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Background: Gene duplication is a dominant mechanism for the evolution of genomes and plays a key role in genome expansion. Gene duplication via retroposition produces RNA-mediated intron-less copies called retrocopies, that may gain regulatory sequence and biological function to generate retrogenes. Retrocopies dynamics have been reported in several model insect species, but there is still a huge knowledge gap about retrocopies dynamics in most insects, and their role in adaptation., Results: In this study, we reported retrocopy dynamics in 40 species of insect pests of plants belonging to six insect orders. We identified a total of 9,930 retrocopies, which is so far the largest set of retrocopies identified in insects. The identified retrocopies were further grouped into 2,599 Retrogenes, 4,578 Chimeras, 1,241 Intact retrocopies, and 1,512 Pseudogene. We also analyzed all the identified retrogenes that were annotated into 506 gene families. The highest number of retrogenes annotated belong to the heat shock proteins superfamily and are present across all the 40 species from the six orders. We found a significant expansion of the heat shock protein superfamily in the studied species. Almost all the retrogenes, including those belonging to heat shock proteins, are under purifying selection. In summary, we report the retrocopies and retrogenes dynamics in a large set of insect pests of plants and the expansion of the heat shock protein family due to retroposition., Conclusion: This study unveils retrocopy dynamics in the insect pests of plants and highlights the evolution of new genes due to retroposition, and their role in important gene families' expansion., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The recent acceleration in genome sequencing targeting previously unexplored parts of the tree of life presents computational challenges. Samples collected from the wild often contain sequences from several organisms, including the target, its cobionts, and contaminants. Effective methods are therefore needed to separate sequences. Though advances in sequencing technology make this task easier, it remains difficult to taxonomically assign sequences from eukaryotic taxa that are not well represented in databases. Therefore, reference-based methods alone are insufficient. Here, I examine how we can take advantage of differences in sequence composition between organisms to identify symbionts, parasites, and contaminants in samples, with minimal reliance on reference data. To this end, I explore data from the Darwin Tree of Life project, including hundreds of high-quality HiFi read sets from insects. Visualizing two-dimensional representations of read tetranucleotide composition learned by a variational autoencoder can reveal distinct components of a sample. Annotating the embeddings with additional information, such as coding density, estimated coverage, or taxonomic labels allows rapid assessment of the contents of a dataset. The approach scales to millions of sequences, making it possible to explore unassembled read sets, even for large genomes. Combined with interactive visualization tools, it allows a large fraction of cobionts reported by reference-based screening to be identified. Crucially, it also facilitates retrieving genomes for which suitable reference data are absent., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Insect vectors significantly threaten global agriculture by transmitting numerous plant viruses. Various measures, from conventional insecticides to genetic engineering, are used to mitigate this threat. However, none provide complete resistance. Therefore, researchers are looking for novel control options. In recent years with the advancements in genomic technologies, genomes and transcriptomes of various insect vectors have been generated. However, the lack of knowledge about gene functions hinders the development of novel strategies to restrict virus spread. RNA interference (RNAi) is widely used to elucidate gene functions, but its variable efficacy hampers its use in managing insect vectors and plant viruses. Genome editing has the potential to overcome these challenges and has been extensively used in various insect pest species. This review summarizes the progress and potential of genome editing in plant virus vectors and its application as a functional genomic tool to elucidate virus-vector interactions. We also discuss the major challenges associated with editing genes of interest in insect vectors., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

For two decades, DNA barcoding and, more recently, DNA metabarcoding have been used for molecular species identification and estimating biodiversity. Despite their growing use, few studies have systematically evaluated these methods. This study aims to evaluate the efficacy of barcoding methods in identifying species and estimating biodiversity, by assessing their consistency with traditional morphological identification and evaluating how assignment consistency is influenced by taxonomic group, sequence similarity thresholds and geographic distance. We first analysed 951 insect specimens across three taxonomic groups: butterflies, bumblebees and parasitic wasps, using both morphological taxonomy and single-specimen COI DNA barcoding. An additional 25,047 butterfly specimens were identified by COI DNA metabarcoding. Finally, we performed a systematic review of 99 studies to assess average consistency between insect species identity assigned via morphology and COI barcoding and to examine the distribution of research effort. Species assignment consistency was influenced by taxonomic group, sequence similarity thresholds and geographic distance. An average assignment consistency of 49% was found across taxonomic groups, with parasitic wasps displaying lower consistency due to taxonomic impediment. The number of missing matches doubled with a 100% sequence similarity threshold and COI intraspecific variation increased with geographic distance. Metabarcoding results aligned well with morphological biodiversity estimates and a strong positive correlation between sequence reads and species abundance was found. The systematic review revealed an 89% average consistency and also indicated taxonomic and geographic biases in research effort. Together, our findings demonstrate that while problems persist, barcoding approaches offer robust alternatives to traditional taxonomy for biodiversity assessment., (© 2024 The Author(s). Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Field-collected specimens were used to obtain nine high-quality genome assemblies from a total of 10 insect species native to prairies and savannas of central Illinois (USA): Mellilla xanthometata (Lepidoptera: Geometridae), Stenolophus ochropezus (Coleoptera: Carabidae), Forcipata loca (Hemiptera: Cicadellidae), Coelinius sp. (Hymenoptera: Braconidae), Thaumatomyia glabra (Diptera: Chloropidae), Brachynemurus abdominalus (Neuroptera: Myrmeleontidae), Catonia carolina (Hemiptera: Achilidae), Oncometopia orbona (Hemiptera: Cicadellidae), Flexamia atlantica (Hemiptera: Cicadellidae) and Stictocephala bisonia (Hemiptera: Membracidae). Sequencing library preparation from single specimens was successful despite extremely small DNA yields (<0.1 μg) for some samples. Additional sequencing and assembly workflows were adapted to each sample depending on the initial DNA yield. PacBio circular consensus (CCS/HiFi) or continuous long reads (CLR) libraries were used to sequence DNA fragments up to 50 kb in length, with Illumina sequenced linked-reads (TellSeq libraries) and Omni-C libraries used for scaffolding and gap-filling. Assembled genome sizes ranged from 135 MB to 3.2 GB. The number of assembled scaffolds ranged from 47 to >13,000, with the longest scaffold per assembly ranging from ~23 to 439 Mb. Genome completeness was high, with BUSCO scores ranging from 85.5% completeness for the largest genome (Stictocephala bisonia) to 98.8% completeness for the smallest genome (Coelinius sp.). The unique content was estimated using RepeatMasker and GenomeScope2, which ranged from 50.7% to 75.8% and roughly decreased with increasing genome size. Structural annotation predicted a range of 19,281-72,469 protein models for sequenced species. Sequencing costs per genome at the time ranged from US$3-5k, averaged ~1600 CPU-hours on a high-performance cluster and required approximately 14 h of bioinformatics analyses with samples using PacBio HiFi data. Most assemblies would benefit from further manual curation to correct possible scaffold misjoins and translocations suggested by off-diagonal or depleted signals in Omni-C contact maps., (© 2024 The Author(s). Molecular Ecology Resources published by John Wiley & Sons Ltd.)

The evolution of insects has been marked by the appearance of key body plan innovations that promoted the outstanding ability of this lineage to adapt to new habitats, boosting the most successful radiation in animals. To understand the evolution of these new structures, it is essential to investigate which genes and gene regulatory networks participate during the embryonic development of insects. Great efforts have been made to fully understand gene expression and gene regulation during the development of holometabolous insects, in particular Drosophila melanogaster. Conversely, functional genomics resources and databases in other insect lineages are scarce. To provide a new platform to study gene regulation in insects, we generated ATAC-seq for the first time during the development of the mayfly Cloeon dipterum, which belongs to Paleoptera, the sister group to all other winged insects. With these comprehensive datasets along six developmental stages, we characterized pronounced changes in accessible chromatin between early and late embryogenesis. The application of ATAC-seq in mayflies provides a fundamental resource to understand the evolution of gene regulation in insects., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Annotation of newly sequenced genomes frequently includes genes, but rarely covers important non-coding genomic features such as the cis -regulatory modules-e.g., enhancers and silencers-that regulate gene expression. Here, we begin to remedy this situation by developing a workflow for rapid initial annotation of insect regulatory sequences, and provide a searchable database resource with enhancer predictions for 33 genomes. Using our previously developed SCRMshaw computational enhancer prediction method, we predict over 2.8 million regulatory sequences along with the tissues where they are expected to be active, in a set of insect species ranging over 360 million years of evolution. Extensive analysis and validation of the data provides several lines of evidence suggesting that we achieve a high true-positive rate for enhancer prediction. One, we show that our predictions target specific loci, rather than random genomic locations. Two, we predict enhancers in orthologous loci across a diverged set of species to a significantly higher degree than random expectation would allow. Three, we demonstrate that our predictions are highly enriched for regions of accessible chromatin. Four, we achieve a validation rate in excess of 70% using in vivo reporter gene assays. As we continue to annotate both new tissues and new species, our regulatory annotation resource will provide a rich source of data for the research community and will have utility for both small-scale (single gene, single species) and large-scale (many genes, many species) studies of gene regulation. In particular, the ability to search for functionally related regulatory elements in orthologous loci should greatly facilitate studies of enhancer evolution even among distantly related species., Competing Interests: HA, ET, KD, JR, TD, XH, YT, MH No competing interests declared, (© 2024, Asma et al.)

Zoraptera (also called "angel insects") is one of the most unexplored insect orders. However, it holds promise for understanding the evolution of insect karyotypes and genome organization given its status as an early branching group of Polyneoptera and Pterygota (winged insects) during the Paleozoic. Here, we provide karyotype descriptions of three Zorapteran species: Brazilozoros huxleyi (2n♂; ♀ = 42; 42), B. kukalovae (2n♂; ♀ = 43; 44) and Latinozoros cacaoensis (2n♂; ♀ = 36; 36). These species represent two of the four recently recognized Zorapteran subfamilies. Contrary to an earlier suggestion that Zoraptera has holocentric chromosomes, we found karyotypes that were always monocentric. Interestingly, we detected both X0 ( B. kukalovae ) and XY ( B. huxleyi , L. cacaoensis ) sex chromosome systems. In addition to conventional karyotype descriptions, we applied fluorescent in situ hybridization for the first time in Zoraptera to map karyotype distributions of 18S rDNA, histone H3 genes, telomeres and (CAG) n and (GATA) n microsatellites. This study provides a foundation for cytogenetic research in Zoraptera., Competing Interests: The authors declare there are no competing interests., (©2024 Jankásek et al.)

Agricultural insect pests (AIPs) are widely successful in adapting to natural and anthropogenic stressors, repeatedly overcoming population bottlenecks and acquiring resistance to intensive management practices. Although they have been largely overlooked in evolutionary studies, AIPs are ideal systems for understanding rapid adaptation under novel environmental conditions. Researchers have identified several genomic mechanisms that likely contribute to adaptive stress responses, including positive selection on de novo mutations, polygenic selection on standing allelic variation and phenotypic plasticity (e.g., hormesis). However, new theory suggests that stress itself may induce epigenetic modifications, which may confer heritable physiological changes (i.e., stress-resistant phenotypes). In this perspective, we discuss how environmental stress from agricultural management generates the epigenetic and genetic modifications that are associated with rapid adaptation in AIPs. We summarise existing evidence for stress-induced evolutionary processes in the context of insecticide resistance. Ultimately, we propose that studying AIPs offers new opportunities and resources for advancing our knowledge of stress-induced evolution., (© 2024 Royal Entomological Society.)

Insect cuticle exhibits a wide array of micro- and nano-structures in terms of size, form, and function. However, the investigation of cellular mechanisms of morphogenesis has centered around a small number of structure types and organisms. The recent expansion of the taxa studied, and subsequent discoveries prompt us to revisit well-known models, like the one for bristle morphogenesis. In addition, common themes are emerging in the morphogenesis of cuticular structures, such as the polyploidy of precursor cells, the role of pigments and cuticular proteins in controlling chitin deposition in space and time, and the role of the apical extracellular matrix in defining the shape of the developing structure. Understanding how these structures are synthesized in biological systems holds promise for bioinspired design., Competing Interests: Declaration of Competing Interest The author declares no competing or financial interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

While genomic resources for social insects have vastly increased over the past two decades, we are still far from understanding the genetic and molecular basis of eusociality. Here, we briefly review three scientific advancements that, when integrated, can be highly synergistic for advancing our knowledge of the genetics and evolution of eusocial traits. Population genomics provides a natural way to quantify the strength of natural selection on coding and regulatory sequences, highlighting genes that have undergone adaptive evolution during the evolution or maintenance of eusociality. Genome-wide association studies (GWAS) can be used to characterize the complex genetic architecture underlying eusocial traits and identify candidate causal variants. Concurrently, CRISPR/Cas9 enables the precise manipulation of gene function to both validate genotype-phenotype associations and study the molecular biology underlying interesting traits. While each approach has its own advantages and disadvantages, which we discuss herein, we argue that their combination will ultimately help us better understand the genetics and evolution of eusocial behavior. Specifically, by triangulating across these three different approaches, researchers can directly identify and study loci that have a causal association with key phenotypes and have evidence of positive selection over the relevant timescales associated with the evolution and maintenance of eusociality in insects., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Insects represent the most diverse animal group, yet previous phylogenetic analyses based on morphological and molecular data have failed to agree on the evolutionary relationships of early insects and their six-legged relatives (together constituting the clade Hexapoda). In particular, the phylogenetic positions of the three early-diverging hexapod lineages-the coneheads (Protura), springtails (Collembola), and two-pronged bristletails (Diplura)-have been debated for over a century, with alternative topologies implying drastically different scenarios of the evolution of the insect body plan and hexapod terrestrialization. We addressed this issue by sampling all hexapod orders and experimenting with a broad range of across-site compositional heterogeneous models designed to tackle ancient divergences. Our analyses support Protura as the earliest-diverging hexapod lineage ("Protura-sister") and Collembola as a sister group to Diplura, a clade corresponding to the original composition of Entognatha, and characterized by the shared possession of internal muscles in the antennal flagellum. The previously recognized 'Elliplura' hypothesis is recovered only under the site-homogeneous substitution models with partial supermatrices. Our cross-validation analysis shows that the site-heterogeneous CAT-GTR model, which recovers "Protura-sister," fits significantly better than homogeneous models. Furthermore, the morphologically unusual Protura are also supported as the earliest-diverging hexapod lineage by other lines of evidence, such as mitogenomes, comparative embryology, and sperm morphology, which produced results similar to those in this study. Our backbone phylogeny of hexapods will facilitate the exploration of the underpinnings of hexapod terrestrialization and megadiversity., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Symbiotic relationships with diverse microorganisms are crucial for many aspects of insect biology. However, while our understanding of insect taxonomic diversity and the distribution of insect species in natural communities is limited, we know much less about their microbiota. In the era of rapid biodiversity declines, as researchers increasingly turn towards DNA-based monitoring, developing and broadly implementing approaches for high-throughput and cost-effective characterization of both insect and insect-associated microbial diversity is essential. We need to verify whether approaches such as high-throughput barcoding, a powerful tool for identifying wild insects, would permit subsequent microbiota reconstruction in these specimens., Methods: High-throughput barcoding ("megabarcoding") methods often rely on non-destructive approaches for obtaining template DNA for PCR amplification by leaching DNA out of insect specimens using alkaline buffers such as HotSHOT. This study investigated the impact of HotSHOT on microbial abundance estimates and the reconstructed bacterial community profiles. We addressed this question by comparing quantitative 16S rRNA amplicon sequencing data for HotSHOT-treated or untreated specimens of 16 insect species representing six orders and selected based on the expectation of limited variation among individuals., Results: We find that in 13 species, the treatment significantly reduced microbial abundance estimates, corresponding to an estimated 15-fold decrease in amplifiable 16S rRNA template on average. On the other hand, HotSHOT pre-treatment had a limited effect on microbial community composition. The reconstructed presence of abundant bacteria with known significant effects was not affected. On the other hand, we observed changes in the presence of low-abundance microbes, those close to the reliable detection threshold. Alpha and beta diversity analyses showed compositional differences in only a few species., Conclusion: Our results indicate that HotSHOT pre-treated specimens remain suitable for microbial community composition reconstruction, even if abundance may be hard to estimate. These results indicate that we can cost-effectively combine barcoding with the study of microbiota across wild insect communities. Thus, the voucher specimens obtained using megabarcoding studies targeted at characterizing insect communities can be used for microbiome characterizations. This can substantially aid in speeding up the accumulation of knowledge on the microbiomes of abundant and hyperdiverse insect species., Competing Interests: The authors declare that they have no competing interests., (© 2024 Andriienko et al.)

The ability to control gene expression is pivotal in genetic engineering and synthetic biology. However, in most nonmodel and pest insect species, empirical evidence for predictable modulation of gene expression levels is lacking. This knowledge gap is critical for genetic control systems, particularly in mosquitoes, where transgenic methods offer novel routes for pest control. Commonly, the choice of RNA polymerase II promoter (Pol II) is the primary method for controlling gene expression, but the options are limited. To address this, we developed a systematic approach to characterize modifications in translation initiation sequences (TIS) and 3' untranslated regions (UTR) of transgenes, enabling the creation of a toolbox for gene expression modulation in mosquitoes and potentially other insects. The approach demonstrated highly predictable gene expression changes across various cell lines and 5' regulatory sequences, representing a significant advancement in mosquito synthetic biology gene expression tools.

Unraveling the phylogeographic histories of species remains a key endeavor for comprehending the evolutionary processes contributing to the rich biodiversity and high endemism found in East Asia. In this study, we explored the phylogeographic patterns and demographic histories of three endemic fishfly and dobsonfly species ( Neochauliodes formosanus , Protohermes costalis , and Neoneuromus orientalis ) belonging to the holometabolan order Megaloptera. These species, which share a broad and largely overlapping distribution, were analyzed using comprehensive mitogenomic data. Our findings revealed a consistent influence of vicariance on the population isolation of Neoc. formosanus and P. costalis between Hainan, Taiwan, and the East Asian mainland during the early Pleistocene, potentially hindering subsequent colonization of the later diverged Neon. orientalis to these islands. Additionally, we unveiled the dual function of the major mountain ranges in East Asia, serving both as barriers and conduits, in shaping the population structure of all three species. Notably, we demonstrated that these co-distributed species originated from Southwest, Southern, and eastern Central China, respectively, then subsequently migrated along multi-directional routes, leading to their sympatric distribution on the East Asian mainland. Furthermore, our results highlighted the significance of Pleistocene land bridges along the eastern coast of East Asia in facilitating the dispersal of mountain-dwelling insects with low dispersal ability. Overall, this study provides novel insight into the synergistic impact of Pleistocene geological and climatic events in shaping the diversity and distribution of aquatic insects in East Asia.

MicroRNAs (miRNAs) are small, non-coding RNAs that are expressed in a tissue- and temporal-specific manner during development. They have been found to be highly conserved during the evolution of different species. miRNAs regulate the expression of several genes in various organisms, with some regulating the expression of multiple genes with similar or completely unrelated functions. Frequent disease and insect pest infestations severely limit agricultural development. Thus, cultivating resistant crops via miRNA-directed gene regulation in plants, insects, and pathogens is an important aspect of modern breeding practices. To strengthen the application of miRNAs in sustainable agriculture, plant endogenous or exogenous miRNAs have been used for plant breeding. Consequently, the development of biological pesticides based on miRNAs has become an important avenue for future pest control methods. However, selecting the appropriate miRNA according to the desired target traits in the target organism is key to successfully using this technology for pest control. This review summarizes the progress in research on miRNAs in plants and other species involved in regulating plant disease and pest resistance pathways. We also discuss the molecular mechanisms of relevant target genes to provide new ideas for future research on pest and disease resistance and breeding in plants.

Main Conclusion: The review offers insights into the current state of research on insect pest-resistant GM crops and the regulations governing the cultivation of GM crops in India. India has a rich crop diversity of more than 160 major and minor crops through its diverse agroclimatic conditions. Insect pests alone cause around USD 36 billion in crop loss annually in India. The last two decades witnessed considerable progress in managing insect pests by adopting innovative techniques including transgenics. In research, significant advancement has been brought in insect pest-resistant transgenics in India since its inception in 2002. However, any events have not been endorsed owing to biosafety impediments, except Bt cotton reaching the commercial release stage. A landmark decision to exempt certain types of gene-edited plants from genetically modified organism (GMO) regulations offers great promise for developing novel insect-resistant crops in India. The article reviews the current research on insect pest-resistant transgenics and its regulations in India., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Insects have evolved complex and diverse visual systems in which light-sensing protein molecules called "opsins" couple with a chromophore to form photopigments. Insect photopigments group into three major gene families based on wavelength sensitivity: long wavelength (LW), short wavelength (SW), and ultraviolet wavelength (UV). In this study, we identified 123 opsin sequences from whole-genome assemblies across 25 caddisfly species (Insecta: Trichoptera). We discovered the LW opsins have the most diversity across species and form two separate clades in the opsin gene tree. Conversely, we observed a loss of the SW opsin in half of the trichopteran species in this study, which might be associated with the fact that caddisflies are active during low-light conditions. Lastly, we found a single copy of the UV opsin in all the species in this study, with one exception: Athripsodes cinereus has two copies of the UV opsin and resides within a clade of caddisflies with colorful wing patterns., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Osmoregulation, the physiological regulation of water and ion balance, is vital for the survival of both aquatic and terrestrial insects. In freshwater aquatic insects, such as those within the Lampyridae family, this function is important due to the natural variation of aquatic habitats. Aquaporins play a key role in this process by facilitating the rapid transport of water molecules across cell membranes, maintaining cellular water balance, and adapting to changes in external salinity. In this study, I investigate the genetic diversity and expression levels of aquaporins in Elateroidea, particularly focusing on the Lampyridae family, using transcriptomic data and in silico analyses. The results reveal the diversity of aquaporins and compare gene expression patterns between freshwater aquatic Lampyridae and terrestrial Elateroidea species, such as Lycidae, Phengodidae, and Elateridae. Phylogenetic analyses identify seven distinct clades of aquaporins and uncovered gene duplication events related to the diversification of Elateridae and Lampyridae. A comparative abundance analysis indicated higher aquaporin expression in aquatic fireflies, aligning with the need for efficient osmoregulation in aquatic environments. Additionally, stage-specific expression patterns in Aspisoma lineatum (Neotropical firefly) and Aquatica lateralis (Paleartic firefly) suggest species-specific strategies for coping with osmotic challenges during development. This study provides insights into the evolutionary adaptations of aquaporins in Elateroidea, highlighting their importance in both aquatic and terrestrial insect physiology., (© 2024 Wiley Periodicals LLC.)

Molt-based transitions in form are a central feature of insect life that have enabled adaptation to diverse and changing environments. The endocrine regulation of these transitions is well established, but an understanding of their genetic regulation has only recently emerged from insect models. The pupal and adult stages of metamorphosing insects are determined by the stage specifying transcription factors broad-complex (br) and Ecdysone inducible protein 93 (E93), respectively. A probable larval determinant, chronologically inappropriate metamorphosis (chinmo), has just recently been characterized. Expression of these three transcription factors in the metamorphosing insects is regulated by juvenile hormone with ecdysteroid hormones, and by mutual repression between the stage-specific transcription factors. This review explores the hypothesis that variations in the onset, duration, and tissue-specific expression of chinmo, br, and E93 underlie other polyphenisms that have arisen throughout insects, including the castes of social insects, aquatic stages of mayflies, and the neoteny of endoparasites. The mechanisms that constrain how chinmo, br, and E93 expression may vary will also constrain the ways that insect life history may evolve. I find that four types of expression changes are associated with novel insect forms: (1) heterochronic shift in the turnover of expression, (2) expansion or contraction of expression, (3) tissue-specific expression, and (4) redeployment of stage-specific expression. While there is more to be learned about chinmo, br, and E93 function in diverse insect taxa, the studies outlined here show that insect stages are modular units in developmental time and a substrate for evolutionary forces to act upon., (© 2024 The Author(s). Evolution & Development published by Wiley Periodicals LLC.)

Interrogating the ecological and geographic factors that influence population divergence dynamics can reveal why some groups of organisms diversify more prolifically than others. One such group is the heathers (Erica, Ericaceae), the largest plant genus in the Cape Floristic Region. We study Erica abietina, a highly variable species complex with four subspecies differing in geographic range, habitat and pollination syndrome. We test for population differentiation, hybridisation, introgression and pollinator-driven divergence using genotyping-by-sequencing on samples across the entire distribution. We find five variably distinct genetic groups, with one subspecies comprising two independent lineages that are geographically isolated and occur on different soil types. Phylogenetic analysis suggests two independent shifts between bird and insect pollination, with accompanying genetic divergence. However, for one pair of populations with different pollinators, we uncover several individuals of hybrid origin at a site of sympatry. These results suggest that floral differentiation driven by divergent selection acts in concert with geographic isolation to maintain reproductive isolation and promote speciation. Our investigations reveal a highly dynamic system whose diversity has been shaped by a variety of interacting forces. We suggest that such a system could be a model for much of the diversification of the Cape flora., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

AbstractRepeatable macroevolutionary patterns provide hope for rules in biology, especially when we can decipher the underlying mechanisms. Here we synthesize natural history, genetic adaptations, and toxin sequestration in herbivorous insects that specialize on plants with cardiac glycoside defenses. Work on the monarch butterfly provided a model for evolution of the "sequestering specialist syndrome," where specific amino acid substitutions in the insect's Na + /K + -ATPase are associated with (1) high toxin resistance (target site insensitivity [TSI]), (2) sequestration of toxins, and (3) aposematic coloration. We evaluate convergence for these traits within and between Lepidoptera, Coleoptera, Diptera, Hemiptera, Hymenoptera, and Orthoptera, encompassing hundreds of toxin-adapted species. Using new and existing data on ∼28 origins of specialization, we show that the monarch model evolved independently in five taxonomic orders (but not Diptera). An additional syndrome occurs in five orders (all but Hymenoptera): aposematic sequesterers with modest to medium TSI. Indeed, all sequestering species were aposematic, and all but one had at least modest TSI. Additionally, several species were aposematic nonsequesterers (potential Batesian mimics), and this combination evolved in species with a range of TSI levels. Finally, we identified some biases among these strategies within taxonomic orders. Biodiversity in this microcosm of life evolved repeatedly with a high degree of similarity across six taxonomic orders, yet we identified alternative trait combinations as well as lineage-specific outcomes.

Despite major breeding efforts by various national and international agencies, yields for the ~40 million hectares of maize, the major food crop in sub-Saharan Africa, have stagnated at <2 tons/ha/year for the past decade, one-third the global average. Breeders have succeeded in breeding increased yield with a modicum of tolerance to some single-weed or pathogen stresses. There has been minimal adoption of these varieties because introgressing polygenic yield and tolerance traits into locally adapted material is very challenging. Multiple traits to deal with pests (weeds, pathogens, and insects) are needed for farmer acceptance, because African fields typically encounter multiple pest constraints. Also, maize has no inherent resistance to some of these pest constraints, rendering them intractable to traditional breeding. The proposed solution is to simultaneously engineer multiple traits into one genetic locus. The dominantly inherited multi-pest resistance trait single locus can be bred simply into locally adapted, elite high-yielding material, and would be valuable for farmers, vastly increasing maize yields, and allowing for more than regional maize sufficiency. © 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.)

Trichoptera (caddisfly) phylogeny provides an interesting example of aquatic insect evolution, with rich ecological diversification, especially for underwater architecture. Trichoptera provide numerous critical ecosystem services and are also one of the most important groups of aquatic insects for assessing water quality. The phylogenetic relationships of Trichoptera have been debated for nearly a century. In particular, the phylogenetic position of the "cocoon-makers" within Trichoptera has long been contested. Here, we designed a universal single-copy orthologue and sets of ultraconserved element markers specific for Trichoptera for the first time. Simultaneously, we reconstructed the phylogenetic relationship of Trichoptera based on genome data from 111 species, representing 29 families and 71 genera. Our phylogenetic inference clarifies the probable phylogenetic relationships of "cocoon-makers" within Integripalpia. Hydroptilidae is considered as the basal lineage within Integripalpia, and the families Glossosomatidae, Hydrobiosidae, and Rhyacophilidae formed a monophyletic clade, sister to the integripalpian subterorder Phryganides. The resulting divergence time and ancestral state reconstruction suggest that the most recent common ancestor of Trichoptera appeared in the early Permian and that diversification was strongly correlated with habitat change., 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 Inc. All rights reserved.)

Twisted-wing insects (Strepsiptera) are an enigmatic order of parasites with unusual life histories and striking sexual dimorphism. Males emerge from hosts as free-living winged adults, while females from most species remain as endoparasites that retain larval traits. Due to scarce genomic data and phylogenetic controversies, Strepsiptera was only recently placed as the closest living relative to beetles (Coleoptera). Here, we report the first PacBio HiFi genome assembly of the strepsipteran Xenos peckii (Xenidae). This de novo assembly size is 72.1 Mb, with a BUSCO score of 87.4%, N50 of 7.3 Mb, 23.4% GC content, and 38.41% repeat content. We identified 8 contigs that contain >75% of the assembly and reflect the haploid chromosome number reported from karyotypic data, and 3 contigs that exhibit sex chromosome coverage patterns. Additionally, the mitochondrial genome is 16,111 bp long and has 37 genes. This long-read assembly for Strepsiptera reveals a miniature genome and provides a unique tool to understand complex genome evolution associated with a parasitic lifestyle and extreme sexual dimorphism., (© 2024. The Author(s).)

Background: Adenosine-to-inosine (A-to-I) RNA editing is a co-/post-transcriptional modification introducing A-to-G variations in RNAs. There is extensive discussion on whether the flexibility of RNA editing exerts a proteomic diversification role, or it just acts like hardwired mutations to correct the genomic allele. Eusocial insects evolved the ability to generate phenotypically differentiated individuals with the same genome, indicating the involvement of epigenetic/transcriptomic regulation., Methods: We obtained the genomes of 104 Hymenoptera insects and the transcriptomes of representative species. Comparative genomic analysis was performed to parse the evolutionary trajectory of a regulatory Ile > Met auto-recoding site in Adar gene., Results: At genome level, the pre-editing Ile codon is conserved across a node containing all eusocial hymenopterans. At RNA level, the editing events are confirmed in representative species and shows considerable condition-specificity. Compared to random expectation, the editable Ile codon avoids genomic substitutions to Met or to uneditable Ile codons, but does not avoid mutations to other unrelated amino acids., Conclusions: The flexibility of Adar auto-recoding site in Hymenoptera is selectively maintained, supporting the flexible RNA editing hypothesis. We proposed a new angle to view the adaptation of RNA editing, providing another layer to explain the great phenotypical plasticity of eusocial insects., (© 2024. The Author(s).)

Biopesticides based on RNA interference (RNAi) took a major step forward with the first registration of a sprayable RNAi product, which targets the world's most damaging potato pest. Proactive resistance management is needed to delay the evolution of resistance by pests and sustain the efficacy of RNAi biopesticides., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Fluctuations in temperature are recognized as a potent driver of selection pressure, fostering genomic variations that are crucial for the adaptation and survival of organisms under selection. Notably, water temperature is a pivotal factor influencing aquatic organism persistence. By comprehending how aquatic organisms respond to shifts in water temperature, we can understand their potential physiological adaptations to environmental change in one or multiple species. This, in turn, contributes to the formulation of biologically relevant guidelines for the landscape scale transcriptome profile of organisms in lotic systems. Here, we investigated the distinct responses of seven stream stonefly species, collected from four geographical regions across Japan, to variations in temperature, including atmospheric and water temperatures. We achieved this by assessing the differences in gene expression through RNA-sequencing within individual species and exploring the patterns of community-genes among different species. We identified 735 genes that exhibited differential expressions across the temperature gradient. Remarkably, the community displayed expression levels differences of respiration and metabolic genes. Additionally, the diversity in molecular functions appeared to be linked to spatial variation, with water temperature differences potentially contributing to the overall functional diversity of genes. We found 22 community-genes with consistent expression patterns among species in response to water temperature variations. These genes related to respiration, metabolism and development exhibited a clear gradient providing robust evidence of divergent adaptive responses to water temperature. Our findings underscore the differential adaptation of stonefly species to local environmental conditions, suggesting that shared responses in gene expression may occur across multiple species under similar environmental conditions. This study emphasizes the significance of considering various species when assessing the impacts of environmental changes on aquatic insect communities and understanding potential mechanisms to cope with such changes., (© 2024 Wiley Periodicals LLC.)

Declines in insect populations have gained formidable attention. Given their crucial role in the ecosystem, the causes of declining insect populations must be investigated. However, the insect clade has been associated with low extinction and high diversification rates. It is unlikely that insects underwent mass extinctions in the past. However, the pace of current climate change could make insect populations vulnerable to extinction. We propose genome size (GS) and transposable elements (TEs) to be rough estimates to assess extinction risk. Larger GS and/or proliferating TEs have been associated with adaptation in rapid climate change scenarios. We speculate that unstable, stressful environmental conditions are strongly associated with GS and TE expansion, which could be further correlated with adaptations. Alternately, stressful conditions trigger TE bursts that are not purged in smaller populations. GS and TE loads could be indicators of small effective populations in the wild, likely experiencing bottlenecks or drastic climatic perturbations, which calls for an urgent assessment of extinction risk., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Insect-protected soybean (SIP) that produces the Cry1A.105 and Cry2Ab2 insecticidal crystal proteins has been developed to provide protection from feeding damage caused by targeted lepidopteran insect pests. Typically, as part of environmental risk assessment (ERA), plant characterization is conducted, and the data submitted to regulatory agencies prior to commercialization of genetically modified (GM) crops. The objectives of this research were to: (a) compare soybean with and without the SIP trait in plant characterization field trials designed to fulfill requirements for submissions to global regulatory agencies and address China-specific considerations and (b) compare risk assessment conclusions across regions and the methodologies used in the field trials. The soybean with and without the SIP trait in temperate, tropical, and subtropical germplasm were planted in replicated multi-location trials in the USA (in 2012 and 2018) and Brazil (in 2013/2014 and 2017/2018). Agronomic, phenotypic, plant competitiveness, and survival characteristics were assessed for soybean entries with and without the SIP trait. Regardless of genetic background, growing region, season, or testing methodology, the risk assessment conclusions were the same: the evaluated insect-protected soybean did not differ from conventional soybean in evaluated agronomic, phenotypic, competitiveness, and survival characteristics indicating no change in plant pest/weed potential. These results reinforce the concept of data transportability across global regions, different seasons, germplasm, and methodologies that should be considered when assessing environmental risks of GM crops., (© 2024. The Author(s).)

Insect-pests infestation greatly affects global agricultural production and is projected to become more severe in upcoming years. There is concern about pesticide application being ineffective due to insect resistance and environmental toxicity. Reduced effectiveness of Bt toxins also made the scientific community shift toward alternative strategies to control devastating agricultural pests. With the advent of host-delivered RNA interference, also known as host-induced gene silencing, targeted insect genes have been suppressed through genetic engineering tools to deliver a novel insect-pest resistance strategy for combating a number of agricultural pests. This review recapitulates the possible mechanism of host-delivered RNA interference (HD-RNAi), in particular, the silencing of target genes of insect-pests. We emphasize the development of the latest strategies against evolving insect targets including designing of artificial microRNAs, vector constructs, and the benefit of using plastid transformation to transform target RNA-interfering genes. Advantages of using HD-RNAi over other small RNA delivery modes and also the supremacy of HD-RNAi over the CRISPR-Cas system particularly for insect resistance have been described. However, the broader application of this technology is restricted due to its several limitations. Using artificial miRNA designs, the host-delivered RNAi + Bt combinatorial approach and chloroplast transformation can overcome limitations of RNAi. With careful design and delivery approaches, RNAi promises to be extremely valuable and effective plant protection strategy to attain durable insect-pest resistance in crops., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Oxidative phosphorylation, essential for energy metabolism and linked to the regulation of longevity, involves mitochondrial and nuclear genes. The functions of these genes and their evolutionary rate covariation (ERC) have been extensively studied, but little is known about whether other nuclear genes not targeted to mitochondria evolutionarily and functionally interact with mitochondrial genes. Here we systematically examined the ERC of mitochondrial and nuclear benchmarking universal single-copy ortholog (BUSCO) genes from 472 insects, identifying 75 non-mitochondria-targeted nuclear genes. We found that the uncharacterized gene CG11837-a putative ortholog of human DIMT1-regulates insect lifespan, as its knockdown reduces median lifespan in five diverse insect species and Caenorhabditis elegans, whereas its overexpression extends median lifespans in fruit flies and C. elegans and enhances oxidative phosphorylation gene activity. Additionally, DIMT1 overexpression protects human cells from cellular senescence. Together, these data provide insights into the ERC of mito-nuclear genes and suggest that CG11837 may regulate longevity across animals., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The rapid advancement of sequencing technologies has revolutionized our understanding of the phylogeny and evolution of insects, enabling researchers to generate extensive molecular data with unprecedented detail [...].

When inferring the evolution of a gene/gene family, it is advisable to use all available coding sequences (CDS) from as many species genomes as possible in order to infer and date all gene duplications and losses. Nowadays, this means using hundreds or even thousands of CDSs, which makes the inferred phylogenetic trees difficult to visualize and interpret. Therefore, it is useful to have an automated way of collapsing large phylogenetic trees according to a taxonomic term decided by the user (family, class, or order, for instance), in order to highlight the minimal set of sequences that should be used to recapitulate the full history of the gene/gene family being studied at that taxonomic level, that can be refined using additional software. Here we present the Phylogenetic Tree Collapser (PTC) program (https://github.com/pegi3s/phylogenetic-tree-collapser), a flexible tool for automated tree collapsing using taxonomic information, that can be easily used by researchers without a background in informatics, since it only requires the installation of Docker, Podman or Singularity. The utility of PTC is demonstrated by addressing the evolution of the ascorbic acid synthesis pathway in insects. A Docker image is available at Docker Hub (https://hub.docker.com/r/pegi3s/phylogenetic-tree-collapser) with PTC installed and ready-to-run., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Insects are established models for understanding host-pathogen interactions and innate immune mechanisms. The innate immune system in insects is highly efficient in recognizing and opposing pathogens that cause detrimental effects during infection. The cuticular layer which covers the superficial layer of the insect body participates in host defense and wound healing by inducing innate immune responses. Previous studies have started to address the involvement of cuticular genes in conferring resistance to insect pathogens, particularly those that infect by disrupting the insect cuticle. For example, the cuticular gene Transglutaminase (TG) in Drosophila melanogaster plays a structural role in cuticle formation and blood coagulation and also possesses immune properties against pathogenic infection. However, more information is becoming available about the immune function of other cuticular gene families in insects. In this review, we aim to highlight the recent advances in insect cuticular immunity and address the necessity of pursuing further research to fill the existing gaps in this important field of insect immunology. This information will lead to novel strategies for the efficient management of agricultural insect pests and vectors of plant and human disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mallick and Eleftherianos.)

Kempnyia (Plecoptera: Perlidae) is an endemic genus of Brazilian stoneflies that has 36 valid species and is distributed primarily in the Atlantic Forest and the mountainous areas of Central Brazil, particularly in Goiás and Tocantins states. Despite being the Brazilian genus with the most DNA sequences available on GenBank, integrative studies on the genus began only recently, in 2014. In this context, herein we studied the morphology and molecular data of Kempnyia specimens deposited in the Aquatic Biology Laboratory (UNESP, Assis) and the Entomology Museum of the Federal University of Viçosa (UFVB, Viçosa) collections. For the integrative approach adopted, in addition to studying the specimens morphologically, we used sequences of the COI mitochondrial gene combined with the following species delimitation methods: Automatic Barcode Gap Discovery (ABGD), both primary (ABGDp) and recursive (ABGDr) partitions; Assemble Species by Automatic Partitioning (ASAP); Poisson Tree Processes (PTP) and the Bayesian implementation of the Poisson Tree Processes (bPTP). As a result, we provided 28 new COI sequences of 21 species and support the description of four new species, namely, K. guarani sp. nov., K. tupiniquim sp. nov., K. una sp. nov., and K. zwickii sp. nov., consequently increasing the known diversity of the genus to 40 species. We also discuss the morphological variations observed in other species of the genus and provide several new geographic records. Therefore, our study brings new insights into the values of intra- and interspecific molecular divergence within Kempnyia, serving as a basis for new studies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 de Almeida 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 significance of entomological evidence in inferring the time, location and cause of death has been demonstrated both theoretically and practically. With the advancement of sequencing technologies, reports have emerged on necrophagous insects' nuclear genomes, transcriptomes, proteomes and mitochondrial genomes. However, within the field of forensic entomology, there is currently no available database that can integrate, store and share the resources of necrophagous insects. The absence of a database poses an inconvenience to the application of entomological evidence in judicial practice and hampers the development of the forensic entomology discipline. Given this, we have developed the Home Of Forensic Entomology database, encompassing 10 core functional modules: Home, Browse, Mitochondria, Proteome, JBrowse, Search, BLAST, Tools, Case base and Maps. Notably, the 'Tools' module enables multiple sequence alignment analysis (Muscle), homologous protein prediction (Genewise), primer design (Primer), large-scale genomic analysis (Lastz), Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, as well as expression profiling (PCA Analysis, Hcluster and Correlation Heatmap). In addition, the present database also works as an interactive platform for researchers by sharing forensic entomological case reports and uploading data and material. This database provides potential visitors with a comprehensive function for multi-omics data analysis, offers substantial references to researchers and criminal scene investigators and facilitates the utilization of entomological evidence in court. Database URL: http://ihofe.com/., (© The Author(s) 2024. Published by Oxford University Press.)

Caddisflies (Trichoptera) are among the most diverse groups of freshwater animals with more than 16 000 described species. They play a fundamental role in freshwater ecology and environmental engineering in streams, rivers and lakes. Because of this, they are frequently used as indicator organisms in biomonitoring programmes. Despite their importance, key questions concerning the evolutionary history of caddisflies, such as the timing and origin of larval case making, remain unanswered owing to the lack of a well-resolved phylogeny. Here, we estimated a phylogenetic tree using a combination of transcriptomes and targeted enrichment data for 207 species, representing 48 of 52 extant families and 174 genera. We calibrated and dated the tree with 33 carefully selected fossils. The first caddisflies originated approximately 295 million years ago in the Permian, and major suborders began to diversify in the Triassic. Furthermore, we show that portable case making evolved in three separate lineages, and shifts in diversification occurred in concert with key evolutionary innovations beyond case making.

Insect cytochromes P450 (CYP450s) are key enzymes responsible for a wide array of oxidative transformations of both endogenous and exogenous substrates. However, there is currently no a universal guideline established for heterologous expression of membrane-bound CYP450s, which hampers their downstream biochemical and structural studies. In this study, we conducted large-scale screening of protein overexpression in Escherichia coli using 71 insect CYP450 sequences and optimized the expression of a difficult-to-express CYP450 (CYP6HX3) using eight different optimizations, including selection of host strains and expression vectors, alternative of leader signal peptides, and N-terminal modifications. We confirmed that 1) Only insect CYP450s belonging to the CYP347 family could be expressed with N-terminal fusion of ompA 2+ signal peptide in E. coli expression system. 2) E. coli Lemo 21 (DE3) effectively improved the expression of CYP6HX3 in the plasma membrane. 3) A brick-red appearance occurred frequently in the expressed thallus or membrane proteins, but this phenomenon could not necessarily indicate successful overexpression of target CYP450s. These findings provide new insights into the recombinant expression of insect CYP450s in E. coli systems and will facilitate the theoretical approaches for functional expression and production of eukaryotic CYP450s., 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.)

Gene regulatory network (GRN) comprises complicated yet intertwined gene-regulator relationships. Understanding the GRN dynamics will unravel the complexity behind the observed gene expressions. Insect gene regulation is often complicated due to their complex life cycles and diverse ecological adaptations. The main interest of this review is to have an update on the current mathematical modelling methods of GRNs to explain insect science. Several popular GRN architecture models are discussed, together with examples of applications in insect science. In the last part of this review, each model is compared from different aspects, including network scalability, computation complexity, robustness to noise and biological relevancy., 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 FibH gene, crucial for silk spinning in insects, encodes a protein that significantly influences silk fiber mechanics. Due to its large size and repetitive sequences, limited known sequences of insect FibH impede comprehensive understanding. Here, we analyzed 114 complete FibH gene sequences from Lepidoptera (71 moths, 24 butterflies) and 13 Trichoptera, revealing single-copy FibH in most species, with 2-3 copies in Hesperinae and Heteropterinae (subfamily of skippers). All FibH genes are structured with two exons and one intron (39-45 bp), with the second exon being notably longer. Moths exhibit higher GC content in FibH compared to butterflies and Trichoptera. The FibH composition varies among species, with moths and butterflies favoring Ala, Gly, Ser, Pro, Gln, and Asn, while Trichoptera FibH is enriched in Gly, Ser, and Arg, and has less Ala. Unique to Trichoptera FibH are Tyr, Val, Arg, and Trp, whereas Lepidoptera FibH is marked by polyAla (polyalanine), polySer (polyserine), and the hexapeptide GAGSGA. A phylogenetic analysis suggests that Lepidoptera FibH evolved from Trichoptera, with skipper FibH evolving from Papilionoidea. This study substantially expands the FibH repertoire, providing a foundation for the development of artificial silk.

Phenotypic variation within species can affect the ecological dynamics of populations and communities. Characterizing the genetic variation underlying such effects can help parse the roles of genetic evolution and plasticity in "eco-evolutionary dynamics" and inform how genetic variation may shape patterns of evolution. Here, we employ genome-wide association (GWA) methods in Timema cristinae stick insects and their co-occurring arthropod communities to identify genetic variation associated with community-level traits. Previous studies have shown that maladaptation (i.e., imperfect crypsis) of T. cristinae can reduce the abundance and species richness of other arthropods due to an increase in bird predation. Whether genetic variation that is independent of crypsis has similar effects is unknown and was tested here using genome-wide genotyping-by-sequencing data of stick insects, arthropod community information, and GWA mapping with Bayesian sparse linear mixed models. We find associations between genetic variation in stick insects and arthropod community traits. However, these associations disappear when host-plant traits are accounted for. We thus use path analysis to disentangle interrelationships among stick-insect genetic variation, host-plant traits, and community traits. This revealed that host-plant size has large effects on arthropod communities, while genetic variation in stick insects has a smaller, but still significant effect. Our findings demonstrate that (1) genetic variation in a species can be associated with community-level traits but that (2) interrelationships among multiple factors may need to be analyzed to disentangle whether such associations represent causal relationships. This work helps to build a framework for genomic studies of eco-evolutionary dynamics., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Evolutionary Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Holistic insect monitoring needs scalable techniques to overcome taxon biases, determine species abundances, and gather functional traits for all species. This requires that we address taxonomic impediments and the paucity of data on abundance, biomass and functional traits. We here outline how these data deficiencies could be addressed at scale. The workflow starts with large-scale barcoding (megabarcoding) of all specimens from mass samples obtained at biomonitoring sites. The barcodes are then used to group the specimens into molecular operational taxonomic units that are subsequently tested/validated as species with a second data source (e.g. morphology). New species are described using barcodes, images and short diagnoses, and abundance data are collected for both new and described species. The specimen images used for species discovery then become the raw material for training artificial intelligence identification algorithms and collecting trait data such as body size, biomass and feeding modes. Additional trait data can be obtained from vouchers by using genomic tools developed by molecular ecologists. Applying this pipeline to a few samples per site will lead to greatly improved insect monitoring regardless of whether the species composition of a sample is determined with images, metabarcoding or megabarcoding. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Molecular methods are currently some of the best-suited technologies for implementation in insect monitoring. However, the field is developing rapidly and lacks agreement on methodology or community standards. To apply DNA-based methods in large-scale monitoring, and to gain insight across commensurate data, we need easy-to-implement standards that improve data comparability. Here, we provide three recommendations for how to improve and harmonize efforts in biodiversity assessment and monitoring via metabarcoding: (i) we should adopt the use of synthetic spike-ins, which will act as positive controls and internal standards; (ii) we should consider using several markers through a multiplex polymerase chain reaction (PCR) approach; and (iii) we should commit to the publication and transparency of all protocol-associated metadata in a standardized fashion. For (i), we provide a ready-to-use recipe for synthetic cytochrome c oxidase spike-ins, which enable between-sample comparisons. For (ii), we propose two gene regions for the implementation of multiplex PCR approaches, thereby achieving a more comprehensive community description. For (iii), we offer guidelines for transparent and unified reporting of field, wet-laboratory and dry-laboratory procedures, as a key to making comparisons between studies. Together, we feel that these three advances will result in joint quality and calibration standards rather than the current laboratory-specific proof of concepts. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Aquatic macroinvertebrates, including many aquatic insect orders, are a diverse and ecologically relevant organismal group yet they are strongly affected by anthropogenic activities. As many of these taxa are highly sensitive to environmental change, they offer a particularly good early warning system for human-induced change, thus leading to their intense monitoring. In aquatic ecosystems there is a plethora of biotic monitoring or biomonitoring approaches, with more than 300 assessment methods reported for freshwater taxa alone. Ultimately, monitoring of aquatic macroinvertebrates is used to calculate ecological indices describing the state of aquatic systems. Many of the methods and indices used are not only hard to compare, but especially difficult to scale in time and space. Novel DNA-based approaches to measure the state and change of aquatic environments now offer unprecedented opportunities, also for possible integration towards commonly applicable indices. Here, we first give a perspective on DNA-based approaches in the monitoring of aquatic organisms, with a focus on aquatic insects, and how to move beyond traditional point-based biotic indices. Second, we demonstrate a proof-of-concept for spatially upscaling ecological indices based on environmental DNA, demonstrating how integration of these novel molecular approaches with hydrological models allows an accurate evaluation at the catchment scale. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

As large cities begin to overrun their landfill capacities, they begin to look for alternative locations to handle the waste stream. Seeing an opportunity to bring in revenue, rural communities offer to handle municipal waste in their landfills. However, many rural communities are also places of agricultural production, which are vulnerable to attacks by invasive insect species, which could be present in green yard waste, the component of municipal waste most likely to contain agriculturally harmful insect species. We used environmental DNA (eDNA) to determine whether green yard waste could be a pathway for invasive insect species to enter and establish in the landfill-receiving agricultural community. We identified several target species that could be in green yard waste coming from Vancouver, BC, Canada, to Central Washington State, USA. We sampled green yard waste from 3 sites every 2 weeks from June to October in 2019 and 2020. DNA was extracted from the nearly 400 samples and subjected to amplification with COI barcoding primers followed by sequencing to identify target insects in the samples. Sequence analyses identified 3 species from the target list: 2 species that are pests of deciduous tree fruits and a generalist root-feeding crop pest. This eDNA technique was useful in identifying potential invasive species in green yard waste and may prove to be an important tool informing policy on the movement of biological material across borders and stemming the spread of invasive species., (Published by Oxford University Press on behalf of Entomological Society of America 2024.)

The adaptation of insects to environments relies on a sophisticated set of behaviors controlled by molecular and physiological processes. Over the past several decades, accumulating studies have unveiled the roles of non-coding RNAs (ncRNAs) in regulating insect behaviors. ncRNAs assume particularly pivotal roles in the behavioral plasticity of insects by rapidly responding to environmental stimuli. ncRNAs also contribute to the maintenance of homeostasis of insects by fine-tuning the expression of target genes. However, a comprehensive review of ncRNAs' roles in regulating insect behaviors has yet to be conducted. Here, we present the recent progress in our understanding of how ncRNAs regulate various insect behaviors, including flight and movement, social behavior, reproduction, learning and memory, and feeding. We refine the intricate mechanisms by which ncRNAs modulate the function of neural, motor, reproductive, and other physiological systems, as well as gene expression in insects like fruit flies, social insects, locusts, and mosquitos. Furthermore, we discuss potential avenues for future studies in ncRNA-mediated insect behaviors., (© 2024. Science China Press.)