapid two-component transient expression assay that was used to identify a gene promoter active in the larval salivary glands of the green blow fly, Lucilia sericata. Sterile L.sericata maggots are widely used for wound debridement. A larval salivary gland gene promoter could be used to make maggots that secrete factors for enhanced wound therapy. Embryos from a line that carry a tetracycline transactivator (tTA)-activated red fluorescent protein gene were injected with plasmid DNA with the tTA gene driven by a constitutive or tissue-specific gene promoter. The hatched larvae were reared on diet and then examined for red fluorescence. A promoter from the LsCG30371 gene was active in the larval salivary glands. The tissue-specificity of the promoter was subsequently confirmed with stable transgenic lines that carried the LsCG30371-tTA gene. The relatively rapid transient expression assay could potentially be used to determine the tissue-specificity of other gene promoters. Further, the stable LsCG30371-tTA lines could be used to make sterile maggots that secrete factors from the salivary glands for enhanced wound healing., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Zoology's dark matter comprises hyperdiverse, poorly known taxa that are numerically dominant but largely unstudied, even in temperate regions where charismatic taxa are well understood. Dark taxa are everywhere, but high diversity, abundance, and small size have historically stymied their study. We demonstrate how entomological dark matter can be elucidated using high-throughput DNA barcoding ("megabarcoding"). We reveal the high abundance and diversity of scuttle flies (Diptera: Phoridae) in Sweden using 31,800 specimens from 37 sites across four seasonal periods. We investigate the number of scuttle fly species in Sweden and the environmental factors driving community changes across time and space., Results: Swedish scuttle fly diversity is much higher than previously known, with 549 putative specie) detected, compared to 374 previously recorded species. Hierarchical Modelling of Species Communities reveals that scuttle fly communities are highly structured by latitude and strongly driven by climatic factors. Large dissimilarities between sites and seasons are driven by turnover rather than nestedness. Climate change is predicted to significantly affect the 47% of species that show significant responses to mean annual temperature. Results were robust regardless of whether haplotype diversity or species-proxies were used as response variables. Additionally, species-level models of common taxa adequately predict overall species richness., Conclusions: Understanding the bulk of the diversity around us is imperative during an era of biodiversity change. We show that dark insect taxa can be efficiently characterised and surveyed with megabarcoding. Undersampling of rare taxa and choice of operational taxonomic units do not alter the main ecological inferences, making it an opportune time to tackle zoology's dark matter., (© 2024. The Author(s).)

The genus Merodon Meigen (Diptera: Syrphidae) is one of the most species-rich hoverfly genera distributed across the Palaearctic and Afrotropical regions. In the Palaearctic, the genus Merodon boasts 195 described species, while its Afrotropical region pales in comparison, with a mere 17 species documented thus far. As a result of 8 years of fieldwork conducted in the Republic of South Africa, in this paper, we present the description of 11 new species for science with a description of immature stages for 2 species, which increases the diversity of this genus in the Afrotropical region by remarkable 39%. These revelations are based on integrating morphology, molecular analysis (COI gene and 28S rRNA) and geometric morphometry. All described species belong to the Merodon planifacies subgroup, the Merodon desuturinus lineage and, within that, to the Afrotropical Merodon melanocerus group. Additionally, we provide an illustrated key to 15 species belonging to the subgroup, a detailed discussion on relevant taxonomic characters, a morphological diagnosis, a distribution map and clarification of the association between M. capi complex and host plants from the genus Merwilla., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

Background: Flystrike, primarily caused by Lucilia cuprina, is a major health and welfare issue for sheep wool industries. Current chemical-based controls can have limited effectiveness due to the emergence of resistance in the parasite. RNA interference (RNAi), which uses double-stranded RNA (dsRNA) as a trigger molecule, has been successfully investigated for the development of innovative pest control strategies. Although RNAi offers great potential, the efficient identification, selection of target genes and delivery of dsRNA represent challenges to be overcome for the successful application of RNAi for control of L. cuprina., Results: A primary L. cuprina (blowfly) embryo cell line (BFEC) was established and confirmed as being derived from L. cuprina eggs by PCR and amplicon sequencing. The BFECs were successfully transfected with plasmids and messenger RNA (mRNA) expressing fluorescent reporter proteins and dsRNA using lipid-based transfection reagents. The transfection of dsRNA into BEFC in this study suggested decreased mRNA levels of target gene expression, which suggested RNAi-mediated knockdown. Three of the dsRNAs identified in this study resulted in reductions of in target gene mRNA levels in BFEC and loss of biological fitness by L. cuprina larvae in a feeding bioassay., Conclusion: This study confirms that the novel BFEC cell line can be used to improve the efficacy of dsRNA-mediated screening to accelerate the identification of potential target genes in the development of RNAi mediated control approaches for L. cuprina. The research models established in this study are encouraging with respect to the use of RNAi as a blowfly control method, however further improvement and validation are required for field applicationsnot prefect, and could be ongoing developing. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Black soldier fly (BSF; Hermetia illucens) is a promising insect species for food and feed production as its larvae can convert different organic waste to high-value protein. Selective breeding is one way to optimize production, but the potential of breeding is only starting to be explored and not yet utilized for BSF. To assist in monitoring a captive population and implementing a breeding program, genomics tools are imperative. We conducted whole genome sequencing of two captive populations separated by geographical distance - Denmark (DK) and Texas, USA (TX). Various population genetics analyses revealed a moderate genetic differentiation between two populations. Moreover, we observed higher inbreeding in the DK population, and the detection of a subpopulation within DK population aligned well with the recent foundation of the DK population from two captive populations. Additionally, we generated gene ontology annotation and variants annotation for wider potential applications. Our findings establish a robust marker set for research in population genetics, facilitating the monitoring of inbreeding and laying the groundwork for practical breeding programs for BSF., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Dr. Tomberlin has a significant financial interest in EVO Conversion Systems LLC. This conflict of interest is managed by a plan submitted to and approved by Texas A&M University and Texas A&M AgriLife., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Invasive species often undergo demographic bottlenecks that cause a decrease in genetic diversity and associated reductions in population fitness. Despite this, they manage to thrive in novel environments. Investigating the effects of inbreeding and genetic bottlenecks on population fitness for invasive species is, therefore, key to understanding how they may survive in new environments. We used the blowfly Calliphora vicina (Sciences, Mathématiques et Physique, 1830, 2, 1), which is native to Europe and was introduced to Australia and New Zealand, to examine the effects of genetic diversity on population fitness. We first collected 59 samples from 15 populations across New Zealand and one in Australia, and used 20,501 biallelic SNPs to investigate population genomic diversity, structure and admixture. We then explored the impacts of repeated experimental bottlenecks on population fitness by creating inbred and outbred lines of C. vicina and measuring a variety of fitness traits. In wild-caught samples, we found low overall genetic diversity, signals of genetic admixture and limited (<3%) genetic differentiation between North and South Island populations, with genetic links between the South Island and Australia. Following experimental bottlenecks, we found significant reductions in fitness for inbred lines. However, fitness effects were not felt equally across all phenotypic traits. Moreover, they were not enough to cause population collapse in any experimental line, suggesting that C. vicina (when under relaxed selection, as in laboratory settings) may be able to compensate for population bottlenecks even when highly inbred. Our results demonstrate the value of a tractable experimental system for investigating processes that may facilitate or hamper biological invasion., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

Purpose: Human ophthalmomyiasis is a rare ocular parasitosis that results from the accidental infestation of dipteran larvae of several species, including Oestrus ovis (Linnaeus, 1758). This study aims to present the fourth documented human case of ophthalmomyiasis in Mexico, identifying the etiological agent through morphological and molecular analyses. Additionally, we investigated the phylogenetic position and genetic distances among different specimens globally characterized based on mitochondrial Cox1 sequences., Methods: A total of five larval specimens were extracted from the patient's eye, with two specimens allocated for identification based on morphological features using a stereomicroscope, and the remaining three preserved in absolute ethanol, one of them used for subsequent analysis using molecular methods. The mitochondrial Cox1 region was amplified and sequenced using automated Sanger sequencing. The resulting sequence was deposited in GenBank under accession number OR440699 and subjected to BlastN analysis against 35 other Cox1 sequences of O. ovis from GenBank. The identity and phylogenetic position of the strains were further explored using parsimony and maximum likelihood phylogenetic methods., Results: Morphological examination of the larval specimens extracted from the patient's eye unequivocally identified them as O. ovis species. BlastN analysis and comprehensive phylogenetic investigations involving a total of 36 Cox1 sequences confirmed the taxonomic identity of the larvae. Notably, our sequence was positioned within the cluster formed by the Brazilian and two Iranian samples. This finding underscores a shared genetic ancestry among these distinct geographical isolates and provides valuable insights into the evolutionary relationships within O. ovis populations., Conclusion: The presence of O. ovis infestation in Mexico City suggests potential shifts in environmental conditions favoring fly proliferation, highlighting the need for vigilance in urban healthcare settings., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The black soldier fly (BSF), Hermetia illucens, has the ability to efficiently bioremediate organic waste into usable bio-compounds. Understanding the impact of domestication and mass rearing on fitness and production traits is therefore important for sustainable production. This study aimed to assess patterns of genomic diversity and its association to phenotypic development across early generations of mass rearing under two selection strategies: selection for greater larval mass (SEL lines) and no direct artificial selection (NS lines). Genome-wide single nucleotide polymorphism (SNP) data were generated using 2bRAD sequencing, while phenotypic traits relating to production and population fitness were measured. Declining patterns of genomic diversity were observed across three generations of captive breeding, with the lowest diversity recorded for the F3 generation of both selection lines, most likely due to founder effects. The SEL cohort displayed statistically significantly greater larval weight com the NS lines with pronounced genetic and phenotypic directional changes across generations. Furthermore, lower genetic and phenotypic diversity, particularly for fitness traits, were evident for SEL lines, illustrating the trade-off between selecting for mass and the resulting decline in population fitness. SNP-based heritability was significant for growth, but was low or non-significant for fitness traits. Genotype-phenotype correlations were observed for traits, but individual locus effect sizes where small and very few of these loci demonstrated a signature for selection. Pronounced genetic drift, due to small effective population sizes, is likely overshadowing the impacts of selection on genomic diversity and consequently phenotypic development. The results hold particular relevance for genetic management and selective breeding for BSF in future., (© 2024 The Author(s). Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Penicillidia dufourii (Westwood 1834) is a specialized parasite categorized under family Nycteribiidae that prefers to parasitize the body surface of various bats under the genus Myotis. Many species of the family Nycteribiidae are carriers of various pathogens; however, research on P. dufourii remains scarce, and studies on its molecular identification and population genetic structure are still lacking. In this study, the complete mitochondrial genome of P. dufourii was elucidated for the first time using Illumina sequencing. The mitochondrial genome is 15,354 bp in size and encodes approximately 37 genes, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region. Analysis of 13 protein-coding genes revealed that UUA, UCA, CGA, and GGA were the most common codons, while nad4L had the fastest evolutionary rate and cox1 the slowest. Phylogenetic analysis based on the mitochondrial genome indicated that P. dufourii is clustered with other species of the family Nycteribiidae and is most closely related to Nycteribia parvula and Phthiridium szechuanum., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: The black soldier fly (BSF), Hermetia illucens, is known for nutrient-recycling through the bioconversion of organic waste into protein-rich insect larvae that can be processed into an animal feed ingredient. However, information on species distribution and its genetic structure in Iran is scarce., Methods and Results: We directed a survey on the Caspian Sea coast, with a reconstructing demographic relationships study using two parts of mitochondrial cytochrome C oxidase 1 (COI) gene (barcode and 3' end regions) and nuclear internal transcribed spacer 2 (ITS2) to identify BSF' genetic diversity in retrospect to the global diversity and the potential origin of the Iranian BSF population. Larvae and adults were recovered from highly decomposed poultry manure, in May 2020. Sequence analysis of both regions of COI gene (about 1500 bp) revealed a single haplotype, identical to that of haplotype C, a worldwide commercial strain originated from Nearctic, Palearctic, or African biogeographic regions. However, the ITS2 locus was confirmed to be invariable across samples from diverse biogeographic regions., Conclusion: The results proved the presence of BSF in north of Iran. However, it is not possible to determine with certainty when and where this species first established in Iran, and they have likely been released to nature due to the existence of companies importing and breeding such flies. Due to heavy international trading, the introduction and settlement of this fly in the southern coasts of the country is highly suggested., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Boukan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Episyrphus balteatus can provide dual ecosystem services including pest control and pollination, which the larvae are excellent predators of aphid pest whereas adults are efficient pollinator. In this study, we assembled a high-quality genome of E. balteatus from northern China geographical population at the chromosome level by using Illumina, PacBio long reads, and Hi-C technologies. The 467.42 Mb genome was obtained from 723 contigs, with a contig N50 of 9.16 Mb and Scaffold N50 of 118.85 Mb, and 90.25% (431.75 Mb) of the assembly was anchored to 4 pseudo-autosomes and one pseudo-heterosome. In total, 14,848 protein-coding genes were annotated, and 95.14% of genes were fully represented in NR, GO, KEGG databases. Besides, we also obtained the mitochondrial genome of E. balteatus of 16, 837 bp in length with 37 typical mitochondrial genes. Overall, this high-quality genome is valuable for evolutionary and genetic studies of E. balteatus and other Syrphidae hoverfly species., (© 2024. The Author(s).)

Linseed, also known as flax is an important oilseed crop with many potential uses in paint, textile, food and pharmaceutical industries. Susceptibility to bud fly (Dasyneura lini Barnes) infestation is a serious biotic concern leading to severe yield penalty in linseed. Protease inhibitors (PIs) are potential candidates that activate during the insect-pest attack and modulate the resistance. In the present study, we explored the PI candidates in the linseed genome and a total of 100 LuPI genes were identified and grouped into five distinct subgroups. The analysis of cis-acting elements revealed that almost all LuPI promoters contain several regulatory elementary related to growth and development, hormonal regulation and stress responses. Across the subfamilies of PIs, the specific domains are consistently found conserved in all protein sequences. The tissue-specific in-silico expression pattern via RNA-seq revealed that all the genes were regulated during different stress. The expression through qRT-PCR of 15 genes revealed the significant up-regulation of LuPI-24, LuPI-40, LuPI-49, LuPI-53, and LuPI-63 upon bud fly infestation in resistant genotype EC0099001 and resistant check variety Neela. This study establishes a foundation resource for comprehending the structural, functional, and evolutionary dimensions of protease inhibitors in linseed., (© 2024. The Author(s).)

Shifts in pollinator occurrence and their pollen transport effectiveness drive the evolution of mating systems in flowering plants. Understanding the genomic basis of these changes is essential for predicting the persistence of a species under environmental changes. We investigated the genomic changes in Brassica rapa over nine generations of pollination by hoverflies associated with rapid morphological evolution toward the selfing syndrome. We combined a genotyping-by-sequencing (GBS) approach with a genome-wide association study (GWAS) to identify candidate genes, and assessed their functional role in the observed morphological changes by studying mutations of orthologous genes in the model plant Arabidopsis thaliana. We found 31 candidate genes involved in a wide range of functions from DNA/RNA binding to transport. Our functional assessment of orthologous genes in A. thaliana revealed that two of the identified genes in B. rapa are involved in regulating the size of floral organs. We found a protein kinase superfamily protein involved in petal width, an important trait in plant attractiveness to pollinators. Moreover, we found a histone lysine methyltransferase (HKMT) associated with stamen length. Altogether, our study shows that hoverfly pollination leads to rapid evolution toward the selfing syndrome mediated by polygenic changes., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

This study presents the sequencing and annotation of mitochondrial genomes from five Rhingia species of the family Syrphidae, focusing on codon bias. Each species possessed 22 tRNAs genes, 13 protein-coding genes, 2 rRNAs genes, and a control region, without any observed gene rearrangements. Nucleotide composition analysis revealed a higher AT content compared with GC content, indicating AT enrichment. Neutrality plot, Parity rule 2 bias, and effective number of codons plot analyses collectively indicated that natural selection primarily influences the codon usage bias in the five Rhingia species. Relative synonymous codon usage analysis identified the optimal codons for Rhingia binotata, R. fromosana, R. campestris, R. louguanensis, and R. xanthopoda as 10, 14, 10, 11, and 12, respectively, all ending with A/U and exhibiting AT preference. Phylogenetic analysis, based on maximum likelihood and Bayesian inference methods applied to three datasets, confirmed the monophyly of Rhingia. In conclusion, this research establishes a foundation for understanding the phylogenetic evolution and codon usage patterns in Rhingia, offering valuable for future studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aphidoletes aphidimyza is widely recognized as an effective predator of aphids in agricultural systems. However, there is limited understanding of its predation mechanisms. In this study, we generated a high-quality chromosome level of the A. aphidimyza genome by combining PacBio, Illumina, and Hi-C data. The genome has a size of 192.08 Mb, with a scaffold N50 size of 46.85 Mb, and 99.08% (190.35 Mb) of the assembly is located on four chromosomes. The BUSCO analysis of our assembly indicates a completeness of 97.8% (n = 1,367), including 1,307 (95.6%) single-copy BUSCOs and 30 (2.2%) duplicated BUSCOs. Additionally, we annotated a total of 13,073 protein-coding genes, 18.43% (35.40 Mb) repetitive elements, and 376 non-coding RNAs. Our study is the first time to report the chromosome-scale genome for the species of A. aphidimyza. It provides a valuable genomic resource for the molecular study of A. aphidimyza., (© 2024. The Author(s).)

The larvae of Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae), the swede midge, targets the meristem of brassica crops where they induce the formation of galls and disrupt seed and vegetable production. Previously, we examined the salivary gland transcriptome of newly-hatched first instar larvae as they penetrated the host and initiated gall formation. Here we examine the salivary gland and midgut transcriptome of third instar larvae and provide evidence for cooperative nutrient acquisition beginning with secretion of enzymes and feeding facilitators followed by gastrointestinal digestion. Sucrose, presumably obtained from the phloem, appeared to be a major nutrient source as several α-glucosidases (sucrases, maltases) and β-fructofuranosidases (invertases) were identified. Genes encoding β-fructofuranosidases/invertases were among the most highly expressed in both tissues and represented two distinct gene families that may have originated via horizontal gene transfer from bacteria. The importance of the phloem as a nutrient source is underscored by the expression of genes encoding regucalcin and ARMET (arginine-rich mutated in early stages of tumor) which interfere with calcium signalling and prevent sieve tube occlusion. Lipids, proteins, and starch appear to serve as a secondary nutrient sources. Genes encoding enzymes involved in the detoxification of glucosinolates (myrosinases, arylsulfatases, and glutathione-S-transferases) were expressed indicative of Brassicaceae host specialization. The midgut expressed simple peritrophins and mucins typical of those found in Type II peritrophic matrices, the first such description for a gall midge., (© 2024 His Majesty the King in Right of Canada and The Author(s). Archives of Insect Biochemistry and Physiology published by Wiley Periodicals LLC. Reproduced with the permission of the Minister of Agriculture and Agri‐Food Canada.)

Despite being endemic in Iraq, no reports have been published in the past 10 years to update the molecular epidemiology of the Old World screwworm fly (OWSF), Chrysomya bezziana, in this country. In the present study, 130 sheep from 10 Iraqi governorates were found infected with C. bezziana larvae, whose identities were PCR-confirmed based on the cytochrome b (Cytb) gene, and 23 isolates from various tested governorates were successfully sequenced. Although most isolates (n = 20) belonged to the common haplotype circulating in Iraq, two new haplotypes were detected. Significant changes in OWSF epidemiology in Iraq were also suggested, since infestations were detected, for the first time, in Nineveh governorate. Isolates of the present study were combined to those previously published from Iraq and worldwide, collected after searching the GenBank, and various genetic and population structure analyses were conducted. These isolates displayed a great statistically significant value when tested for the purifying (negative) selection, suggesting the limited occurrence of genetic variations, which was evidenced by the high sequence conservation (C = 0.937) value detected. A few isolates from Africa were revealed during our search, and clustered in a separate lineage other than that of the Asian isolates. The latter displayed different genetic variation patterns when compared. For example, isolates from geographically separate regions, e.g., the Gulf Arab countries and South-Eastern Asia had marked genetic differences. On the other hand, isolates from regions with close geographic proximity (the Gulf Arab countries and Iran) had limited genetic subdivision. This is not the case when comparing isolates from 10 islands in the Indonesian Archipelago. Populations from Sumatra and Sumba were isolated and displayed high genetic variations toward the other populations. On the contrary, populations from Sulawesi, Lombok and Sumbawa displayed limited genetic variations. This is particularly important, since it can help detecting the dynamics of establishing the sterile insect technique over various regions as an effective control strategy against the OWSFs., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Recent studies on oestroidean Diptera (Brachycera) are providing a comprehensive and nuanced understanding of the evolutionary history of this remarkably diverse clade of holometabolous insects. The Oestroidea, which includes formidable pests such as various blowflies, botflies, and flesh flies that infest livestock, pets and humans, are mostly composed of beneficial species that act as scavengers or parasitoids on various pest insects. In our research, we used genomic methods to elucidate the phylogenetic position of Nesodexia corsicana Villeneuve, 1911 (Diptera: Calliphoridae), a mysterious oestroid species endemic to Corsica and characterized by distinctive morphological features that have puzzled taxonomists for years. Contrary to initial hypotheses, our results place Nesodexia Villeneuve, 1911 within the Calliphoridae subfamily Rhinophorinae, a small lineage of terrestrial isopod parasitoids. Through detailed morphological analysis of adults of both sexes and eggs, we uncovered significant insights consistent with our phylogenomic reconstruction. The unique morphological features of the species, coupled with its restricted and fragmented habitat, highlight its potential conservation importance. We delineated the area of occupancy for N. corsicana and assessed its "threatened" category using specific IUCN Red List criteria. In addition, we mapped the available habitat within its range and determined potential key biodiversity areas (KBA) triggered by N. corsicana. New potential KBAs are only partially covered by the Corsican Regional Park. Finally, we mapped the distribution of habitats on the island to assess the potential distribution of the species beyond its currently known geographic range., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

In forensic entomology, determining the age of a larva from a body to estimate time since death is commonly performed through the measurement of a physical trait. Gene expression has been studied as an alternative age estimation approach, but the storage conditions required for these studies are different than those used in forensic entomological casework. Studies analyzing gene expression prioritize the preservation of RNA, which requires fresh tissue and ultra-cold storage. Casework, in contrast, utilizes hot water killing specimens that may not be analyzed for a long period after collection. In the current study, the impact of hot water killing on gene expression was assessed for larval samples of the forensically important blow fly, Cochliomyia macellaria. Successful amplification of the sex-determining gene, transformer, was tested across larvae ranging in size from 3.22 to 16.85 mm in length after storage times of 1-2 weeks, 4-5 weeks, and 8-9 weeks at 4°C in RNAlater. Larvae hot water killed were processed in tandem with larvae stored live to allow for a direct assessment of the impact of boiling on gene expression. As expected, the transformer gene was successfully amplified in all larvae stored live. For the hot water-killed larvae, the success rate was only slightly lower, with 3 out of 75 larvae not generating a sex-specific band pattern. The results show gene expression can be used for hot water-killed samples, though future work across different genes, species, and extending to quantitative gene expression methods is needed., (© 2024 American Academy of Forensic Sciences.)

Horseflies from the Tabanidae family play a significant role in traditional Chinese medicine to treat various health conditions, including coronary heart disease, stroke, headaches, liver cirrhosis, psoriasis, and hepatic carcinoma. There are 27 species of Tabaninae (Tabanidae) used as medicine, and they showed high morphological similarities with those for which medicinal properties have not been reported. Nonetheless, there have been reports suggesting that medicinal crude drugs sometimes contain irrelevant or false species, impacting the drug's efficacy. In this current study, we collected 14 batches, totaling 13,528 individuals, from various provinces in China. Instead of "classic" DNA barcoding strategy, we employed a high-throughput metabarcoding approach to assess the biological composition of crude drug mixtures derived from horseflies. Our analysis identified 40 Amplicon Sequence Variants (ASVs) with similarity percentages ranging from 92% to 100% with 12 previously reported species. Species delimitation methods revealed the presence of 11 Molecular Operational Taxonomic Units (MOTUs), with ten belonging to the Tabanus genus and one to Hybomitra. Tabanus sp6 displayed the highest relative abundance, and its ASVs showed close resemblance to Tabanus pleski. Our investigations revealed that the medicinal batches were biologically composed of 6 to 12 species. Some batches contained ASVs that closely resembled species previously associated with false Tabanus species. In conclusion, our findings offer valuable insights into the biological composition of crude drugs derived from horseflies and have the potential to enhance the quality of these traditional medicines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: Livestock droppings cause some environmental problems, but they have the potential to be used as effective biomass resources. The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is suitable for efficiently processing such resources. By using BSF larvae for the disposal of livestock droppings, we can obtain two valuable products: protein resources and organic fertilizer. However, there is insufficient research on the digestive enzymes suitable for processing this waste. Here, we aimed to construct an efficient BSF processing system using livestock droppings, and we explored the digestive enzymes involved in this process., Results: First, we investigated the characteristics of transcripts expressed in the midgut of BSF larvae and found that immune response-related genes were expressed in the midgut. Then, we investigated digestive enzymes and identified a novel serine protease, HiBrachyurin, whose mRNA was highly expressed in the posterior midgut when BSF larvae fed on horse droppings. Despite the low protein content of horse droppings, larvae that fed on horse droppings accumulated more protein than those in the other groups. Therefore, HiBrachyurin may contribute to digestibility in the early stage of protein degradation in BSF larvae fed on horse droppings., (© 2024. The Author(s).)

Background: African swine fever (ASF) is a highly contagious and severe haemorrhagic disease of Suidae, with mortalities that approach 100 percent. Several studies suggested the potential implication of non-biting dipterans in the spread of ASFV in pig farms due to the identification of the ASFV DNA. However, to our knowledge, no study has evaluated the viral DNA load in non-biting dipterans collected in outbreak farms and no risk factors have been analysed. In this context, our study aimed to analyse the risk factors associated with the presence of non-biting dipterans collected from ASF outbreaks in relation to the presence and load of viral DNA., Methods: Backyard farms (BF), type A farms (TAF), and commercial farms (CF), were targeted for sampling in 2020. In 2021, no BF were sampled. Each farm was sampled only once. The identification of the collected flies to family, genus, or species level was performed based on morphological characteristics using specific keys and descriptions. Pools were made prior to DNA extraction. All extracted DNA was tested for the presence of the ASFV using a real-time PCR protocol. For this study, we considered every sample with a CT value of 40 as positive. The statistical analysis was performed using Epi Info 7 software (CDC, USA)., Results: All collected non-biting flies belonged to five families: Calliphoridae, Sarcophagidae, Fanniidae, Drosophilidae, and Muscidae. Of the 361 pools, 201 were positive for the presence of ASFV DNA. The obtained CT values of the positive samples ranged from 21.54 to 39.63, with a median value of 33.59 and a mean value of 33.56. Significantly lower CT values (corresponding to higher viral DNA load) were obtained in Sarcophagidae, with a mean value of 32.56; a significantly higher number of positive pools were noticed in August, mean value = 33.12., Conclusions: Our study brings compelling evidence of the presence of the most common synanthropic flies near domestic pig farms carrying ASFV DNA, highlighting the importance of strengthening the biosecurity measures and protocols for prevention of the insect life cycle and distribution., (© 2024. The Author(s).)

Few studies have examined the genetic population structure of vector-borne microparasites in wildlife, making it unclear how much these systems can reveal about the movement of their associated hosts. This study examined the complex host–vector–microbe interactions in a system of bats, wingless ectoparasitic bat flies (Nycteribiidae), vector-borne microparasitic bacteria ( Bartonella ) and bacterial endosymbionts of flies ( Enterobacterales ) across an island chain in the Gulf of Guinea, West Africa. Limited population structure was found in bat flies and Enterobacterales symbionts compared to that of their hosts. Significant isolation by distance was observed in the dissimilarity of Bartonella communities detected in flies from sampled populations of Eidolon helvum bats. These patterns indicate that, while genetic dispersal of bats between islands is limited, some non-reproductive movements may lead to the dispersal of ectoparasites and associated microbes. This study deepens our knowledge of the phylogeography of African fruit bats, their ectoparasites and associated bacteria. The results presented could inform models of pathogen transmission in these bat populations and increase our theoretical understanding of community ecology in host–microbe systems.

Contamination of food products with mycotoxins such as aflatoxin B 1 (AFB 1 ) poses a severe risk to human health. Larvae of the black soldier fly (BSFL), Hermetia illucens (Diptera: Stratiomyidae), can successfully metabolize AFB 1 without any negative consequences on their survival or growth. Organic waste streams contaminated with mycotoxins can be upcycled into protein-rich BSFL as an alternative feed for livestock and the left-over feed residue into nutrient-rich crop fertilizers. However, the underlying mechanisms that allow BSFL to metabolize AFB 1 are unknown. In this study, five-day-old BSFL were fed with either a control or an AFB 1 -spiked (20 μg/kg) diet to elucidate the underlying mechanisms. Larval samples were collected at three timepoints (6 h, 24 h and 72 h) and subjected to RNA-Seq analysis to determine gene expression patterns. Provision of an AFB 1 -spiked diet resulted in an up-regulation of 357 and a down-regulation of 929 unique genes. Upregulated genes include multiple genes involved in AFB 1 metabolism in other (insect) species. Downregulated genes were generally involved in the insects' growth, development, and immunity. BSFL possesses a diverse genetic arsenal that encodes for enzymes capable of metabolizing AFB 1 without trade-offs on larval survival. In conclusion, the adverse impact of AFB 1 exposure on immunity-related processes is observed in the transcriptomic response, and is indicative of a trade-off between detoxification and immune responses., Competing Interests: Declaration of competing interest None, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Accurate insect identification is critical to the estimation of time of colonization (TOC) and post-mortem interval (PMI) in medicolegal death investigations. DNA testing is advantageous because it enables the identification of immature specimens that may not be identified based on morphology alone. We describe here a simplified DNA barcoding method for identifying relevant species that may be implemented by forensic genetics laboratories. A cytochrome oxidase (COI) fragment is analyzed after PCR amplification with a single primer set. The method is effective for many species commonly encountered in death investigations in the USA: members of blowfly genera Calliphora, Chrysomya, Cochliomyia, Lucilia, and Phormia; members of the flesh fly genera Blaesoxipha, Oxysarcodexia, Ravinia, and Sarcophaga; and the scuttle fly Megaselia scalaris. We tested the method on specimens with verified identifications and used it to build a collection of reference sequences from specimens collected in Harris County, Texas. We show here the correct identification of larvae, pupae, and pupal exuviae from the medicolegal casework., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The world faces a major infectious disease challenge. Interest in the discovery, design, or development of antimicrobial peptides (AMPs) as an alternative approach for the treatment of bacterial infections has increased. Insects are a good source of AMPs which are the main effector molecules of their innate immune system. Black Soldier Fly Larvae (BSFL) are being developed for large-scale rearing for food sustainability, waste reduction and as sustainable animal and fish feed. Bioinformatic studies have suggested that BSFL have the largest number of AMPs identified in insects. However, most AMPs identified in BSF have not yet undergone antimicrobial evaluation but are promising leads to treat critical infections., Results: Jg7197.t1, Jg7902.t1 and Jg7904.t1 were expressed into the haemolymph of larvae following infection with Salmonella enterica serovar Typhimurium and were predicted to be AMPs using the computational tool ampir. The genes encoding these proteins were within 2 distinct clusters in chromosome 1 of the BSF genome. Following removal of signal peptides, predicted structures of the mature proteins were superimposed, highlighting a high degree of structural conservation. The 3 AMPs share primary sequences with proteins that contain a Kunitz-binding domain; characterised for inhibitory action against proteases, and antimicrobial activities. An in vitro antimicrobial screen indicated that heterologously expressed SUMO-Jg7197.t1 and SUMO-Jg7902.t1 did not show activity against 12 bacterial strains. While recombinant SUMO-Jg7904.t1 had antimicrobial activity against a range of Gram-negative and Gram-positive bacteria, including the serious pathogen Pseudomonas aeruginosa., Conclusions: We have cloned and purified putative AMPs from BSFL and performed initial in vitro experiments to evaluate their antimicrobial activity. In doing so, we have identified a putative novel defensin-like AMP, Jg7904.t1, encoded in a paralogous gene cluster, with antimicrobial activity against P. aeruginosa., (© 2024. The Author(s).)

Aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mediate the responses of adaptive metabolism to various xenobiotics. Here, we found that BoAhR and BoARNT are highly expressed in the midgut of Bradysia odoriphaga larvae. The expression of BoAhR and BoARNT was significantly increased after exposure to imidacloprid and phoxim. The knockdown of BoAhR and BoARNT significantly decreased the expression of CYP6SX1 and CYP3828A1 as well as P450 enzyme activity and caused a significant increase in the sensitivity of larvae to imidacloprid and phoxim. Exposure to β-naphthoflavone (BNF) significantly increased the expression of BoAhR , BoARNT , CYP6SX1, and CYP3828A1 as well as P450 activity and decreased larval sensitivity to imidacloprid and phoxim. Furthermore, CYP6SX1 and CYP3828A1 were significantly induced by imidacloprid and phoxim, and the silencing of these two genes significantly reduced larval tolerance to imidacloprid and phoxim. Taken together, the BoAhR / BoARNT pathway plays key roles in larval tolerance to imidacloprid and phoxim by regulating the expression of CYP6SX1 and CYP3828A1 .

Trypanosomatids are obligate parasites of animals, predominantly insects and vertebrates, and flowering plants. Monoxenous species, representing the vast majority of trypanosomatid diversity, develop in a single host, whereas dixenous species cycle between two hosts, of which primarily insect serves as a vector. To explore in-depth the diversity of insect trypanosomatids including their co-infections, sequence profiling of their 18S rRNA gene was used for true bugs (Hemiptera; 18% infection rate) and flies (Diptera; 10%) in Cuba. Out of 48 species (molecular operational taxonomic units) belonging to the genera Vickermania (16 spp.), Blastocrithidia (7), Obscuromonas (4), Phytomonas (5), Leptomonas / Crithidia (5), Herpetomonas (5), Wallacemonas (2), Kentomonas (1), Angomonas (1) and two unnamed genera (1 + 1), 38 species have been encountered for the first time. The detected Wallacemonas and Angomonas species constitute the most basal lineages of their respective genera, while Vickermania emerged as the most diverse group. The finding of Leptomonas seymouri , which is known to rarely infect humans, confirms that Dysdercus bugs are its natural hosts. A clear association of Phytomonas with the heteropteran family Pentatomidae hints at its narrow host association with the insect rather than plant hosts. With a focus on multiple infections of a single fly host, using deep Nanopore sequencing of 18S rRNA, we have identified co-infections with up to 8 trypanosomatid species. The fly midgut was usually occupied by several Vickermania species, while Herpetomonas and/or Kentomonas species prevailed in the hindgut. Metabarcoding was instrumental for analysing extensive co-infections and also allowed the identification of trypanosomatid lineages and genera.

The black soldier fly (BSF), Hermetia illucens, has gained traction recently as a means to achieve closed-loop production cycles. BSF can subsist off mammalian waste products and their consumption of such waste in turn generates compost that can be used in agricultural operations. Their environmental impact is minimal and BSF larvae are edible, with a nutritional profile high in protein and other essential vitamins. Therefore, it is conceivable to use BSF as a mechanism for both reducing organic waste and maintaining a low-impact food source for animal livestock or humans. The main drawback to BSF as a potential human food source is they are deficient in fat-soluble vitamins such as Vitamins A, D, and E. While loading BSF with essential vitamins may be achieved via diet-based interventions, this undercuts the goal of a closed-loop as specialized diets would require additional supply chains. An alternative is to genetically engineer BSF that can synthesize these essential vitamins. Here we describe a BSF line that has been engineered with the two main carotenoid biosynthetic genes, CarRA and CarB for production of provitamin carotenoids within the Vitamin A family. Our data describe the manipulation of the BSF genome to insert transgenes for expression of functional protein products., Competing Interests: Declaration of competing interest The authors declare that the research was conducted with no potential conflicts of interest. Echelon Biosciences is a commercial entity with no current products associated with this research and no other commercial or financial relationships related to this project beyond the subcontractor., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Hippoboscid flies are bloodsucking arthropods that can transmit pathogenic microorganisms and are therefore potential vectors for pathogens such as Bartonella spp. These Gram-negative bacteria can cause mild-to-severe clinical signs in humans and animals; therefore, monitoring Bartonella spp. prevalence in louse fly populations appears to be a useful prerequisite for zoonotic risk assessment., Methods: Using convenience sampling, we collected 103 adult louse flies from four ked species (Lipoptena cervi, n = 22; Lipoptena fortisetosa, n = 61; Melophagus ovinus, n = 12; Hippobosca equina, n = 8) and the pupae of M. ovinus (n = 10) in the federal state of Saxony, Germany. All the samples were screened by polymerase chain reaction (PCR) for Bartonella spp. DNA, targeting the citrate synthase gene (gltA). Subsequently, PCRs targeting five more genes (16S, ftsZ, nuoG, ribC and rpoB) were performed for representatives of revealed gltA genotypes, and all the PCR products were sequenced to identify the Bartonella (sub)species accurately., Results and Conclusions: The overall detection rates for Bartonella spp. were 100.0%, 59.1%, 24.6% and 75.0% in M. ovinus, L. cervi, L. fortisetosa and H. equina, respectively. All the identified bartonellae belong to the Bartonella schoenbuchensis complex. Our data support the proposed reclassification of the (sub)species status of this group, and thus we conclude that several genotypes of B. schoenbuchensis were detected, including Bartonella schoenbuchensis subsp. melophagi and Bartonella schoenbuchensis subsp. schoenbuchensis, both of which have previously validated zoonotic potential. The extensive PCR analysis revealed the necessity of multiple PCR approach for proper identification of the ruminant-associated bartonellae., (© 2024 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Wolbachia , a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Diaphorina citri Kuwayama, the vector of Candidatus Liberibacter asiaticus ( C Las) that causes citrus greening disease, is naturally infected with Wolbachia ( w Di). However, the interaction between wDi and D. citri remains poorly understood. In this study, we performed a pan-genome analysis using 65 w Di genomes to gain a comprehensive understanding of w Di. Based on average nucleotide identity (ANI) analysis, we classified the w Di strains into Asia and North America strains. The ANI analysis, principal coordinates analysis (PCoA), and phylogenetic tree analysis supported that the D. citri in Florida did not originate from China. Furthermore, we found that a significant number of core genes were associated with metabolic pathways. Pathways such as thiamine metabolism, type I secretion system, biotin transport, and phospholipid transport were highly conserved across all analyzed w Di genomes. The variation analysis between Asia and North America w Di showed that there were 39,625 single-nucleotide polymorphisms (SNPs), 2153 indels, 10 inversions, 29 translocations, 65 duplications, 10 SV-based insertions, and 4 SV-based deletions. The SV-based insertions and deletions involved genes encoding transposase, phage tail tube protein, ankyrin repeat (ANK) protein, and group II intron-encoded protein. Pan-genome analysis of w Di contributes to our understanding of the geographical population of w Di, the origin of hosts of D. citri , and the interaction between w Di and its host, thus facilitating the development of strategies to control the insects and huanglongbing (HLB).

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. coprophila are presented here. These protocols will be useful for the rapidly increasing number of laboratories studying B. coprophila to take advantage of its unique biological features, which include (1) a monopolar spindle in male meiosis I; (2) non-disjunction of the X dyad in male meiosis II; (3) chromosome imprinting to distinguish maternal from paternal homologs; (4) germ line-limited (L) chromosomes; (5) chromosome elimination (paternal chromosomes in male meiosis I; one to two X chromosomes in early embryos; L chromosomes from the soma in early embryos); (6) sex determination by the mother (there is no Y chromosome); and (7) developmentally regulated DNA amplification at the DNA puff loci in larval salivary gland polytene chromosomes. It is now possible to explore these many unique features of chromosome mechanics by using the recent advances in sequencing and assembly of the B. coprophila genome and the development of transformation methodology for genomic engineering. The growing scientific community that uses B. coprophila for research will benefit from the protocols described here for mating the flies (phenotypic markers for mothers that will have only sons or only daughters; details of mass mating for biochemical experiments), checking embryo hatch, feeding larvae, and other comments on its rearing.

The Australian sheep blowfly, Lucilia cuprina dorsalis , is a major sheep ectoparasite causing subcutaneous myiasis (flystrike), which can lead to reduced livestock productivity and, in severe instances, death of the affected animals. It is also a primary colonizer of carrion, an efficient pollinator, and used in maggot debridement therapy and forensic investigations. In this study, we report the complete mitochondrial (mt) genome of L. c. dorsalis from the Northern Territory (NT), Australia, where sheep are prohibited animals, unlike the rest of Australia. The mt genome is 15,943 bp in length, comprising 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region. The gene order of the current mt genome is consistent with the previously published L. cuprina mt genomes. Nucleotide composition revealed an AT bias, accounting for 77.5% of total mt genome nucleotides. Phylogenetic analyses of 56 species/taxa of dipterans indicated that L. c. dorsalis and L. sericata are the closest among all sibling species of the genus Lucilia , which helps to explain species evolution within the family Luciliinae. This study provides the first complete mt genome sequence for L. c. dorsalis derived from the NT, Australia to facilitate species identification and the examination of the evolutionary history of these blowflies.

The Hessian fly, Mayetiola destructor (Say), is one of the most important insect pest plaguing wheat (Triticum aestivum, L) producers across the United States and around the world. Genetic resistance is the stalwart for control of Hessian fly. However, new genotypes (biotypes) arise in deployment of wheat containing resistance genes, so field populations must be evaluated periodically to provide information on the efficacy of those deployed genes. Louisiana (LA), with its diverse agricultural landscape, is not exempt from the challenges posed by this destructive pest. We previously documented the resistance response of wheat lines harboring Hessian fly resistance (H) genes against field populations collected in 2008 from across the southeastern United States, including Iberville Parish, LA. In the spring of 2023, we reevaluated the resistance response of 27 H genes from the field populations collected from Iberville Parish, LA, and compared the results with those observed in 2008. Sixteen H genes showed comparable resistance to the field populations from both years. While 3 of the H genes, H11, H23, and H24, showed a significant decrease in resistance, 2 genes, H16 and H31, had marked increase in resistance. Furthermore, 6 additional H genes were evaluated in 2023, with 4 showing >70% resistance. Our results clearly identify a total of 20 H genes that are moderate to highly effective against the 2023 Hessian fly population from Iberville Parish, LA. The resistance response documented in this study offers valuable information to wheat breeders in the region for effective management of this insect pest., (Published by Oxford University Press on behalf of Entomological Society of America 2024.)

Transgenic insect strains with tetracycline repressible (Tet-Off) female-lethal genes provide significant advantages over traditional sterile insect techniques for insect population control, such as reduced diet and labor costs and more efficient population suppression. Tet-Off systems are suppressed by tetracycline-class antibiotics, most commonly tetracycline (Tc) or doxycycline (Dox), allowing for equal sex ratio colonies of transgenic insects when reared with Tc or Dox and male-only generations in their absence. Dox is a more stable molecule and has increased uptake than Tc, which could be advantageous in some insect mass-rearing systems. Here, we evaluated the suitability of Dox for rearing Tet-Off female-lethal strains of Australian sheep blowfly, Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae), and New World screwworm, Cochliomyia hominivorax (Coquerel, 1858) (Diptera: Calliphoridae), and the effects of dosage on strain performance. For both species, colonies were able to be maintained with mixed-sex ratios at much lower dosages of Dox than Tc. Biological yields of C. hominivorax on either antibiotic were not significantly different. Reduction of Dox dosages in C. hominivorax diet did not affect biological performance, though rearing with 10 or 25 μg/mL was more productive than 50 μg/mL. Additionally, C. hominivorax mating performance and longevity were equal on all Dox dosages. Overall, Dox was a suitable antibiotic for mass-rearing Tet-Off female-lethal L. cuprina and C. hominivorax and was functional at much lower dosages than Tc., (Published by Oxford University Press on behalf of Entomological Society of America 2024.)

The larvae of black soldier fly (BSFL) convert organic waste into insect proteins used as feedstuff for livestock and aquaculture. BSFL production performance is considerably reduced during winter season. Herein, the intraspecific diversity of ten commercial BSF colonies collected in China was evaluated. The Bioforte colony was subjected to selective breeding at 12 °C and 16 °C to develop cold-tolerant BSF with improved production performance. After breeding for nine generations, the weight of larvae, survival rate, and the dry matter conversion rate significantly increased. Subsequently, intestinal microbiota in the cold-tolerant strain showed that bacteria belonging to Morganella, Dysgonomonas, Salmonella, Pseudochrobactrum, and Klebsiella genera were highly represented in the 12 °C bred, while those of Acinetobacter, Pseudochrobactrum, Enterococcus, Comamonas, and Leucobacter genera were significantly represented in the 16 °C bred group. Metagenomic revealed that several animal probiotics of the Enterococcus and Vagococcus genera were greatly enriched in the gut of larvae bred at 16 °C. Moreover, bacterial metabolic pathways including carbohydrate, lipid, amino acids, and cofactors and vitamins, were significantly increased, while organismal systems and human diseases was decreased in the 16 °C bred group. Transcriptomic analysis revealed that the upregulated differentially expressed genes in the 16 °C bred groups mainly participated in Autophagy-animal, AMPK signaling pathway, mTOR signaling pathway, Wnt signaling pathway, FoxO signaling pathway, Hippo signaling pathway at day 34 under 16 °C conditions, suggesting their significant role in the survival of BSFL. Taken together, these results shed lights on the role of intestinal microflora and gene pathways in the adaptation of BSF larvae to cold stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The Hessian fly (HF), Mayetiola destructor (Diptera: Cecidomyiidae), is one of the most devastating insect pests of cereals including wheat, barley, and rye. Although wheat is the preferred host for HF, this continuously evolving pest has been emerging as a threat to barley production. However, characterization and identification of genetic resistance to HF has not been conducted in barley. In the present study, we used a genome-wide association study (GWAS) to identify barley resistance loci to HF using a geographically diverse set of 234 barley accessions. The results showed that around 90% of barley lines were highly susceptible, indicating a significant vulnerability to HF in barley, and a total of 29 accessions were resistant, serving as potential resistance resources. GWAS with a mixed linear model revealed two marker-trait associations, both on chromosome 4H. The resistance loci and associated markers will facilitate barley improvement and development for breeders. In addition, our results are fundamental for genetic studies to understand the HF resistance mechanism in barley., Competing Interests: The author(s) declare no conflict of interest.

This study aims to study the morphological and molecular characterization of (Pseudolynchia canariensis; Macquart, 1839)in the Al-Baha region of Saudi Arabia. Ninety-four pigeons were obtained from traditional pigeon breeding farms of the Al-Baha region, and fly samples were collected. Taxonomic keys were used to define the morphology of flies, whereas molecular characteristics were identified based on cytochrome c oxidase subunit 1. The rate of Pseudolynchia canariensis infestation in the examined pigeons was 44.5%. The genetic sequences of the fly samples were deposited in GenBank (accession number OQ073507). The match rate between the fly samples from the present study and those previously recorded in GenBank (accession numbers: EF531220, OM073981, and MW853922) displayed 99.66%. This study demonstrates that Pseudolynchia canariensis is common in the Al-Baha region; thus, further studies are required to detect other species from the same genus and their geographical distribution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

As an environmental factor, temperature impacts the distribution of species and influences interspecific competition. The molecular chaperones encoded by small heat shock proteins (sHsps) are essential for rapid, appropriate responses to environmental stress. This study focuses on Hsp20.8 , which encodes a temperature-responsive sHsp in Liriomyza trifolii , an insect pest that infests both agricultural and ornamental crops. Hsp20.8 expression was highest at 39℃ in L. trifolii pupae and adults, and expression levels were greater in pupae than in adults. Recombinant Hsp20.8 was expressed in Escherichia coli and conferred a higher survival rate than the empty vector to bacterial cells exposed to heat stress. RNA interference experiments were conducted using L. trifolii adults and prepupae and the knockdown of Hsp20.8 expression increased mortality in L. trifolii during heat stress. The results expand our understanding of sHsp function in Liriomyza spp. and the ongoing adaptation of this pest to climate change. In addition, this study is also important for predicting the distribution of invasive species and proposing new prevention and control strategies based on temperature adaptation.

Hypodermosis in Cervus elaphus was studied in the Riaño Regional Hunting Reserve, Province of León, north-western Spain. One hundred and ten red deer were examined for the presence of warble fly larvae. They were analyzed by PCR analysis of the COI region of mt-DNA and identified as Hypoderma actaeon. The prevalence of larvae was 42.7% with a mean intensity of 12.5 ± 18 (range 1-80) warbles/deer infested. The distribution of larvae in the infested animals showed an aggregated/overdispersed pattern (aggregation index = 25.84), where the larvae are not randomly or uniformly distributed, but strongly aggregated among their hosts. Larvae were found in all three states. First and second-instars were observed mainly in the autumn until the end of winter (November-March) and third-instars in late winter until mid-spring (March-May). The adult animals and the males had a higher prevalence than the young and the females, finding statistically significant differences only according to the sex of the animals. Seasonal variations were observed in the prevalence with the highest number of infested animals in winter and autumn, but not in terms of the mean intensity of parasites. Additionally, we assessed the presence of anti-Hypoderma antibodies in serum by means of indirect ELISA tests, using a crude larval extract (CLE) and a purified fraction the hypodermin C (HyC) obtained from first instars of Spanish isolates of Hypoderma lineatum (cattle). These findings confirm that H. actaeon is widely distributed in northern Spain, and provide new information about its chronobiology in mountainous Atlantic ecosystems from southwestern Europe., (© 2023. The Author(s).)

Meiotic drivers that act during spermatogenesis derive a transmission advantage by disabling sperm that do not carry the driver, often leading to substantially reduced overall sperm number and function. A new study by Bates et al. shows no sperm deficit for a driver in a stalk-eyed fly, in contrast to a related species. This observed sperm compensation is possibly due to secondary testes-expanding mutations linked to the driving genomic locus., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE). All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In Earth's history warm and cold periods have alternated. Especially, during the Pleistocene, the alternation between these different climatic conditions has led to frequent range expansions and retractions of many species: while thermophilic species dispersed during warm periods, cold adapted species retracted to cold refugia and vice versa. After the last Pleistocene cycle many cold adapted taxa found refuges in relict habitats in mountain ranges. One example for such a cold adapted relict is the flightless snow fly Chionea araneoides (Dalman, 1816). It can be found in lower mountain ranges of Central Europe exclusively in stone runs and stony accumulations which provide cold microclimates. Imagines develop only in winter. They have strongly restricted ranges and hence experienced strong isolation predicting that local populations may show local adaptation and hence also genetic differentiation. We investigated this for several middle mountain ranges of Germany using the COI barcoding gene. Our analyses revealed two distinct lineages, one in the Bavarian Forest and a second one in all other more northern locations up to Scandinavia. These lineages likely go back to post-Pleistocene isolation and should be studied in more detail in the future, also to confirm the taxonomic status of both lineages. Further, we confirmed former records of the species for Germany and report new records for the federal states of Saxony, Lower Saxony, Saxony-Anhalt and Thuringia. Finally, we provide the first evidence of two types of males for the species, a small and a larger male type., (© 2024. The Author(s).)

The black soldier fly (BSF), Hermetia illucens, has the potential to serve as a valuable resource for waste bioconversion due to the ability of the larvae to thrive in a microbial-rich environment. Being an ecological decomposer, the survival of BSF larvae (BSFL) relies on developing an efficient defense system. Cathepsin L (CTSL) is a cysteine protease that plays roles in physiological and pathological processes. In this study, the full-length of CTSL was obtained from BSF. The 1,020-bp open reading frame encoded a preprotein of 339 amino acids with a predicted molecular weight of 32 kDa. The pro-domain contained the conserved ERFNIN, GNYD, and GCNGG motifs, which are all characteristic of CTSL. Homology revealed that the deduced amino acid sequence of BSF CTSL shared 74.22-72.99% identity with Diptera flies. Immunohistochemical (IHC) analysis showed the CTSL was predominantly localized in the gut, especially in the midgut. The mRNA expression of CTSL in different larval stages was analyzed by quantitative real-time PCR (RT-qPCR), which revealed that CTSL was expressed in the second to sixth instar, with the highest expression in the fifth instar. Following an immune challenge in vivo using Escherichia coli (E. coli), CTSL mRNA was significantly up-regulated at 6 h post-stimulation. The Z-Phe-Arg-AMC was gradually cleaved by the BSFL extract after 3 h post-stimulation. These results shed light on the potential role of CTSL in the defense mechanism that helps BSFL to survive against pathogens in a microbial-rich environment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chiang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The level of resistance to radiation and the developmental and molecular responses can vary between species, and even between developmental stages of one species. For flies (order: Diptera), prior studies concluded that the fungus gnat Bradysia (Sciara) coprophila (sub-order: Nematocera) is more resistant to irradiation-induced mutations that cause visible phenotypes than the fruit fly Drosophila melanogaster (sub-order: Brachycera). Therefore, we characterized the effects of and level of resistance to ionizing radiation on B. coprophila throughout its life cycle. Our data show that B. coprophila embryos are highly sensitive to even low doses of gamma-irradiation, whereas late-stage larvae can tolerate up to 80 Gy (compared to 40 Gy for D. melanogaster) and still retain their ability to develop to adulthood, though with a developmental delay. To survey the genes involved in the early transcriptional response to irradiation of B. coprophila larvae, we compared larval RNA-seq profiles with and without radiation treatment. The up-regulated genes were enriched for DNA damage response genes, including those involved in DNA repair, cell cycle arrest, and apoptosis, whereas the down-regulated genes were enriched for developmental regulators, consistent with the developmental delay of irradiated larvae. Interestingly, members of the PARP and AGO families were highly up-regulated in the B. coprophila radiation response. We compared the transcriptome responses in B. coprophila to the transcriptome responses in D. melanogaster from 3 previous studies: whereas pathway responses are highly conserved, specific gene responses are less so. Our study lays the groundwork for future work on the radiation responses in Diptera., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chloropidae, commonly known as grass flies, represent the most taxonomically diverse family of Diptera Carnoidea, comprising over 3000 described species worldwide. Previous phylogenetic studies of this family have predominantly relied on morphological characters, with mitochondrial genomes being reported in a few species. This study presents 11 newly sequenced mitochondrial genomes (10 Chloropidae and 1 Milichiidae) and provides the first comprehensive comparative analysis of mitochondrial genomes for Chloropidae. Apart from 37 standard genes and the control region, three conserved intergenic sequences across Diptera Cyclorrhapha were identified in all available chloropid mitochondrial genomes. Evolutionary rates within Chloropidae exhibit significant variation across subfamilies, with Chloropinae displaying higher rates than the other three subfamilies. Phylogenetic relationships based on mitochondrial genomes were inferred using maximum likelihood and Bayesian methods. The monophyly of Chloropidae and all four subfamilies is consistently strongly supported, while subfamily relationships within Chloropidae remain poorly resolved, possibly due to rapid evolution.

Parasitoids have utilized a variety of strategies to counteract host defense. They are in different taxonomic status and exhibit phenotypic and genetic diversity, and thus are thought to evolve distinct anti-defense mechanisms. In this study, we investigated the performance of two closely related parasitoids, Exorista japonica and Exorista sorbillans (Diptera: Tachinidae) that are biological control agents in agriculture and major insect pests in sericulture, on the host Bombyx mori. We show that the host is more susceptible to E. sorbillans infection while relatively resistant to E. japonica infection. Moreover, the expression levels of host antimicrobial peptides (AMPs) genes are repressed at early infection and induced at late infection of E. japonica, while AMPs are over-expressed at early infection and return to normal levels at late infection of E. sorbillans. In parallel, Toll and IMD pathway genes are generally induced at late infection of E. japonica, whereas these genes are up-regulated at early infection and down-regulated at late infection of E. sorbillans. Activating of host Toll/IMD pathways and AMPs expression by lipopolysaccharide (LPS) represses the larval growth of E. sorbillans. Conversely, inhibiting host Toll/IMD pathways by RNA interference significantly promotes E. japonica development. Therefore, the Toll/IMD pathways are required in the host for defense against infection of dipteran parasitoids. Overall, our study provides the new insight into the diversified host-parasitoid interactions, and offers a theoretical basis for further studies of the adaptive mechanism of dipteran parasitoids., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)