RNAi efficiency is enhanced through knockdown of double-stranded RNA-degrading enzymes in butterfly Papilio xuthus.

The efficiency of RNA interference (RNAi) has always limited the research on the phenotype innovation of Lepidoptera insects. Previous studies have found that double-stranded RNA-degrading enzyme (dsRNase) is an important factor in RNAi efficiency, but there have been no relevant reports in butterflies (Papilionoidea). Papilio xuthus is one of the important models in butterflies with an extensive experimental application value. To explore the effect of dsRNase in the RNAi efficiency on butterflies, six dsRNase genes (PxdsRNase 1-6) were identified in P. xuthus genome, and their dsRNA-degrading activities were subsequently detected by ex vivo assays. The result shows that the dsRNA-degrading ability of gut content (<1 h) was higher than hemolymph content (>12 h). We then investigated the expression patterns of these PxdsRNase genes during different tissues and developmental stages, and related RNAi experiments were carried out. Our results show that different PxdsRNase genes had different expression levels at different developmental stages and tissues. The expression of PxdsRNase2, PxdsRNase3, and PxdsRNase6 were upregulated significantly through dsGFP injection, and PxdsRNase genes can be silenced effectively by injecting their corresponding dsRNA. RNAi-of-RNAi studies with PxEbony, which acts as a reporter gene, observed that silencing PxdsRNase genes can increase RNAi efficiency significantly. These results confirm that silencing dsRNase genes can improve RNAi efficiency in P. xuthus significantly, providing a reference for the functional study of insects such as butterflies with low RNAi efficiency.

A working model for the formation of Robertsonian chromosomes.

Robertsonian chromosomes form by fusion of two chromosomes that have centromeres located near their ends, known as acrocentric or telocentric chromosomes. This fusion creates a new metacentric chromosome and is a major mechanism of karyotype evolution and speciation. Robertsonian chromosomes are common in nature and were first described in grasshoppers by the zoologist W. R. B. Robertson more than 100 years ago. They have since been observed in many species, including catfish, sheep, butterflies, bats, bovids, rodents and humans, and are the most common chromosomal change in mammals. Robertsonian translocations are particularly rampant in the house mouse, Mus musculus domesticus, where they exhibit meiotic drive and create reproductive isolation. Recent progress has been made in understanding how Robertsonian chromosomes form in the human genome, highlighting some of the fundamental principles of how and why these types of fusion events occur so frequently. Consequences of these fusions include infertility and Down's syndrome. In this Hypothesis, I postulate that the conditions that allow these fusions to form are threefold: (1) sequence homology on non-homologous chromosomes, often in the form of repetitive DNA; (2) recombination initiation during meiosis; and (3) physical proximity of the homologous sequences in three-dimensional space. This Hypothesis highlights the latest progress in understanding human Robertsonian translocations within the context of the broader literature on Robertsonian chromosomes.

Impacts of fire and prospects for recovery in a tropical peat forest ecosystem.

Uncontrolled fires place considerable burdens on forest ecosystems, compromising our ability to meet conservation and restoration goals. A poor understanding of the impacts of fire on ecosystems and their biodiversity exacerbates this challenge, particularly in tropical regions where few studies have applied consistent analytical techniques to examine a broad range of ecological impacts over multiyear time frames. We compiled 16 y of data on ecosystem properties (17 variables) and biodiversity (21 variables) from a tropical peatland in Indonesia to assess fire impacts and infer the potential for recovery. Burned forest experienced altered structural and microclimatic conditions, resulting in a proliferation of nonforest vegetation and erosion of forest ecosystem properties and biodiversity. Compared to unburned forest, habitat structure, tree density, and canopy cover deteriorated by 58 to 98%, while declines in species diversity and abundance were most pronounced for trees, damselflies, and butterflies, particularly for forest specialist species. Tracking ecosystem property and biodiversity datasets over time revealed most to be sensitive to recurrent high-intensity fires within the wider landscape. These megafires immediately compromised water quality and tree reproductive phenology, crashing commercially valuable fish populations within 3 mo and driving a gradual decline in threatened vertebrates over 9 mo. Burned forest remained structurally compromised long after a burn event, but vegetation showed some signs of recovery over a 12-y period. Our findings demonstrate that, if left uncontrolled, fire may be a pervasive threat to the ecological functioning of tropical forests, underscoring the importance of fire prevention and long-term restoration efforts, as exemplified in Indonesia.

A genetic atlas for the butterflies of continental Canada and United States.

Multi-locus genetic data for phylogeographic studies is generally limited in geographic and taxonomic scope as most studies only examine a few related species. The strong adoption of DNA barcoding has generated large datasets of mtDNA COI sequences. This work examines the butterfly fauna of Canada and United States based on 13,236 COI barcode records derived from 619 species. It compiles i) geographic maps depicting the spatial distribution of haplotypes, ii) haplotype networks (minimum spanning trees), and iii) standard indices of genetic diversity such as nucleotide diversity (π), haplotype richness (H), and a measure of spatial genetic structure (GST). High intraspecific genetic diversity and marked spatial structure were observed in the northwestern and southern North America, as well as in proximity to mountain chains. While species generally displayed concordance between genetic diversity and spatial structure, some revealed incongruence between these two metrics. Interestingly, most species falling in this category shared their barcode sequences with one at least other species. Aside from revealing large-scale phylogeographic patterns and shedding light on the processes underlying these patterns, this work also exposed cases of potential synonymy and hybridization.

Testing of arctic insect hemolymph as a secondary agent in applied cryopreservation.

BACKGROUND: Cold hardiness of insects from extremely cold regions is based on a principle of natural cryoprotection, which is associated with physiological mechanisms provided by cryoprotectants. OBJECTIVE: Since arctic cold-hardy insects are producers of highly effective cryoprotectants, in this study, the hemolymph of Aporia crataegi L. and Upis ceramboides L. from an extremely cold area (Yakutia) was tested as a secondary component of cryoprotective agents (CPA) for cryopreservation (-80 degree C) of human peripheral blood lymphocytes and skin fibroblasts. MATERIALS AND METHODS: Lymphocytes and skin fibroblasts were treated with various combinations of DMSO and hemolymph extract and step-wise cooled to -80 degree C. Post-cryopreservation cell viability was assessed by vital staining and morphological appearance. RESULTS: Viability was higher when cells were frozen with a mixture containing DMSO and Upis ceramboides hemolymph compared to the cells frozen in DMSO, while cells frozen with DMSO and Aporia crataegi hemolymph did not survive. The fact that hemolymph of not every cold-resistant insect can be used as a secondary agent along with DMSO indicates that only a unique combination of hemolymph components and its compatibility with cells might result in a positive effect. CONCLUSION: Although the use of insect hemolymph as a complementary agent in applied cryopreservation is a problem in terms of practical application, such studies could initiate new trends in the search for the most successful hemolymph-like cryoprotectant systems. https://doi.org/10.54680/fr24210110712.

What one genus of showy moths can say about migration, adaptation, and wing pattern.

The Ornate Moth, Utetheisa ornatrix, has served as a model species in chemical ecology studies for decades. Like in the widely publicized stories of the Monarch and other milkweed butterflies, the Ornate Moth and its relatives are tropical insects colonizing whole continents assisted by their chemical defenses. With the recent advances in genomic techniques and evo-devo research, it is becoming a model for studies in other areas, from wing pattern development to phylogeography, from toxicology to epigenetics. We used a genomic approach to learn about Utetheisa's evolution, detoxification, dispersal abilities, and wing pattern diversity. We present an evolutionary genomic analysis of the worldwide genus Utetheisa, then focusing on U. ornatrix. Our reference genome of U. ornatrix reveals gene duplications in the regions possibly associated with detoxification abilities, which allows them to feed on toxic food plants. Finally, comparative genomic analysis of over 100 U. ornatrix specimens from the museum with apparent differences in wing patterns suggest the potential roles of cortex and lim3 genes in wing pattern formation of Lepidoptera and the utility of museum-preserved collection specimens for wing pattern research.

A macroevolutionary role for chromosomal fusion and fission in Erebia butterflies.

The impact of large-scale chromosomal rearrangements, such as fusions and fissions, on speciation is a long-standing conundrum. We assessed whether bursts of change in chromosome numbers resulting from chromosomal fusion or fission are related to increased speciation rates in Erebia, one of the most species-rich and karyotypically variable butterfly groups. We established a genome-based phylogeny and used state-dependent birth-death models to infer trajectories of karyotype evolution. We demonstrated that rates of anagenetic chromosomal changes (i.e., along phylogenetic branches) exceed cladogenetic changes (i.e., at speciation events), but, when cladogenetic changes occur, they are mostly associated with chromosomal fissions rather than fusions. We found that the relative importance of fusion and fission differs among Erebia clades of different ages and that especially in younger, more karyotypically diverse clades, speciation is more frequently associated with cladogenetic chromosomal changes. Overall, our results imply that chromosomal fusions and fissions have contrasting macroevolutionary roles and that large-scale chromosomal rearrangements are associated with bursts of species diversification.

Transcriptome-based analysis reveals a crucial role of the 20E/HR3 pathway in the diapause of Pieris rapae.

Pieris rapae is among the most damaging pests globally, and diapause makes it highly resistant to environmental stresses, playing a crucial role in the survival and reproduction of P. rapae while exacerbating the challenges of pest management and control. However, the mechanisms of its diapause regulation remain poorly understood. This research used RNA sequencing to profile the transcriptomes of three diapause phases (induction and preparation, initiation, maintenance) and synchronous nondiapause phases in P. rapae. During each comparison phase, 759, 1045, and 4721 genes were found to be differentially expressed. Among these, seven clock genes and seven pivotal hormone synthesis and metabolism genes were identified as having differential expression patterns in diapause type and nondiapause type. The weighted gene co-expression network analysis (WGCNA) revealed the red and blue modules as pivotal for diapause initiation, while the grey module was identified to be crucial to diapause maintenance. Meanwhile, the hub genes HDAC11, METLL16D, Dyw-like, GST, and so on, were identified within these hub modules. Moreover, an ecdysone downstream nuclear receptor gene, HR3, was found to be a shared transcription factor across all three phases. RNA interference of HR3 resulted in delayed pupal development, indicating its involvement in regulating pupal dipause in P. rapae. The further hormone assays revealed that the 20-hydroxyecdysone (20E) titer in diapause type pupae was lower than that in nondiapause type pupae, which exhibited a similar trend to HR3. When 20E was injected into diapause pupae, the HR3 expression levels were improved, and the pupal diapause were broken. These results indicate that the 20E/HR3 pathway is a critical pathway for the diapause regulation of P. rapae, and perturbing this pathway by ecdysone treatment or RNAi would result in the disruption of diapause. These findings provide initial insights into the molecular mechanisms of P. rapae diapause and suggest the potential use of ecdysone analogs and HR3 RNAi pesticides, which specifically target to diapause, as a means of pest control in P. rapae.

Changes in landscape and climate in Mexico and Texas reveal small effects on migratory habitat of monarch butterflies (Danaus plexippus).

The decline of the iconic monarch butterfly (Danaus plexippus) in North America has motivated research on the impacts of land use and land cover (LULC) change and climate variability on monarch habitat and population dynamics. We investigated spring and fall trends in LULC, milkweed and nectar resources over a 20-year period, and ~ 30 years of climate variables in Mexico and Texas, U.S. This region supports spring breeding, and spring and fall migration during the annual life cycle of the monarch. We estimated a - 2.9% decline in milkweed in Texas, but little to no change in Mexico. Fall and spring nectar resources declined < 1% in both study extents. Vegetation greenness increased in the fall and spring in Mexico while the other climate variables did not change in both Mexico and Texas. Monarch habitat in Mexico and Texas appears relatively more intact than in the midwestern, agricultural landscapes of the U.S. Given the relatively modest observed changes in nectar and milkweed, the relatively stable climate conditions, and increased vegetation greenness in Mexico, it seems unlikely that habitat loss (quantity or quality) in Mexico and Texas has caused large declines in population size or survival during migration.

Food and time: dietary plasticity of different sources of a generalist insect herbivore.

Painted lady butterflies (Vanessa cardui L., Nymphalidae) are generalist herbivores and serve as a model system across several fields of biology. While it has been demonstrated that V. cardui caterpillars can develop on different host plants, much of this work has been done on commercially sourced caterpillars, which could limit our understanding of wild V. cardui populations. In this study, we sought to explore possible differences in how commercial and wild V. cardui caterpillars may respond to feeding on different host plants, and subsequently, how their diet impacts immune response and survival. Here, we analyzed performance, survival, and immune response of wild and commercially sourced V. cardui caterpillars over several generations on diets that consisted of either 1 of 4 different host plant species or a mixed diet including all 4 species. Qualitatively, we observed that wild larvae had a better larval performance and hemocyte counts compared to the commercial larvae. The results demonstrate that both wild and commercially sourced caterpillars grew and survived best on the same diet treatments (mallow, narrowleaf plantain, and a mixed diet) during development across generations. Immune responses showed similar patterns across host plants between wild and commercial populations, with individuals showing lowered immune responses on dandelion and lupine and higher ones on mallow, plantain and the mixed diet; although the relative rankings on those 3 diets varied. Survival also demonstrated similar patterns, in that individuals reared on dandelion and lupine had the lowest survival.

Genome Assembly and Annotation of the Dark-Branded Bushbrown Butterfly Mycalesis mineus (Nymphalidae: Satyrinae).

We report a high-quality genome draft assembly of the dark-branded bushbrown, Mycalesis mineus, a member of the Satyrinae subfamily of nymphalid butterflies. This species is emerging as a promising model organism for investigating the evolution and development of phenotypic plasticity. Using 45.99 Gb of long-read data (N50 = 11.11 kb), we assembled a genome size of 497.4 Mb for M. mineus. The assembly is highly contiguous and nearly complete (96.8% of Benchmarking Universal Single-Copy Orthologs lepidopteran genes were complete and single copy). The genome comprises 38.71% of repetitive elements and includes 20,967 predicted protein-coding genes. The assembled genome was super-scaffolded into 28 pseudo-chromosomes using a closely related species, Bicyclus anynana, with a chromosomal-level genome as a template. This valuable genomic tool will advance both ongoing and future research focused on this model organism.

Evidence of Nonrandom Patterns of Functional Chromosome Organization in Danaus plexippus.

Our understanding on the interplay between gene functionality and gene arrangement at different chromosome scales relies on a few Diptera and the honeybee, species with quality reference genome assemblies, accurate gene annotations, and abundant transcriptome data. Using recently generated 'omic resources in the monarch butterfly Danaus plexippus, a species with many more and smaller chromosomes relative to Drosophila species and the honeybee, we examined the organization of genes preferentially expressed at broadly defined developmental stages (larva, pupa, adult males, and adult females) at both fine and whole-chromosome scales. We found that developmental stage-regulated genes do not form more clusters, but do form larger clusters, than expected by chance, a pattern consistent across the gene categories examined. Notably, out of the 30 chromosomes in the monarch genome, 12 of them, plus the fraction of the chromosome Z that corresponds to the ancestral Z in other Lepidoptera, were found enriched for developmental stage-regulated genes. These two levels of nonrandom gene organization are not independent as enriched chromosomes for developmental stage-regulated genes tend to harbor disproportionately large clusters of these genes. Further, although paralogous genes were overrepresented in gene clusters, their presence is not enough to explain two-thirds of the documented cases of whole-chromosome enrichment. The composition of the largest clusters often included paralogs from more than one multigene family as well as unrelated single-copy genes. Our results reveal intriguing patterns at the whole-chromosome scale in D. plexippus while shedding light on the interplay between gene expression and chromosome organization beyond Diptera and Hymenoptera.

Chromosome-Level Assembly and Annotation of the Pearly Heath Coenonympha arcania Butterfly Genome.

We present the first chromosome-level genome assembly and annotation of the pearly heath Coenonympha arcania, generated with a PacBio HiFi sequencing approach and complemented with Hi-C data. We additionally compare synteny, gene, and repeat content between C. arcania and other Lepidopteran genomes. This reference genome will enable future population genomics studies with Coenonympha butterflies, a species-rich genus that encompasses some of the most highly endangered butterfly taxa in Europe.

Different ecological demands shape differences in population structure and behaviour among the two generations of the small pearl-bordered fritillary.

The population structure and behaviour of univoltine butterfly species have been studied intensively. However, much less is known about bivoltine species. In particular, in-depth studies of the differences in population structure, behaviour, and ecology between these two generations are largely lacking. Therefore, we here present a mark-release-recapture study of two successive generations of the fritillary butterfly Boloria selene performed in eastern Brandenburg (Germany). We revealed intersexual and intergenerational differences regarding behaviour, dispersal, population characteristics, and protandry. The observed population densities were higher in the second generation. The flight activity of females decreased in the second generation, but remained unchanged in males. This was further supported by the rate of wing decay. The first generation displayed a linear correlation between wing decay and passed time in both sexes, whereas the linear correlation was lost in second-generation females. The proportion of resting individuals in both sexes increased in the second generation, as well as the number of nectaring females. The choice of plant genera used for nectaring seems to be more specialised in the first and more opportunistic in the second generation. The average flight distances were generally higher for females than for males and overall higher in the first generation. Predictions of long-distance movements based on the inverse power function were also generally higher in females than in males but lower in the first generation. Additionally, we found protandry only in the first but not in the second generation, which might correlate with the different developmental pathways of the two generations. These remarkable differences between both generations might reflect an adaptation to the different ecological demands during the flight season and the different tasks they have, i.e., growth in the spring season; dispersal and colonisation of new habitats during the summer season.

Some taxonomic notes on Capila translucida (Leech, 1894) and C. hainana Crowley, 1900 (Hesperiidae, Pyrginae).

Based on a comparison of the 658 bp COI gene sequence and adult morphology, the intraspecific variability of Capila translucida (Leech, 1894) is clarified, and both C. hainana hainana Crowley, 1900 syn. n. and C. hainana sinorientalis Huang & Ding, 1994 syn. n., of which the males are still unknown so far, are considered as junior subjective synonyms of C. translucida. The taxonomic history of the involved taxa is reviewed. Adults and genitalia of both sexes of C. translucida are illustrated.

Molecular data and morphological characters clarify the taxonomic status of Pintara heringi pieridoides (Liu & Gu, 1994) (Hesperiidae, Pyrginae).

We found Albiphasma heringi (Mell, 1922) and A. pieridoides (Liu & Gu, 1994) to be conspecific by the 658 bp COI gene sequences and male genitalia characters. Considering the distinguishable wing patterns and allopatric distribution of the two taxa, we treat pieridoides as a subspecies of heringi. Therefore, the genus Albiphasma Huang, Chiba, Wang and Fan, 2016, which was established for heringi and pieridoides, becomes monotypic, and in light of morphological similarities and close genetic distance between heringi and Pintara bowringi (Joicey & Talbot, 1921), we propose its synonymy with Pintara Evans, 1932. The adults and male genitalia of both P. heringi heringi (Mell, 1922) and P. heringi pieridoides (Liu & Gu, 1994) are illustrated.

A new subspecies of Aporia procris Leech, 1890 hidden in Sichuan, China (Lepidoptera, Pieridae).

Further information on the distribution of Aporia procris Leech, 1890 is provided. The population of A. procris from the upper Dadu River, W. Sichuan, previously recognized as ssp. yaozhui Huang, 2021, is treated as a new subspecies, viz., A. p. huangsiyaoi ssp. nov., based upon evidence from external features, with its genital and molecular characters given.

A new species of the genus Celaenorrhinus Hbner, [1819] from Hainan, China (Lepidoptera, Hesperiidae).

A new species, Celaenorrhinus qianhanae Li & Liu sp. nov., is described from Hainan, China. Its unique genital characteristics and wing markings are compared with other congeneric species from this island and adjacent regions, including Vietnam and China, etc. In addition, its COI gene is sequenced for molecular analysis, and the secondary sexual character, hair tufts on hind tibiae, is discussed for comparison.

A genetic cause of male mate preference.

A gene for mate preference has been shared between hybridizing butterfly species.

A biogeographical appraisal of the threatened South East Africa Montane Archipelago ecoregion.

Recent biological surveys of ancient inselbergs in southern Malawi and northern Mozambique have led to the discovery and description of many species new to science, and overlapping centres of endemism across multiple taxa. Combining these endemic taxa with data on geology and climate, we propose the 'South East Africa Montane Archipelago' (SEAMA) as a distinct ecoregion of global biological importance. The ecoregion encompasses 30 granitic inselbergs reaching > 1000 m above sea level, hosting the largest (Mt Mabu) and smallest (Mt Lico) mid-elevation rainforests in southern Africa, as well as biologically unique montane grasslands. Endemic taxa include 127 plants, 45 vertebrates (amphibians, reptiles, birds, mammals) and 45 invertebrate species (butterflies, freshwater crabs), and two endemic genera of plants and reptiles. Existing dated phylogenies of endemic animal lineages suggests this endemism arose from divergence events coinciding with repeated isolation of these mountains from the pan-African forests, together with the mountains' great age and relative climatic stability. Since 2000, the SEAMA has lost 18% of its primary humid forest cover (up to 43% in some sites)-one of the highest deforestation rates in Africa. Urgently rectifying this situation, while addressing the resource needs of local communities, is a global priority for biodiversity conservation.