Genomic resources of two aquatic Lepidoptera, Elophila obliteralis and Hyposmocoma kahamanoa, reveal similarities with Trichoptera in amino acid composition of major silk genes.

While most species of butterflies and moths (Lepidoptera) have entirely terrestrial life histories, ∼0.5% of the described species are known to have an aquatic larval stage. Larvae of aquatic Lepidoptera are similar to caddisflies (Trichoptera) in that they use silk to anchor themselves to underwater substrates or to build protective cases. However, the physical properties and genetic elements of silks in aquatic Lepidoptera remain unstudied, as most research on lepidopteran silk has focused on the commercially important silkworm, Bombyx mori. Here, we provide high-quality PacBio HiFi genome assemblies of two distantly-related aquatic Lepidoptera species (Elophila obliteralis (Pyraloidea: Crambidae) and Hyposmocoma kahamanoa (Gelechioidea: Cosmopterigidae)). As a step toward understanding the evolution of underwater silk in aquatic Lepidoptera, we used our two genome assemblies and compared them to published genetic data of aquatic and terrestrial Lepidoptera. Sequences of the primary silk protein, h-fibroin in aquatic moths have conserved termini and share a basic motif structure with terrestrial Lepidoptera. However, these sequences were similar to aquatic Trichoptera in that the percentage of positively and negatively charged amino acids was much higher than in terrestrial Lepidoptera, indicating a possible adaptation of silks to aquatic environments.

Evolutionary genomics of three agricultural pest moths reveals rapid evolution of host adaptation and immune-related genes.

BACKGROUND: Understanding the genotype of pest species provides an important baseline for designing integrated pest management (IPM) strategies. Recently developed long-read sequence technologies make it possible to compare genomic features of nonmodel pest species to disclose the evolutionary path underlying the pest species profiles. Here we sequenced and assembled genomes for 3 agricultural pest gelechiid moths: Phthorimaea absoluta (tomato leafminer), Keiferia lycopersicella (tomato pinworm), and Scrobipalpa atriplicella (goosefoot groundling moth). We also compared genomes of tomato leafminer and tomato pinworm with published genomes of Phthorimaea operculella and Pectinophora gossypiella to investigate the gene family evolution related to the pest species profiles. RESULTS: We found that the 3 solanaceous feeding species, P. absoluta, K. lycopersicella, and P. operculella, are clustered together. Gene family evolution analyses with the 4 species show clear gene family expansions on host plant-associated genes for the 3 solanaceous feeding species. These genes are involved in host compound sensing (e.g., gustatory receptors), detoxification (e.g., ABC transporter C family, cytochrome P450, glucose-methanol-choline oxidoreductase, insect cuticle proteins, and UDP-glucuronosyl), and digestion (e.g., serine proteases and peptidase family S1). A gene ontology enrichment analysis of rapid evolving genes also suggests enriched functions in host sensing and immunity. CONCLUSIONS: Our results of family evolution analyses indicate that host plant adaptation and pathogen defense could be important drivers in species diversification among gelechiid moths.

CRISPR-based diagnostics detects invasive insect pests.

Rapid identification of organisms is essential for many biological and medical disciplines, from understanding basic ecosystem processes, disease diagnosis, to the detection of invasive pests. CRISPR-based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR-based diagnostic developed with the universal cytochrome-oxidase 1 gene (CO1). The CO1 gene is the most sequenced gene among Animalia, and therefore our approach can be adopted to detect nearly any animal. We tested the approach on three difficult-to-identify moth species (Keiferia lycopersicella, Phthorimaea absoluta and Scrobipalpa atriplicella) that are major invasive pests globally. We designed an assay that combines recombinase polymerase amplification (RPA) with CRISPR for signal generation. Our approach has a much higher sensitivity than real-time PCR assays and achieved 100% accuracy for identification of all three species, with a detection limit of up to 120 fM for P. absoluta and 400 fM for the other two species. Our approach does not require a sophisticated laboratory, reduces the risk of cross-contamination, and can be completed in less than 1 h. This work serves as a proof of concept that has the potential to revolutionize animal detection and monitoring.

CRISPR-based diagnostics detects invasive insect pests.

Rapid identification of organisms is essential across many biological and medical disciplines, from understanding basic ecosystem processes and how organisms respond to environmental change, to disease diagnosis and detection of invasive pests. CRISPR-based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR-based diagnostic developed with the universal cytochrome-oxidase 1 gene (CO1). The CO1 gene is the most sequenced gene among Animalia, and therefore our approach can be adopted to detect nearly any animal. We tested the approach on three difficult-to-identify moth species (Keiferia lycopersicella, Phthorimaea absoluta, and Scrobipalpa atriplicella) that are major invasive pests globally. We designed an assay that combines recombinase polymerase amplification (RPA) with CRISPR for signal generation. Our approach has a much higher sensitivity than other real time-PCR assays and achieved 100% accuracy for identification of all three species, with a detection limit of up to 120 fM for P. absoluta and 400 fM for the other two species. Our approach does not require a lab setting, reduces the risk of cross-contamination, and can be completed in less than one hour. This work serves as a proof of concept that has the potential to revolutionize animal detection and monitoring.

A global phylogeny of butterflies reveals their evolutionary history, ancestral hosts and biogeographic origins.

Butterflies are a diverse and charismatic insect group that are thought to have evolved with plants and dispersed throughout the world in response to key geological events. However, these hypotheses have not been extensively tested because a comprehensive phylogenetic framework and datasets for butterfly larval hosts and global distributions are lacking. We sequenced 391 genes from nearly 2,300 butterfly species, sampled from 90 countries and 28 specimen collections, to reconstruct a new phylogenomic tree of butterflies representing 92% of all genera. Our phylogeny has strong support for nearly all nodes and demonstrates that at least 36 butterfly tribes require reclassification. Divergence time analyses imply an origin ~100 million years ago for butterflies and indicate that all but one family were present before the K/Pg extinction event. We aggregated larval host datasets and global distribution records and found that butterflies are likely to have first fed on Fabaceae and originated in what is now the Americas. Soon after the Cretaceous Thermal Maximum, butterflies crossed Beringia and diversified in the Palaeotropics. Our results also reveal that most butterfly species are specialists that feed on only one larval host plant family. However, generalist butterflies that consume two or more plant families usually feed on closely related plants.

Mezcal worm in a bottle: DNA evidence suggests a single moth species.

Mezcals are distilled Mexican alcoholic beverages consumed by many people across the globe. One of the most popular mezcals is tequila, but there are other forms of mezcal whose production has been part of Mexican culture since the 17th century. It was not until the 1940-50s when the mezcal worm, also known as the "tequila worm", was placed inside bottles of non-tequila mezcal before distribution. These bottled larvae increased public attention for mezcal, especially in Asia, Europe, and the United States. Despite these larvae gaining global interest, their identity has largely remained uncertain other than that they are larvae of one of three distantly related holometabolous insects. We sequenced the COI gene from larvae in different kinds of commercially available mezcals. All larval DNA that amplified was identified as the agave redworm moth, Comadia redtenbacheri. Those that did not amplify were also confirmed morphologically to be the larva of this species.

Anti-bat ultrasound production in moths is globally and phylogenetically widespread.

Warning signals are well known in the visual system, but rare in other modalities. Some moths produce ultrasonic sounds to warn bats of noxious taste or to mimic unpalatable models. Here, we report results from a long-term study across the globe, assaying moth response to playback of bat echolocation. We tested 252 genera, spanning most families of large-bodied moths, and document anti-bat ultrasound production in 52 genera, with eight subfamily origins described. Based on acoustic analysis of ultrasonic emissions and palatability experiments with bats, it seems that acoustic warning and mimicry are the raison d'être for sound production in most moths. However, some moths use high-duty-cycle ultrasound capable of jamming bat sonar. In fact, we find preliminary evidence of independent origins of sonar jamming in at least six subfamilies. Palatability data indicate that jamming and warning are not mutually exclusive strategies. To explore the possible organization of anti-bat warning sounds into acoustic mimicry rings, we intensively studied a community of moths in Ecuador and, using machine-learning approaches, found five distinct acoustic clusters. While these data represent an early understanding of acoustic aposematism and mimicry across this megadiverse insect order, it is likely that ultrasonically signaling moths comprise one of the largest mimicry complexes on earth.

The genome sequence of the peacock moth, Macaria notata (Linnaeus, 1758).

We present a genome assembly from an individual male Macaria notata (the peacock moth; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 394 megabases in span. The majority of the assembly (99.98%) is scaffolded into 29 chromosomal pseudomolecules with the Z sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

Long-read HiFi sequencing correctly assembles repetitive heavy fibroin silk genes in new moth and caddisfly genomes.

Insect silk is a versatile biomaterial. Lepidoptera and Trichoptera display some of the most diverse uses of silk, with varying strength, adhesive qualities, and elastic properties. Silk fibroin genes are long (>20 Kbp), with many repetitive motifs that make them challenging to sequence. Most research thus far has focused on conserved N- and C-terminal regions of fibroin genes because a full comparison of repetitive regions across taxa has not been possible. Using the PacBio Sequel II system and SMRT sequencing, we generated high fidelity (HiFi) long-read genomic and transcriptomic sequences for the Indianmeal moth (Plodia interpunctella) and genomic sequences for the caddisfly Eubasilissa regina. Both genomes were highly contiguous (N50  = 9.7 Mbp/32.4 Mbp, L50  = 13/11) and complete (BUSCO complete  = 99.3%/95.2%), with complete and contiguous recovery of silk heavy fibroin gene sequences. We show that HiFi long-read sequencing is helpful for understanding genes with long, repetitive regions.

The genome sequence of the sallow kitten, Furcula furcula (Clerck, 1759).

We present a genome assembly from an individual male Furcula furcula (the sallow kitten; Arthropoda; Insecta; Lepidoptera; Notodontidae). The genome sequence is 736 megabases in span. The entire assembly (100%) is scaffolded into 29 chromosomal pseudomolecules, with the Z sex chromosome assembled. The complete mitochondrial genome was also assembled and is 17.2 kilobases in length.

First Annotated Genome of a Mandibulate Moth, Neomicropteryx cornuta, Generated Using PacBio HiFi Sequencing.

We provide a new, annotated genome assembly of Neomicropteryx cornuta, a species of the so-called mandibulate archaic moths (Lepidoptera: Micropterigidae). These moths belong to a lineage that is thought to have split from all other Lepidoptera more than 300 Ma and are consequently vital to understanding the early evolution of superorder Amphiesmenoptera, which contains the order Lepidoptera (butterflies and moths) and its sister order Trichoptera (caddisflies). Using PacBio HiFi sequencing reads, we assembled a highly contiguous genome with a contig N50 of nearly 17 Mb. The assembled genome length of 541,115,538 bp is about half the length of the largest published Amphiesmenoptera genome (Limnephilus lunatus, Trichoptera) and double the length of the smallest (Papilio polytes, Lepidoptera). We find high recovery of universal single copy orthologs with 98.1% of BUSCO genes present and provide a genome annotation of 15,643 genes aided by resolved isoforms from PacBio IsoSeq data. This high-quality genome assembly provides an important resource for studying ecological and evolutionary transitions in the early evolution of Amphiesmenoptera.

Review of recent taxonomic changes to the emerald moths (Lepidoptera: Geometridae: Geometrinae).

BACKGROUND: The subfamily Geometrinae (Lepidoptera: Geometridae), commonly known as emerald moths, is an ecologically diverse group of moths with over 2,500 described species. Many taxonomic and phylogenetic studies of Geometrinae have been undertaken in the past decade, resulting in hundreds of new taxonomic changes since online publication of the most recent checklist in December 2007. NEW INFORMATION: This review synthesises the last 12 years of alpha-taxonomic research in Geometrinae. A comprehensive list of Geometrinae genus- and species-group descriptions, synonymies, combinations and other taxonomic changes, made since 2007, is provided. Since 2007, the known species richness of Geometrinae has increased from 2,529 to 2,642 species; an updated list of all these species is presented in a supplementary spreadsheet.

Phylogenomics reveals the evolutionary timing and pattern of butterflies and moths.

Butterflies and moths (Lepidoptera) are one of the major superradiations of insects, comprising nearly 160,000 described extant species. As herbivores, pollinators, and prey, Lepidoptera play a fundamental role in almost every terrestrial ecosystem. Lepidoptera are also indicators of environmental change and serve as models for research on mimicry and genetics. They have been central to the development of coevolutionary hypotheses, such as butterflies with flowering plants and moths' evolutionary arms race with echolocating bats. However, these hypotheses have not been rigorously tested, because a robust lepidopteran phylogeny and timing of evolutionary novelties are lacking. To address these issues, we inferred a comprehensive phylogeny of Lepidoptera, using the largest dataset assembled for the order (2,098 orthologous protein-coding genes from transcriptomes of 186 species, representing nearly all superfamilies), and dated it with carefully evaluated synapomorphy-based fossils. The oldest members of the Lepidoptera crown group appeared in the Late Carboniferous (∼300 Ma) and fed on nonvascular land plants. Lepidoptera evolved the tube-like proboscis in the Middle Triassic (∼241 Ma), which allowed them to acquire nectar from flowering plants. This morphological innovation, along with other traits, likely promoted the extraordinary diversification of superfamily-level lepidopteran crown groups. The ancestor of butterflies was likely nocturnal, and our results indicate that butterflies became day-flying in the Late Cretaceous (∼98 Ma). Moth hearing organs arose multiple times before the evolutionary arms race between moths and bats, perhaps initially detecting a wide range of sound frequencies before being co-opted to specifically detect bat sonar. Our study provides an essential framework for future comparative studies on butterfly and moth evolution.

Practical rare event sampling for extreme mesoscale weather.

Extreme mesoscale weather, including tropical cyclones, squall lines, and floods, can be enormously damaging and yet challenging to simulate; hence, there is a pressing need for more efficient simulation strategies. Here, we present a new rare event sampling algorithm called quantile diffusion Monte Carlo (quantile DMC). Quantile DMC is a simple-to-use algorithm that can sample extreme tail behavior for a wide class of processes. We demonstrate the advantages of quantile DMC compared to other sampling methods and discuss practical aspects of implementing quantile DMC. To test the feasibility of quantile DMC for extreme mesoscale weather, we sample extremely intense realizations of two historical tropical cyclones, 2010 Hurricane Earl and 2015 Hurricane Joaquin. Our results demonstrate quantile DMC's potential to provide low-variance extreme weather statistics while highlighting the work that is necessary for quantile DMC to attain greater efficiency in future applications.

Developing a vocabulary and ontology for modeling insect natural history data: example data, use cases, and competency questions.

Insects are possibly the most taxonomically and ecologically diverse class of multicellular organisms on Earth. Consequently, they provide nearly unlimited opportunities to develop and test ecological and evolutionary hypotheses. Currently, however, large-scale studies of insect ecology, behavior, and trait evolution are impeded by the difficulty in obtaining and analyzing data derived from natural history observations of insects. These data are typically highly heterogeneous and widely scattered among many sources, which makes developing robust information systems to aggregate and disseminate them a significant challenge. As a step towards this goal, we report initial results of a new effort to develop a standardized vocabulary and ontology for insect natural history data. In particular, we describe a new database of representative insect natural history data derived from multiple sources (but focused on data from specimens in biological collections), an analysis of the abstract conceptual areas required for a comprehensive ontology of insect natural history data, and a database of use cases and competency questions to guide the development of data systems for insect natural history data. We also discuss data modeling and technology-related challenges that must be overcome to implement robust integration of insect natural history data.

Phylogenetics of moth-like butterflies (Papilionoidea: Hedylidae) based on a new 13-locus target capture probe set.

The Neotropical moth-like butterflies (Hedylidae) are perhaps the most unusual butterfly family. In addition to being species-poor, this family is predominantly nocturnal and has anti-bat ultrasound hearing organs. Evolutionary relationships among the 36 described species are largely unexplored. A new, target capture, anchored hybrid enrichment probe set ('BUTTERFLY2.0') was developed to infer relationships of hedylids and some of their butterfly relatives. The probe set includes 13 genes that have historically been used in butterfly phylogenetics. Our dataset comprised of up to 10,898 aligned base pairs from 22 hedylid species and 19 outgroups. Eleven of the thirteen loci were successfully captured from all samples, and the remaining loci were captured from ≥94% of samples. The inferred phylogeny was consistent with recent molecular studies by placing Hedylidae sister to Hesperiidae, and the tree had robust support for 80% of nodes. Our results are also consistent with morphological studies, with Macrosoma tipulata as the sister species to all remaining hedylids, followed by M. semiermis sister to the remaining species in the genus. We tested the hypothesis that nocturnality evolved once from diurnality in Hedylidae, and demonstrate that the ancestral condition was likely diurnal, with a shift to nocturnality early in the diversification of this family. The BUTTERFLY2.0 probe set includes standard butterfly phylogenetics markers, captures sequences from decades-old museum specimens, and is a cost-effective technique to infer phylogenetic relationships of the butterfly tree of life.

Preserving and vouchering butterflies and moths for large-scale museum-based molecular research.

Butterflies and moths (Lepidoptera) comprise significant portions of the world's natural history collections, but a standardized tissue preservation protocol for molecular research is largely lacking. Lepidoptera have traditionally been spread on mounting boards to display wing patterns and colors, which are often important for species identification. Many molecular phylogenetic studies have used legs from pinned specimens as the primary source for DNA in order to preserve a morphological voucher, but the amount of available tissue is often limited. Preserving an entire specimen in a cryogenic freezer is ideal for DNA preservation, but without an easily accessible voucher it can make specimen identification, verification, and morphological work difficult. Here we present a procedure that creates accessible and easily visualized "wing vouchers" of individual Lepidoptera specimens, and preserves the remainder of the insect in a cryogenic freezer for molecular research. Wings are preserved in protective holders so that both dorsal and ventral patterns and colors can be easily viewed without further damage. Our wing vouchering system has been implemented at the University of Maryland (AToL Lep Collection) and the University of Florida (Florida Museum of Natural History, McGuire Center of Lepidoptera and Biodiversity), which are among two of the largest Lepidoptera molecular collections in the world.

Blood, sweat, and tears: a review of the hematophagous, sudophagous, and lachryphagous Lepidoptera.

Although adult Lepidoptera are not often considered medically relevant, some butterflies and moths are notorious for their consumption of mammalian body fluids. These Lepidoptera can be blood-feeding (hematophagous), tear-feeding (lachryphagous), or sweat-feeding (we use the term "sudophagous"). Blood-feeding Lepidoptera have been observed piercing the skin of their hosts during feeding, while tear-feeding Lepidoptera have been observed frequenting the eyes of hosts in order to directly obtain lachrymal fluid. These behaviors have negative human health implications and some potential for disease transmission. In this study, articles concerning feeding behavior of blood, sweat, and tear-feeding Lepidoptera were reviewed, with emphasis on correlations between morphological characters and feeding behaviors. Harmful effects and vector potential of these Lepidoptera are presented and discussed.

Heel neuroma: a case study.

UNLABELLED: This article reviews the diagnosis, pathology, and treatment of plantar heel neuroma, an entity that has previously been described and recognized, yet one that we feel warrants further review because of the prevalence of plantar heel pain that does not respond to the usual array of treatments. We feel that neuroma of the medial calcaneal nerve often goes undiagnosed, and may progress to a severely painful condition that requires surgical intervention. In this article, we describe another case of heel neuroma in an otherwise healthy patient with a history of chronic plantar heel pain that was unresponsive to a wide array of nonsurgical treatments. LEVEL OF CLINICAL EVIDENCE: 4.