The complete mitochondrial genome (mitogenome) of Yponomeuta montanatus is sequenced and compared with other published yponomeutoid mitogenomes. The mitogenome is circular, 15,349 bp long, and includes the typical metazoan mitochondrial genes (13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes) and an A + T-rich region. All 13 protein-coding genes use a typical start codon ATN, the one exception being cox1, which uses CGA across yponomeutoid mitogenomes. Comparative analyses further show that the secondary structures of tRNAs are conserved, including loss of the Dihydorouidine (DHU) arm in trnS1 (AGN), but remarkable nucleotide variation has occurred mainly in the DHU arms and pseudouridine (TψC) loops. A + T-rich regions exhibit substantial length variation among yponomeutoid mitogenomes, and conserved sequence blocks are recognized but some of them are not present in all species. Multiple phylogenetic analyses confirm the position of Y. montanatus in Yponomeutoidea. However, the superfamily-level relationships in the Macroheterocera clade in Lepidoptera recovered herein show considerable difference with that recovered in previous mitogenomic studies, raising the necessity of extensive phylogenetic investigation when more mitogenomes become available for this clade.

We sequenced mitogenomes of five skippers (family Hesperiidae, Lepidoptera) to obtain further insight into the characteristics of butterfly mitogenomes and performed phylogenetic reconstruction using all available gene sequences (PCGs, rRNAs, and tRNAs) from 85 species (20 families in eight superfamilies). The general genomic features found in the butterflies also were found in the five skippers: a high A+T composition (79.3%-80.9%), dominant usage of TAA stop codon, similar skewness pattern in both strands, consistently length intergenic spacer sequence between tRNA(Gln) and ND2 (64-87 bp), conserved ATACTAA motif between tRNA(Ser (UCN)) and ND1, and characteristic features of the A+T-rich region (the ATAGA motif, varying length of poly-T stretch, and poly-A stretch). The start codon for COI was CGA in four skippers as typical, but Lobocla bifasciatus evidently possessed canonical ATG as start codon. All species had the ancestral arrangement tRNA(Asn)/tRNA(Ser (AGN)), instead of the rearrangement tRNA(Ser (AGN))/tRNA(Asn), found in another skipper species (Erynnis). Phylogenetic analyses using all available genes (PCGs, rRNAS, and tRNAs) yielded the consensus superfamilial relationships ((((((Bombycoidea+Noctuoidea+Geometroidea)+Pyraloidea)+Papilionoidea)+Tortricoidea)+Yponomeutoidea)+Hepialoidea), confirming the validity of Macroheterocera (Bombycoidea, Noctuoidea, and Geometroidea in this study) and its sister relationship to Pyraloidea. Within Rhopalocera (butterflies and skippers) the familial relationships (Papilionidae+(Hesperiidae+(Pieridae+((Lycaenidae+Riodinidae)+Nymphalidae)))) were strongly supported in all analyses (0.98-1 by BI and 96-100 by ML methods), rendering invalid the superfamily status for Hesperioidea. On the other hand, current mitogenome-based phylogeny did not find consistent superfamilial relationships among Noctuoidea, Geometroidea, and Bombycoidea and the familial relationships within Bombycoidea between analyses, requiring further taxon sampling in future studies.

We developed a universal method of Lepidoptera molecular sexing. The method is based on comparing the number of copies of the same gene in different sexes. Males of the majority of lepidopteran species have two Z chromosomes, whereas females have only one Z chromosome. Correspondingly, the number of copies of each gene located on this chromosome differs by two times between males and females. For quantitative estimation, we used qPCR. Via multiple alignment of the kettin (a Z chromosome gene) nucleotide sequences, we detected the most conserved fragment and designed primers with broad interspecies specificity for Lepidoptera. Using these primers, we successfully determined the sex of three lepidopteran species belonging to different superfamilies. The developed method is a simple, cost-effective and high-throughput technique for routine sexing. The sex of lepidopteran individuals can be examined at any developmental stage.

To better understand the diversity and phylogeny of Lepidoptera, the complete mitochondrial genome of Choristoneura longicellana (=Hoshinoa longicellana) was determined. It is a typical circular duplex molecule with 15,759bp in length, containing the standard metazoan set of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and an A+T-rich region. All of the inferred tRNA secondary structures show the common cloverleaf pattern, with the exception of trnS1(AGN), which lacks the DHU arm. The rrnL of C. Longicellana is the longest in sequenced lepidopterans. C. Longicellana has the same gene order as all lepidopteran species currently available in GenBank. There are 5 overlapping regions ranging from 1bp to 8bp and 14 intergenic spacers ranging from 1bp to 48bp. In addition, there are four similar tandem macro-satellite regions with the lengths of 101bp, 98bp, 92bp, and 92bp respectively in the A+T-rich regions of C. longicellana. We sampled 89 species representing 13 superfamilies, and reconstructed their relationship among Lepidoptera by Bayesian Inference and Maximum Likelihood analysis. The topology of the two phylogenetic analysis trees is identical roughly, except for Cossoidea in different locations, the positions of Cossoidea, Copromorphoidea, Gelechioidea, Zygaenoidea were not determined based the limited sampling. (Geometroidea+(Noctuoidea+Bombycoidea)) form the Macrolepidoptera "core". Pyraloidea group with the "core" Macrolepidoptera. Papilionoidea are not Macrolepidoptera. The Hesperiidae (represent Hesperioidea) is nested in the Papilionoidea, and closely related to Pieridae and Papilionidae. The well-known relationship of (Nymphalidae+(Riodinidae+Lycaenidae)) is recovered in this paper.

Vision is underpinned by phototransduction, a signaling cascade that converts light energy into an electrical signal. Among insects, phototransduction is best understood in Drosophila melanogaster. Comparison of D. melanogaster against three insect species found several phototransduction gene gains and losses, however, lepidopterans were not examined. Diurnal butterflies and nocturnal moths occupy different light environments and have distinct eye morphologies, which might impact the expression of their phototransduction genes. Here we investigated: 1) how phototransduction genes vary in gene gain or loss between D. melanogaster and Lepidoptera, and 2) variations in phototransduction genes between moths and butterflies. To test our prediction of phototransduction differences due to distinct visual ecologies, we used insect reference genomes, phylogenetics, and moth and butterfly head RNA-Seq and transcriptome data. As expected, most phototransduction genes were conserved between D. melanogaster and Lepidoptera, with some exceptions. Notably, we found two lepidopteran opsins lacking a D. melanogaster ortholog. Using antibodies we found that one of these opsins, a candidate retinochrome, which we refer to as unclassified opsin (UnRh), is expressed in the crystalline cone cells and the pigment cells of the butterfly, Heliconius melpomene. Our results also show that butterflies express similar amounts of trp and trpl channel mRNAs, whereas moths express ∼50× less trp, a potential adaptation to darkness. Our findings suggest that while many single-copy D. melanogaster phototransduction genes are conserved in lepidopterans, phototransduction gene expression differences exist between moths and butterflies that may be linked to their visual light environment.

Lepidoptera are used as a model for the study of insect olfactory proteins. Among them, odorant degrading enzymes (ODEs), that degrade odorant molecules to maintain the sensitivity of antennae, have received less attention. In particular, antennal esterases (AEs; responsible for ester degradation) are crucial for intraspecific communication in Lepidoptera. Currently, transcriptomic and genomic studies have provided AEs in several species. However, efforts in gene annotation, classification, and functional assignment are still lacking. Therefore, we propose to combine evidence at evolutionary, structural, and functional level to update ODEs as well as key information into an easier classification, particularly of AEs. Finally, the kinetic parameters for putative inhibition of ODEs are discussed in terms of its role in future integrated pest management (IPM) strategies.

Conservation of at-risk species requires multi-faceted and carefully-considered management approaches to be successful. For arthropods, the presence of endosymbiotic bacteria, such as Wolbachia (Rickettsiales: Rickettsiaceae), may complicate management plans and exacerbate the challenges faced by conservation managers. Wolbachia poses a substantial and underappreciated threat to the conservation of arthropods because infection may induce a number of phenotypic effects, most of which are considered deleterious to the host population. In this study, the prevalence of Wolbachia infection in lepidopteran species of conservation concern was examined. Using standard molecular techniques, 22 species of Lepidoptera were screened, of which 19 were infected with Wolbachia. This rate is comparable to that observed in insects as a whole. However, this is likely an underestimate because geographic sampling was not extensive and may not have included infected segments of the species' ranges. Wolbachia infections may be particularly problematic for conservation management plans that incorporate captive propagation or translocation. Inadvertent introduction of Wolbachia into uninfected populations or introduction of a new strain may put these populations at greater risk for extinction. Further sampling to investigate the geographic extent of Wolbachia infections within species of conservation concern and experiments designed to determine the nature of the infection phenotype(s) are necessary to manage the potential threat of infection.

Finding plant cultivars that are resistant or tolerant against lepidopteran pests, takes time, effort and is costly. We present here a high throughput leaf-disk consumption assay system, to screen plants for resistance or chemicals for their deterrence. A webcam capturing images at regular intervals can follow the feeding activities of 150 larvae placed into individual cages. We developed a computer program running under an open source image analysis program to analyze and measure the surface of each leaf disk over time. We further developed new statistical procedures to analyze the time course of the feeding activities of the larvae and to compare them between treatments. As a test case, we compared how European corn borer larvae respond to a commercial antifeedant containing azadirachtin, and to quinine, which is a bitter alkaloid for many organisms. As expected, increasing doses of azadirachtin reduced and delayed feeding. However, quinine was poorly effective at the range of concentrations tested (10-5 M to 10-2 M). The model cage, the camera holder, the plugins, and the R scripts are freely available, and can be modified according to the users' needs.

The presence of lignin within biomass impedes the production of liquid fuels. Plants with altered lignin content and composition are more amenable to lignocellulosic conversion to ethanol and other biofuels but may be more susceptible to insect damage where lignin is an important resistance factor. However, reduced lignin lines of switchgrasses still retained insect resistance in prior studies. Therefore, we hypothesized that sorghum lines with lowered lignin content will also retain insect resistance. Sorghum excised leaves and stalk pith Sorghum bicolor (L.) Moench (Poales: Poaceae) from near isogenic brown midrib (bmr) 6 and 12 mutants lines, which have lowered lignin content and increased lignocellulosic ethanol conversion efficiency, were examined for insect resistance relative to wild-type (normal BTx623). Greenhouse and growth chamber grown plant tissues were fed to first-instar larvae of corn earworms, Helicoverpa zea (Boddie) and fall armyworms Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), two sorghum major pests. Younger bmr leaves had significantly greater feeding damage in some assays than wild-type leaves, but older bmr6 leaves generally had significantly less damage than wild-type leaves. Caterpillars feeding on the bmr6 leaves often weighed significantly less than those feeding on wild-type leaves, especially in the S. frugiperda assays. Larvae fed the pith from bmr stalks had significantly higher mortality compared with those larvae fed on wild-type pith, which suggested that bmr pith was more toxic. Thus, reducing lignin content or changing subunit composition of bioenergy grasses does not necessarily increase their susceptibility to insects and may result in increased resistance, which would contribute to sustainable production.

The taxon Lepidoptera is one of the most widespread and recognisable insect orders with 160,000 species worldwide and with more than 20,000 species in Africa. Lepidoptera have a complete metamorphosis and the adults (butterflies and moths) are quite different from the larvae (caterpillars). The purpose of the study was to make an overview of how butterflies/moths and caterpillars are utilised, perceived and experienced in daily life across sub-Saharan Africa.

The complete mitochondrial genome of Casmara patrona (Lepidoptera: Oecophoridae) was sequenced for a future phylogenetic study of Lepidoptera. The circle genome of the moth is 15,393 bp in length with a pronounced base bias of A + T (79.3%), containing 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and a putative control region. The coxI gene had a CGA start codon as most lepidopteran species, other PCGs use the typical ATN codons. All PCGs end with the complete stop codon TAA. Phylogenetic analyses showed that the monophyly of Oecophoridae was highly supported based on the concatenated sequence of the 13 PCGs. In addition, Oecophoridae and Xyloryctidae had the closest relationship.

Apolipoprotein D (ApoD) plays important roles in response to injury, cell differentiation, lifespan extension, and increasing stress resistance. However, the evolutionary mechanism of ApoD in insects remains largely unelucidated. We conducted a comprehensive study of the molecular evolution and functional divergence of ApoD in insects. A type I functional divergence analysis revealed significant differences among insect ApoD homologs, suggesting that they underwent functional divergence. We demonstrated that lepidopteran insects have three genes that are close homologs to ApoD and show divergences in sequence, expression pattern, and protein-protein interaction. Furthermore, positive selection was detected in lepidopteran ApoD2, and positively selected sites were located around the pocket and loop domains, which might result in conformational changes and affect binding properties. Moreover, we showed that the three ApoDs in Bombyx mori were significantly regulated by environmental stress. Thus, this work illustrates the dialectical relationship between genetic diversity and functional conservation of ApoD and highlights its unique functions in the stress response of Lepidoptera.

Northern and mountainous ice sheets have expanded and contracted many times due to ice ages. Consequently, temperate species have been confined to refugia during the glacial periods wherefrom they have recolonized warming northern habitats between ice ages. In this study, we compare the gene CYP405A2 between different populations of the common burnet moth Zygaena filipendulae from across the Western Palearctic region to illuminate the colonization history of this species. These data show two major clusters of Z. filipendulae populations possibly reflecting two different refugial populations during the last ice age. The two types of Z. filipendulae only co-occur in Denmark, Sweden, and Scotland indicating that Northern Europe comprise the hybridization zone where individuals from two different refugia met after the last ice age. Bayesian phylogeographic and ecological clustering analyses show that one cluster probably derives from an Alpe Maritime refugium in Southern France with ancestral expansive tendencies to the British Isles in the west, touching Northern Europe up to Denmark and Sweden, and extending throughout Central Europe into the Balkans, the Peleponnes, and South East Europe. The second cluster encompasses East Anatolia as the source area, from where multiple independent dispersal events to Armenia, to the Alborz mountains in north-western Iran, and to the Zagros mountains in western Iran are suggested. Consequently, the classical theory of refugia for European temperate species in the Iberian, Italian, and Balkan peninsulas does not fit with the data from Z. filipendulae populations, which instead support more Northerly, mountainous refugia.

We obtained and analyzed whole genome data for more than 160 representatives of skipper butterflies (family Hesperiidae) from all known subfamilies, tribes and most distinctive genera. We found that two genera, Katreus Watson, 1893 and Ortholexis Karsch, 1895, which are sisters, are well-separated from all other major phylogenetic lineages and originate near the base of the Hesperiidae tree, prior to the origin of some subfamilies. Due to this ancient origin compared to other subfamilies, this group is described as Katreinae Grishin, subfam. n. DNA sequencing of primary type specimens reveals that Ortholexismelichroptera Karsch, 1895 is not a female of Ortholexisholocausta Mabille, 1891, but instead a female of Ortholexisdimidia Holland, 1896. This finding establishes O.dimidia as a junior subjective synonym of O.melichroptera. Furthermore, we see that Chamunda Evans, 1949 does not originate within Pyrginae Burmeister, 1878, but, unexpectedly, forms an ancient lineage of its own at the subfamily rank: Chamundinae Grishin, subfam. n. Finally, a group of two sister genera, Barca de Nicéville, 1902 and Apostictopterus Leech, [1893], originates around the time Hesperiinae Latreille, 1809 have split from their sister clade. A new subfamily Barcinae Grishin, subfam. n. sets them apart from all other Hesperiidae.

In this research, the complete mitochondrial genome (mitogenome) of Phthorimaea operculella was sequenced and annotated. The mitogenome of P. operculella is 15,269 bp in length and contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosome RNA (12s and 16srRNA) genes and 1 control region. In addition, we used Endoclita signifier as the outgroup to analyze phylogenetic relationship, and the phylogenetic tree showed the sister relationship between P. operculella and Tuta absoluta.

Recently, we reported a novel mode of action in monarch butterfly (Danaus plexippus) larvae exposed to neonicotinoid insecticides: arrest in pupal ecdysis following successful larval ecdysis. In this paper, we explore arrested pupal ecdysis in greater detail and propose adverse outcome pathways to explain how neonicotinoids cause this effect. Using imidacloprid as a model compound, we determined that final-instar monarchs, corn earworms (Helicoverpa zea), and wax moths (Galleria mellonella) showed high susceptibility to arrested pupal ecdysis while painted ladies (Vanessa cardui) and red admirals (Vanessa atalanta) showed low susceptibility. Fall armyworms (Spodoptera frugiperda) and European corn borers (Ostrinia nubilalis) were recalcitrant. All larvae with arrested ecdysis developed pupal cuticle, but with incomplete shedding of larval cuticle and unexpanded pupal appendages; corn earworm larvae successfully developed into adults with unexpanded appendages. Delayed initiation of pupal ecdysis was also observed with treated larvae. Imidacloprid exposure was required at least 26 h prior to pupal ecdysis to disrupt the molt. These observations suggest neonicotinoids may disrupt the function of crustacean cardioactive peptide (CCAP) neurons, either by directly acting on their nicotinic acetylcholine receptors or by acting on receptors of inhibitory neurons that regulate CCAP activity.

The complete mitochondrial genome of Ostrinia kasmirica (Moore, 1888) was sequenced in this study. The circular mitogenome is 15,214 bp in length, containing 37 typical encoded genes and a non-coding control region. The gene organization and nucleotide composition are similar to those of most other sequenced Ostrinia species. All protein-coding genes (PCGs) initiate with ATN and terminate with TAN, except cox1 starts with CGA and cox1, cox2, nad5 terminate with an incomplete codon T. The control region of 308 bp contains three conserved features including the motif 'TTAGA' preceded a poly-T stretch, a microsatellite-like (TA)n element, and a poly-A stretch upstream of trnM. Phylogenetic analysis based on mitogenome sequences revealed that the O. kasmirica (the second species group) was more closely related to the third species group of the genus and the first species group was not at the basal position of this genus as that Mutuura and Munroe indicated.

The complete mitochondrial genome (mitogenome) of Biston thoracicaria (Lepidoptera: Geometridae) is 15,538 bp in length, containing 13 PCGs, 22 tRNAs, two rRNAs, and an A + T-rich region. All PCGs initiate with typical start codon of ATN and share the complete stop codon of TAA, whereas cox1 starts with CGA. The ML analysis was performed using a dataset matrix containing 13 PCGs concatenated from the mitogenomes of Geometridae species. Our study presented the phylogenetic relationship of (Larentiinae + ((Sterrhinae + (Ennominae + Geometrinae))). Within the genera Biston, B. thoracicaria grouped with other species as the sister group.

We obtained and phylogenetically analyzed whole genome shotgun sequences of nearly all species from the tribe Emesidini Seraphim, Freitas Kaminski, 2018 (Riodinidae) and representatives from other Riodinidae tribes. We see that the recently proposed genera Neoapodemia Trujano, 2018 and Plesioarida Trujano García, 2018 are closely allied with Apodemia C. R. Felder, [1865] and are better viewed as its subgenera, new status. Overall, Emesis Fabricius, 1807 and Apodemia (even after inclusion of the two subgenera) are so phylogenetically close that several species have been previously swapped between these two genera. New combinations are: Apodemia (Neoapodemia) zela (Butler, 1870), Apodemia (Neoapodemia) ares (Edwards, 1882), and Apodemia (Neoapodemia) arnacis (Stichel, 1928) (not Emesis); and Emesis phyciodoides (Barnes Benjamin, 1924) (not Apodemia), assigned to each genus by their monophyly in genomic trees with the type species (TS) of the genus. Surprisingly, we find that Emesis emesia Hewitson, 1867 is not grouped with Emesis, but in addition to Apodemia forms a third lineage of similar rank, here named Curvie Grishin, gen. n. (TS: Symmachia emesia Hewitson, 1867). Furthermore, we partition Emesis into 6 subgenera (4 new): Emesis (TS: Hesperia ovidius Fabricius, 1793, a subjective junior synonym of Papilio cereus Linnaeus, 1767), Aphacitis Hübner, [1819] (TS: Papilio dyndima Cramer, [1780], a subjective junior synonym of Papilio lucinda Cramer, [1775]), Poeasia Grishin, subgen. n. (TS: Emesis poeas Godman, [1901]), Mandania Grishin, subgen. n. (TS: Papilio mandana Cramer, [1780]), Brimia Grishin, subgen. n. (TS: Emesis brimo Godman Salvin, 1889), and Tenedia Grishin, subgen. n. (TS: Emesis tenedia C. R. Felder, 1861). Next, genomic comparison of primary type specimens suggests new status for Emesis vimena Schaus, 1928 as a subspecies of Emesis brimo Godman Salvin, 1889, Emesis adelpha Le Cerf, 1958 with E. a. vicaria Le Cerf, 1958 are subspecies of Emesis heteroclita Stichel, 1929, and Emesis tristis Stichel, 1929 is not a synonym of E. brimo vimena but of Emesis lupina Godman Salvin, 1886. A new status of a species is given to the following taxa: Emesis furor A. Butler H. Druce, 1872 (not a subspecies of E. mandana (Cramer, 1780)), Emesis melancholica Stichel, 1916 (not a subspecies of E. lupina Godman Salvin, 1886), Emesis progne (Godman, 1903) (not a subspecies of E. brimo Godman Salvin, 1889), and Emesis opaca Stichel, 1910 (not a synonym of E. lucinda (Cramer, 1775)). Emesis castigata diringeri Gallard 2008 is a subjective junior synonym of E. opaca, new status. Finally, Xanthosa Grishin, gen. n. (TS: Charmona xanthosa Stichel, 1910) is proposed for a sister lineage of Sertania Callaghan Kaminski, 2017 and Befrostia Grishin, gen. n. (TS: Emesis elegia Stichel, 1929) is proposed for a clade without apparent phylogenetic affinities that we place in Befrostiini Grishin, trib. n. In conclusion, genomic data reveal a number of errors in the current classification of Emesidini and allow us to confidently reclassify the tribe partitioning it in three genera: Apodemia, Curvie gen. n. and Emesis.

Exaggerated morphologies have evolved in insects as adaptations to nectar feeding by natural selection. For example, the suctorial mouthparts of butterflies enable these insects to gain access to floral nectar concealed inside deep floral tubes. Proboscis length in Lepidoptera is known to scale with body size, but whether extreme absolute proboscis lengths of nectar feeding butterflies result from a proportional or disproportional increase with body size that differs between phylogenetic lineages remains unknown. We surveyed the range of variation that occurs in scaling relationships between proboscis length and body size against a phylogenetic background among Costa Rican Hesperiidae. We obtained a new record holder for the longest proboscis in butterflies and showed that extremely long proboscides evolved at least three times independently within Neotropical Hesperiidae. We conclude that the evolution of extremely long proboscides results from allometric scaling with body size, as demonstrated in hawk moths. We hypothesize that constraints on the evolution of increasingly long butterfly proboscides may come from (1) the underlying scaling relationships, i.e., relative proboscis length, combined with the butterfly's flight style and flower-visiting behaviour and/or (2) developmental constraints during the pupal phase. Lastly, we discuss why butterflies did not evolve similar scaling relationships as hawk moths.