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The advent of next-generation sequencing technology has allowed for the collection of large portions of the genome for phylogenetic analysis. Hybrid enrichment and transcriptomics are two techniques that leverage next-generation sequencing and have shown much promise. However, methods for processing hybrid enrichment data are still limited. We developed a pipeline for anchored hybrid enrichment (AHE) read assembly, orthology determination, contamination screening, and data processing for sequences flanking the target “probe” region. We apply this approach to study the phylogeny of butterflies and moths (Lepidoptera), a megadiverse group of more than 157,000 described species with poorly understood deep-level phylogenetic relationships. We introduce a new, 855 locus AHE kit for Lepidoptera phylogenetics and compare resulting trees to those from transcriptomes. The enrichment kit was designed from existing genomes, transcriptomes, and expressed sequence tags and was used to capture sequence data from 54 species from 23 lepidopteran families. Phylogenies estimated from AHE data were largely congruent with trees generated from transcriptomes, with strong support for relationships at all but the deepest taxonomic levels. We combine AHE and transcriptomic data to generate a new Lepidoptera phylogeny, representing 76 exemplar species in 42 families. The tree provides robust support for many relationships, including those among the seven butterfly families. The addition of AHE data to an existing transcriptomic dataset lowers node support along the Lepidoptera backbone, but firmly places taxa with AHE data on the phylogeny. Combining taxa sequenced for AHE with existing transcriptomes and genomes resulted in a tree with strong support for (Calliduloidea + Gelechioidea + Thyridoidea) + (Papilionoidea + Pyraloidea + Macroheterocera). To examine the efficacy of AHE at a shallow taxonomic level, phylogenetic analyses were also conducted on a sister group representing a more recent divergence, the Saturniidae and Sphingidae. These analyses utilized sequences from the probe region and data flanking it, nearly doubled the size of the dataset; resulting trees supported new phylogenetics relationships, especially within the Saturniidae and Sphingidae (e.g., Hemarina derived in the latter). We hope that our data processing pipeline, hybrid enrichment gene set, and approach of combining AHE data with transcriptomes will be useful for the broader systematics community.

Diurnal and nocturnal hawkmoths (Sphingidae, Lepidoptera) have three spectral types of receptor sensitive to ultraviolet, blue and green light. As avid flower visitors and pollinators, they use olfactory and visual cues to find and recognise flowers. Moths of the diurnal species Macroglossum stellatarum and the nocturnal species Deilephila elpenor, Hyles lineata and Hyles gallii use and learn the colour of flowers. Nocturnal species can discriminate flowers at starlight intensities when humans and honeybees are colour-blind. M. stellatarum can use achromatic, intensity-related cues if colour cues are absent, and this is probably also true for D. elpenor. Both species can recognise colours even under a changed illumination colour.

Pollinators use their sense of smell to locate flowers from long distances, but little is known about how they are able to discriminate their target odor from a mélange of other natural and anthropogenic odors. Here, we measured the plume from Datura wrightii flowers, a nectar resource for Manduca sexta moths, and show that the scent was dynamic and rapidly embedded among background odors. The moth's ability to track the odor was dependent on the background and odor frequency. By influencing the balance of excitation and inhibition in the antennal lobe, background odors altered the neuronal representation of the target odor and the ability of the moth to track the plume. These results show that the mix of odors present in the environment influences the pollinator's olfactory ability.

Fil: More, Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina

Plant trichomes constitute a first line of defence against insect herbivores. The pre- and post-ingestive defensive functions of glandular trichomes are well documented and include direct toxicity, adhesion, antinutrition and defence gene induction. By contrast, the defensive functions of non-glandular trichomes are less well characterized, although these structures are thought to serve as physical barriers that impede herbivore feeding and movement. We experimentally varied the density of stellate non-glandular trichomes in several ways to explore their pre- and post-ingestive effects on herbivores. Larvae of Manduca sexta (Sphingidae) initiated feeding faster and gained more weight on Solanum carolinense (Solanaceae) leaves having lower trichome densities (or experimentally removed trichomes) than on leaves having higher trichome densities. Adding trichomes to artificial diet also deterred feeding and adversely affected caterpillar growth relative to controls. Scanning electron and light microscopy revealed that the ingestion of stellate trichomes by M. sexta caterpillars caused extensive damage to the peritrophic membrane, a gut lining that is essential to digestion and pathogen isolation. These findings suggest that, in addition to acting as a physical barrier to deter feeding, trichomes can inhibit caterpillar growth and development via post-ingestive effects.

Nectar-feeding animals have among the highest recorded metabolic rates. High aerobic performance is linked to oxidative damage in muscles. Antioxidants in nectar are scarce to nonexistent. We propose that nectarivores use nectar sugar to mitigate the oxidative damage caused by the muscular demands of flight. We found that sugar-fed moths had lower oxidative damage to their flight muscle membranes than unfed moths. Using respirometry coupled with δ13C analyses, we showed that moths generate antioxidant potential by shunting nectar glucose to the pentose phosphate pathway (PPP), resulting in a reduction in oxidative damage to the flight muscles. We suggest that nectar feeding, the use of PPP, and intense exercise are causally linked and have allowed the evolution of powerful fliers that feed on nectar.

• Biotic and abiotic context may affect the intensity of interspecific interactions and subsequently drive locally particular phenotypic selection patterns on interacting traits. • We evaluated the geographical variation of matching traits of the brush-type flowers of Caesalpinia gilliesii and of the proboscis length of its guild of hawkmoth pollinators, as well as their relationship with environmental variables. We assessed the geographical variation of interacting traits (style and filament vs mean proboscis length of the guild of hawkmoths) across seven populations and estimated phenotypic selection on the plant side. • Interacting traits showed similar relationships with environmental variables. Phenotypic selection on the plant side was influenced by proboscis length and by environmental conditions. Mean proboscis length of the guild was shorter than previously recorded for the same study area, thus probably shifting the selective optima of flower length. We observed two presumptive coevolutionary cold spots where one-sided negative directional selection is acting on style length. The lack of selection on the pollinator side should be further confirmed. • We provided joint evidence, mostly lacking, about the geographical variation of selective pressures on the plant side associated with both proboscis length and abiotic conditions. We suggest that recent environmental change may be shifting floral length optima.

Fil: More, Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina

Clanis bilineata Walker (Lepidoptera: Sphingidae), a burgeoning edible insect, is experiencing rising demand in China and other regions. Despite this interest, larval production is currently constrained by the limitations of artificial production technologies, particularly the selection of optimal host plants. This study rigorously evaluated the performance of C . bilineatha larvae on four main host plants: round-leaf soybean, pointed-leaf soybean, black locust, and kudzu. Preference tests demonstrated that the larvae were most attracted to black locust (34.76 ± 4.65%), with subsequent preferences for kudzu (25.00 ± 6.12%), round-leaf soybean (23.17 ± 2.79%), and pointed-leaf soybean (14.02 ± 4.74%). No significant preference differences were noted between round-leaf soybean and either black locust or kudzu. In feeding assays, the larvae exhibited a marked preference for round-leaf soybean (37.36 ± 0.81 g, total feeding amount for larvae), followed by kudzu (37.26 ± 0.82 g), pointed-leaf soybean (35.38 ± 1.31 g), and black locust (28.53 ± 0.81 g). When the larvae were fed on round-leaf soybean, they exhibited significantly higher survival rate (39.33 ± 0.90%), body weight (9.75 ± 0.07 g), total biomass (383.43 ± 7.35 g), pupation rate (87.78 ± 1.73%), and egg production (189.80 ± 1.06 eggs/female) compared to other hosts. These findings uncovered that round-leaf soybean significantly enhances larval performance, suggesting its potential for improving C . bilineata larval production and sustainability in cultivation systems.

Sexual dimorphism in insects often extends beyond size differences, encompassing a range of morphological traits. These sex differences in wing size, body mass, antenna shape, and even the epiphyses of lepidopterans have been studied previously. The epiphysis, located on the foretibiae of lepidopterans, plays a crucial role in removing dust and other debris from the antennae, helping to maintain the sensory functions essential for navigation and communication. Unique to lepidopterans, the epiphysis is particularly important for species with highly developed antennae, such as hawkmoths. This study presents novel insights into the significant sexual dimorphism and allometric trends in the epiphyses of hawkmoths (Lepidoptera: Sphingidae). Through comprehensive morphometric analyses of 564 specimens across two tribes of Macroglossinae, we demonstrated that males typically possess longer epiphyses than females, correlating with antennal length. We observed that tibial length, epiphysis length, and width influence epiphysis positioning on the foretibiae. Our findings suggest that medium-sized epiphyses were a conserved trait in the common ancestor of extant hawkmoths, revealing both interspecific and intraspecific variations. This study not only advances the understanding of sexual dimorphism in lepidopterans but also sheds light on the evolutionary mechanisms driving morphological diversity. These discoveries contribute to a broader understanding of sexual dimorphism in Sphingidae and raise essential questions about the evolutionary pressures driving this variation in lepidopterans.