Genome-Wide Gene Birth-Death Dynamics Are Associated with Diet Breadth Variation in Lepidoptera.

Comparative analyses of gene birth-death dynamics have the potential to reveal gene families that played an important role in the evolution of morphological, behavioral, or physiological variation. Here, we used whole genomes of 30 species of butterflies and moths to identify gene birth-death dynamics among the Lepidoptera that are associated with specialist or generalist feeding strategies. Our work advances this field using a uniform set of annotated proteins for all genomes, investigating associations while correcting for phylogeny, and assessing all gene families rather than a priori subsets. We discovered that the sizes of several important gene families (e.g. those associated with pesticide resistance, xenobiotic detoxification, and/or protein digestion) are significantly correlated with diet breadth. We also found 22 gene families showing significant shifts in gene birth-death dynamics at the butterfly (Papilionoidea) crown node, the most notable of which was a family of pheromone receptors that underwent a contraction potentially linked with a shift to visual-based mate recognition. Our findings highlight the importance of uniform annotations, phylogenetic corrections, and unbiased gene family analyses in generating a list of candidate genes that warrant further exploration.

De-novo long-read genome assembly and annotation of the luna moth (Actias luna) fully resolves repeat-rich silk genes.

We present the first long-read de-novo -assembly and annotation of the luna moth (Actias luna) and provide the full characterization of heavy chain fibroin (h-fibroin)--, a long and highly repetitive gene (>20 Kbp) essential in silk fiber production. There are more than 160,000 described species of moths and butterflies (Lepidoptera), but only within the last five years have we begun to recover high-quality annotated whole genomes across the order which capture h-fibroin. Using PacBio HiFi reads, we produce the first high-quality long-read reference genome for this species. The assembled genome has a length of 532 Mbp, a contig N50 of 16.8 Mbp, an L50 of 14 contigs, and 99.4% completeness (BUSCO). Our annotation using Bombyx mori protein and A.luna RNAseq evidence captured a total of 20,866 genes at 98.9% completeness with 10,267 functionally annotated proteins and a full-length h-fibroin annotation of 2,679 amino acid residues.

Transcriptomic and proteomic elucidation of Z chromosome dosage compensation in Helicoverpa armigera.

Transcriptomic data have been used to study sex chromosome dosage compensation (SCDC) in approximately 10 Lepidoptera ZW species, yielding a consensus compensation pattern of Z ≈ ZZ < AA . $$ \approx \mathrm{ZZ}<\mathrm{AA}. $$ It remains unclear whether this compensation pattern holds when examining more Lepidoptera ZW species and/or using proteomic data to analyse SCDC. Here we combined transcriptomic and proteomic data as well as transcriptional level of six individual Z genes to reveal the SCDC pattern in Helicoverpa armigera, a polyphagous lepidopteran pest of economic importance. Transcriptomic analysis showed that the Z chromosome expression of H. armigera was balanced between male and female but substantially reduced relative to autosome expression, exhibiting an SCDC pattern of Z ≈ ZZ < AA $$ \approx \mathrm{ZZ}<\mathrm{AA} $$ . When using H. amigera midgut proteomic data, the SCDC pattern of this species changed from Z ≈ ZZ < AA $$ \approx \mathrm{ZZ}<\mathrm{AA} $$ at transcriptomic level to Z = ZZ = AA at the proteomic level. RT-qPCR analysis of transcript abundance of six Z genes found that compensation for each Z gene could vary from no compensation to overcompensation, depending on the individual genes and tissues tested. These results demonstrate for the first time the existence of a translational compensation mechanism, which is operating in addition to a translational mechanism, such as has been reported in other lepidopteran species. And the transcriptional compensation mechanism functions to accomplish Z chromosome dosage balance between the sexes (M = F on the Z chromosome), whereas the translation compensation mechanism operates to achieve dosage compensation between Z chromosome and autosome (Z = AA).

The genome sequence of the Marbled White Spot, Protodeltote pygarga (Hufnagel, 1766).

We present a genome assembly from an individual male Protodeltote pygarga (the Marbled White Spot; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 421.1 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.48 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,784 protein coding genes.

A comprehensive gene expression analysis of the unique three-layered cocoon of the cecropia moth, Hyalophora cecropia.

The larvae of the moth Hyalophora cecropia spin silk cocoons with morphologically distinct layers. We investigated the expression of the individual silk protein components of these cocoons in relation to the morphology of the silk gland and its affiliation to the different layers of the cocoon. The study used transcriptomic and proteomic analyses to identify 91 proteins associated with the silk cocoons, 63 of which have a signal peptide indicating their secretory nature. We checked the specificity of their expression in different parts of the SG and the presence of the corresponding protein products in each cocoon layer. Differences were observed among less abundant proteins with unclear functions. The representation of proteins in the inner envelope and intermediate space was similar, except for a higher proportion of probable contaminating proteins, mostly originating from the gut. On the other hand, the outer envelope contains a number of putative enzymes with unclear function. However, the protein most specific to the outer layer has sequence homology to putative serine/threonine kinase-like proteins and some adhesive proteins, and its closest homolog in Bombyx mori was found in the scaffold silk. This research provides valuable insights into the silk production of the cecropia moth, highlighting both similarities and differences to other moth species.

The genome sequence of the Winter Shade, Tortricodes alternella [Denis & Schiffermüller], 1775.

We present a genome assembly from an individual male Tortricodes alternella (the Winter Shade; Arthropoda; None; Lepidoptera; Tortricidae). The genome sequence is 441.2 megabases in span. Most of the assembly is scaffolded into 22 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.73 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,280 protein coding genes.

The InBIO Barcoding Initiative Database: DNA barcodes of Portuguese moths.

Background: The InBIO Barcoding Initiative (IBI) Dataset - DS-IBILP08 contains records of 2350 specimens of moths (Lepidoptera species that do not belong to the superfamily Papilionoidea). All specimens have been morphologically identified to species or subspecies level and represent 1158 species in total. The species of this dataset correspond to about 42% of mainland Portuguese Lepidoptera species. All specimens were collected in mainland Portugal between 2001 and 2022. All DNA extracts and over 96% of the specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources. New information: The authors enabled "The InBIO Barcoding Initiative Database: DNA barcodes of Portuguese moths" in order to release the majority of data of DNA barcodes of Portuguese moths within the InBIO Barcoding Initiative. This dataset increases the knowledge on the DNA barcodes of 1158 species from Portugal belonging to 51 families. There is an increase in DNA barcodes of 205% in Portuguese specimens publicly available. The dataset includes 61 new Barcode Index Numbers. All specimens have their DNA barcodes publicly accessible through BOLD online database and the distribution data can be accessed through the Global Biodiversity Information Facility (GBIF).

Armoured Lepidopteran Caterpillars Preserved in Non-Fossil Resins and What They Tell Us about the Fossil Preservation of Caterpillars.

Resin is a plastic-like product of trees. Older occurrences of such resin are referred to as amber and are considered fossil resin. Younger resins are termed copals. Even younger ones have been dubbed defaunation resins. Non-fossil resins remain in a terminological limbo, often referred to as "sub-fossils". We report two lepidopteran caterpillars preserved in non-fossil resin: one from Madagascar, one from Brazil. Prominent hairs (=setae) and spines (=spine-like setae) of the specimens make it likely that they represent larvae of Erebidae (e.g., tussock moths and others). So far, most known caterpillars preserved in resins are either "naked" or bear protective cases; only few are armoured with spines or hairs. In particular, long-haired caterpillars such as the ones reported here are so far almost absent. Only one specimen with comparable setae has been reported from 15-million-year-old Dominican amber, but no significant details of this specimen are accessible. We briefly also review the record of caterpillars known from the Holocene, recognising that it is very sparse. The new specimens demonstrate that very hairy caterpillars can readily be preserved in resins in fine detail. Furthermore, the specimens increase the known size range of caterpillars preserved in resins, with one measuring more than 12 mm.

Behavioural changes in aposematic Heliconius melpomene butterflies in response to their predatory bird calls.

Prey-predator interactions have resulted in the evolution of many anti-predatory traits. One of them is the ability for prey to listen to predators and avoid them. Although prey anti-predatory behavioural responses to predator auditory cues are well described in a wide range of taxa, studies on whether butterflies change their behaviours in response to their predatory calls are lacking. Heliconius butterflies are unpalatable and form Müllerian mimicry rings as morphological defence strategies against their avian predators. Like many other butterflies in the Nymphalidae family, some Heliconius butterflies possess auditory organs, which are hypothesized to assist with predator detection. Here we test whether Heliconius melpomene change their behaviour in response to their predatory bird calls by observing the behaviour of male and female H. m. plessini exposed to calls of Heliconius avian predators: rufous-tailed jacamar, migratory Eastern kingbird, and resident tropical kingbird. We also exposed them to the calls of the toco toucan, a frugivorous bird as a control bird call, and an amplified greenhouse background noise as a noise control. We found that individuals changed their behaviour in response to jacamar calls only. Males increased their walking and fluttering behaviour, while females did not change their behaviour during the playback of the jacamar call. Intersexual behaviours like courtship, copulation, and abdomen lifting did not change in response to bird calls. Our findings suggest that despite having primary predatory defences like toxicity and being in a mimicry ring, H. m. plessini butterflies changed their behaviour in response to predator calls. Furthermore, this response was predator specific, as H. m. plesseni did not respond to either the Eastern kingbird or the tropical kingbird calls. This suggests that Heliconius butterflies may be able to differentiate predatory calls, and potentially the birds associated with those calls.

Knock-Out of ACY-1 Like Gene in Spodoptera litura Supports the Notion that FACs Improve Nitrogen Metabolism.

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine were originally identified in the regurgitant of Spodoptera exigua larvae. These fatty acid amino acid conjugates (FACs) are known to be elicitors that induce plants to release volatile compounds which in turn attract natural enemies of the larvae such as parasitic wasps. FAC concentrations are regulated by enzymatic biosynthesis and hydrolysis in the intestine of Lepidoptera larvae. It has been proposed that FAC metabolism activates glutamine synthetase and plays an important role in nitrogen metabolism in larvae. In this study, we identified candidate genes encoding a FACs hydrolase in Spodoptera litura using genomic information of various related lepidopteran species in which FACs hydrolases have been reported. We analyzed the importance of FAC hydrolysis on caterpillar performance with CRISPR/Cas9 knock outs. Larvae of strains with an inactive FACs hydrolase excreted FACs in their feces. They absorbed 30% less nitrogen from the diet compared to WT caterpillars resulting in a reduction of their body weight of up to 40% compared to wild type caterpillars. These results suggest that the hydrolysis of FACs is an important metabolism for insects and that FACs are important for larval growth.

Wetland butterfly thriving in abandoned jungle: Neptis rivularis in the Czech Republic.

With ongoing insect declines, species expanding in distribution and abundance deserve attention, as understanding their success may help design conservation strategies for less successful species. Common causes of these successes include warmer climates, novel resources, and exploiting land use change, including land abandonment. These factors affect the nymphalid butterfly Neptis rivularis, developing on Spiraea spp. shrubs and reaching the north-western limits of its trans-Palearctic distribution in Central Europe. We combined mark-recapture, behaviour analysis, and distribution modelling to study N. rivularis in wetlands of the Trebonsko Protected Landscape (IUCN category V). The long-living adults (up to 4 weeks) spent a considerable amount of time searching for partners, ovipositing and nectaring at Spiraea shrubs, alternating this with stays in tree crowns, where they located cool shelters, spent nights, and presumably fed on honeydew. They formed high-density populations (310 adults/ha), exploiting high host plant abundance. They adhered to floodplains and to conditions of relatively mild winters. The ongoing Spiraea encroachment of abandoned alluvial grasslands is, thus, a transient situation, ultimately followed by forest encroachment. Rewilding the habitats by introducing native ungulates presents an opportunity to restore the disturbance regime of the sites. The increased resource supply combined with a warming climate has opened up temperate Europe to colonization by N. rivularis.

Flexural rigidity of hawkmoth antennae depends on the bending direction.

To probe its environment, the flying insect controllably flexes, twists, and maneuvers its antennae by coupling mechanical deformations with the sensory output. We question how the materials properties of insect antennae could influence their performance. A comparative study was conducted on four hawkmoth species: Manduca sexta, Ceratomia catalpae, Manduca quinquemaculata, and Xylophanes tersa. The morphology of the antennae of three hawkmoths that hover while feeding and one putatively non-nectar-feeding hawkmoth (Ceratomia catalpa) do not fundamentally differ, and all the antennae are comb-like (i.e., pectinate), markedly in males but weakly in females. Applying different weights to the free end of extracted cantilevered antennae, we discovered anisotropy in flexural rigidity when the antenna is forced to bend dorsally versus ventrally. The flexural rigidity of male antennae was less than that of females. Compared with the hawkmoths that hover while feeding, Ceratomia catalpae has almost two orders of magnitude lower flexural rigidity. Tensile tests showed that the stiffness of male and female antennae is almost the same. Therefore, the differences in flexural rigidity are explained by the distinct shapes of the antennal pectination. Like bristles in a comb, the pectinations provide extra rigidity to the antenna. We discuss the biological implications of these discoveries in relation to the flight habits of hawkmoths. Flexural anisotropy of antennae is expected in other groups of insects, but the targeted outcome may differ. Our work offers promising new applications of shaped fibers as mechanical sensors. STATEMENT OF SIGNIFICANCE: Insect antennae are blood-filled, segmented fibers with muscles in the two basal segments. The long terminal segment is muscle-free but can be flexed. Our comparative analysis of mechanical properties of hawkmoth antennae revealed a new feature: antenna resistance to bending depends on the bending direction. Our discovery replaces the conventional textbook scenario considering hawkmoth antennae as rigid rods. We showed that the pectinate antennae of hawkmoths behave as a comb in which the bristles resist bending when they come together. This anisotropy of flexural resistance offers a new mode of environmental sensing that has never been explored. The principles we found apply to other insects with non-axisymmetric antennae. Our work offers new applications for shaped fibers that could be designed to sense the flows.

The genome sequence of the White-point, Mythimna albipuncta (Denis & Schiffermüller, 1775).

We present a genome assembly from an individual male Mythimna albipuncta (the White-point; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 698.6 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.38 kilobases in length. Gene annotation of this assembly on Ensembl identified 13,679 protein coding genes.

Survey of Leafminers on American Chestnut and other Castanea spp. (Fagales: Fagaceae) on Long Island, NY.

The effect of the 20th-century functional extinction of the American Chestnut (Fagaceae: Castanea dentata (Marshall) Borkh) on associated herbivorous insects is unknown. These insects include leafminers that spend at least part of their larval phase feeding between the epidermises of leaves. We surveyed leafminers on C. dentata, nonnative Castanea spp., and hybrids on Long Island, NY. We found 10 leafminer species feeding on Castanea spp. A first New York State record was documented for Stigmella castaneaefoliella (Chambers) (Lepidoptera: Nepticulidae). New host records are established for 6 lepidopterans, including a new host genus for Phyllonorycter basistrigella (Clemens) (Lepidoptera: Gracillariidae). We found no significant differences in the mean intensity of S. castaneaefoliella leaf mines on native and nonnative Castanea spp.; however, our sample size was small. Thus, we guardedly conclude that nonnative Castanea spp. can serve as refugia for C. dentata leafminers native to North America while acknowledging that the extent to which nonnative species are utilized requires further investigation.

W chromosome sequences of two bombycid moths provide an insight into the origin of Fem.

Fem is a W-linked gene that encodes a piRNA precursor, and its product, Fem piRNA, is a master factor of female determination in Bombyx mori. Fem has low similarity to any known sequences, and the origin of Fem remains unclear. So far, two hypotheses have been proposed for the origin of Fem: The first hypothesis is that Fem is an allele of Masc, which assumes that the W chromosome was originally a homologous chromosome of the Z chromosome. The second hypothesis is that Fem arose by the transposition of Masc to the W chromosome. To explore the origin of Fem, we determined the W chromosome sequences of B. mori and, as a comparison, a closely relative bombycid species of Trilocha varians with a Fem-independent sex determination system. To our surprise, although the sequences of W and Z chromosomes show no homology to each other, a few pairs of homologues are shared by W and Z chromosomes, indicating the W chromosome of both species originated from Z chromosome. In addition, the W chromosome of T. varians lacks Fem, while the W chromosome of B. mori has over 100 copies of Fem. The high-quality assembly of the W chromosome of B. mori arose the third hypothesis about the origin of Fem: Fem is a chimeric sequence of multiple transposons. More than half of one transcriptional unit of Fem shows a significant homology to RTE-BovB. Moreover, the Fem piRNA-producing region could correspond to the boundary of the two transposons, gypsy and satellite DNA.

Local climate change velocities and evolutionary history explain multidirectional range shifts in a North American butterfly assemblage.

Species are often expected to shift their distributions either poleward or upslope to evade warming climates and colonise new suitable climatic niches. However, from 18-years of fixed transect monitoring data on 88 species of butterfly in the midwestern United States, we show that butterflies are shifting their centroids in all directions, except towards regions that are warming the fastest (southeast). Butterflies shifted their centroids at a mean rate of 4.87 km year-1. The rate of centroid shift was significantly associated with local climate change velocity (temperature by precipitation interaction), but not with mean climate change velocity throughout the species' ranges. Species tended to shift their centroids at a faster rate towards regions that are warming at slower velocities but increasing in precipitation velocity. Surprisingly, species' thermal niche breadth (range of climates butterflies experience throughout their distribution) and wingspan (often used as metric for dispersal capability) were not correlated with the rate at which species shifted their ranges. We observed high phylogenetic signal in the direction species shifted their centroids. However, we found no phylogenetic signal in the rate species shifted their centroids, suggesting less conserved processes determine the rate of range shift than the direction species shift their ranges. This research shows important signatures of multidirectional range shifts (latitudinal and longitudinal) and uniquely shows that local climate change velocities are more important in driving range shifts than the mean climate change velocity throughout a species' entire range.

Genome-wide identification and phylogenetic analysis of the tetraspanin gene family in lepidopteran insects and expression profiling analysis in Helicoverpa armigera.

The tetraspanin gene family encodes cell-surface proteins that span the membrane 4 times and play critical roles in a wide range of biological processes across numerous organisms. Recent findings highlight the involvement of a tetraspanin of the lepidopteran pest Helicoverpa armigera in resistance to Bacillus thuringiensis Cry insecticidal proteins, which are extensively used in transgenic crops. Thus, a better understanding of lepidopteran tetraspanins is urgently needed. In the current study, genome scanning in 10 lepidopteran species identified a total of 283 sequences encoding potential tetraspanins. Based on conserved cysteine patterns in the large extracellular loop and their phylogenetic relationships, these tetraspanins were classified into 8 subfamilies (TspA to TspH). Six ancestral introns were identified within lepidopteran tetraspanin genes. Tetraspanins in TspA, TspB, TspC, and TspD subfamilies exhibit highly similar gene organization, while tetraspanins in the remaining 4 subfamilies exhibited variation in intron loss and/or gain during evolution. Analysis of chromosomal distribution revealed a lepidopteran-specific cluster of 10 to 11 tetraspanins, likely formed by tandem duplication events. Selective pressure analysis indicated negative selection across all orthologous groups, with ω values ranging between 0.004 and 0.362. However, positive selection was identified at 18 sites within TspB5, TspC5, TspE3, and TspF10. Furthermore, spatiotemporal expression analysis of H. armigera tetraspanins demonstrated variable expression levels across different developmental stages and tissues, suggesting diverse functions of tetraspanin members in this globally important insect pest. Our findings establish a solid foundation for subsequent functional investigations of tetraspanins in lepidopteran species.

Characterization of the complete mitochondrial genome of an endemic species in China, Aulocera merlina (Lepidoptera: Nymphalidae: Satyrinae) and phylogenetic analysis within Satyrinae.

The mitochondrial genome (mitogenome) has been extensively used as molecular markers in determining the insect phylogenetic relationships. In order to resolve the relationships among tribes and subtribes of Satyrinae at the mitochondrial genomic level, we obtained the complete mitogenome of Aulocera merlina (Oberthür, 1890) (Lepidoptera: Nymphalidae: Satyrinae) with a size of 15,259 bp. The mitogenome consisted of 37 typical genes, including 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and an A + T-rich region. The gene organization and arrangement were similar to those of all other known Satyrinae mitogenomes. All PCGs were initiated with the canonical codon pattern ATN, except for the cox1 gene, which used an atypical CGA codon. Nine PCGs used the complete stop codon TAA, while the remaining PCGs (cox1, cox2, nad4, and nad5) were terminated with a single T nucleotide. The canonical cloverleaf secondary structures were found in all tRNAs, except for trnS1 which lacked a dihydrouridine arm. The 448 bp A + T-rich region was located between rrnS and trnM, and it included the motif ATAGA followed by a 19-bp poly-T stretch and a microsatellite-like (TA)6 element preceded by the ATTTA motif. The phylogenetic tree, inferred using Bayesian inference and maximum likelihood methods, generated similar tree topologies, revealing well-supported monophyletic groups at the tribe level and recovering the relationship ((Satyrini + Melanitini) + ((Amathusiini + Elymniini) + Zetherini)). The close relationship between Satyrina and Melanargiina within the Satyrini was widely accepted. Additionally, Lethina, Parargina, and Mycalesina were closely related and collectively formed a sister group to Coenonymphina. Moreover, A. merlina was closely related to Oeneis buddha within the Satyrina. These findings will provide valuable information for future studies aiming to elucidate the phylogenetic relationships of Satyrinae.

Insect floral visitors of thermo-Mediterranean shrubland maquis (Ajaccio, Corsica, France).

Background: The Mediterranean Region represents a biodiversity hotspot with a high rate of endemism. In its western part, Corsica Island is notable in terms of biodiversity due to its large surface and its large range of habitats from seaside to alpine biotopes. Amongst diverse groups, insects, notably the main orders of pollinators composed of Coleoptera, Hymenoptera, Diptera and Lepidoptera, represent a good part of the insular richness. New information: Our sampling effort focused on the insects from these four orders visiting flowers in a characteristic thermo-Mediterranean vegetation. Our database is an insight into the Corsican floral visitor biodiversity from three sites separated by a few kilometres in the region of Ajaccio during 13 months over two successive years. In total, 4012 specimens were sampled and 252 species or morpho-species identified from 133 genera and 47 families. Beetles were by far the most abundant order representing about 54% of the sampled specimens. The most diverse order was the Hymenoptera representing 39% of the species. Our continuous survey showed that these orders are temporally dynamic both between years and between seasons in terms of abundance and diversity.

Meiotic drive against chromosome fusions in butterfly hybrids.

Species frequently differ in the number and structure of chromosomes they harbor, but individuals that are heterozygous for chromosomal rearrangements may suffer from reduced fitness. Chromosomal rearrangements like fissions and fusions can hence serve as a mechanism for speciation between incipient lineages, but their evolution poses a paradox. How can rearrangements get fixed between populations if heterozygotes have reduced fitness? One solution is that this process predominantly occurs in small and isolated populations, where genetic drift can override natural selection. However, fixation is also more likely if a novel rearrangement is favored by a transmission bias, such as meiotic drive. Here, we investigate chromosomal transmission distortion in hybrids between two wood white (Leptidea sinapis) butterfly populations with extensive karyotype differences. Using data from two different crossing experiments, we uncover that there is a transmission bias favoring the ancestral chromosomal state for derived fusions, a result that shows that chromosome fusions actually can fix in populations despite being counteracted by meiotic drive. This means that meiotic drive not only can promote runaway chromosome number evolution and speciation, but also that it can be a conservative force acting against karyotypic change and the evolution of reproductive isolation. Based on our results, we suggest a mechanistic model for why chromosome fusion mutations may be opposed by meiotic drive and discuss factors contributing to karyotype evolution in Lepidoptera.