Chromosome-level genome assembly of the snakefly Mongoloraphidia duomilia (Raphidioptera: Raphidiidae).

Raphidioptera (snakeflies) are a holometabolan order with the least species diversity but play a pivotal role in understanding the origin of complete metamorphosis. Here, we provide an annotated, chromosome-level reference genome assembly for an Asian endemic snakefly Mongoloraphidia duomilia (Yang, 1998) of the family Raphidiidae, assembled using PacBio HiFi and Hi-C data from female specimens. The resulting assembly is 653.56 Mb, of which 97.90% is anchored into 13 chromosomes. The scaffold N50 is 53.50 Mb, and BUSCO completeness is 97.80%. Repetitive elements comprise 64.31% of the genome (366.04 Mb). We identified 599 noncoding RNAs and predicted 11,141 protein-coding genes in the genome (97.70% BUSCO completeness). The new snakefly genome will facilitate comparison of genome architecture across Neuropterida and Holometabola and shed light on the ecological and evolutionary transitions between Neuropterida and Coleopterida.

DNA barcoding unveils a high diversity of caddisflies (Trichoptera) in the Mount Halimun Salak National Park (West Java; Indonesia).

Background: Trichoptera are one of the most diverse groups of freshwater insects worldwide and one of the main bioindicators for freshwater quality. However, in many areas, caddisflies remain understudied due to lack of taxonomic expertise. Meanwhile, globally increasing anthropogenic stress on freshwater streams also threatens Trichoptera diversity. Methods: To assess the Trichoptera diversity of the area within and around the Mount Halimun Salak National Park (MHSNP or Taman Nasional Gunung Halimun Salak) in West Java (Indonesia), we conducted a molecular-morphological study on Trichoptera diversity using larvae from a benthic survey and adults from hand-netting. In addition to morphological identification, we applied four different molecular taxon delimitation approaches (Generalized Mixed Yule Coalescent, Bayesian Poisson Tree Processes, Automatic Barcode Gap Discovery and Assemble Species by Automatic Partitioning) based on DNA barcoding of Cytochrome-C-Oxidase I (COI). Results: The molecular delimitation detected 72 to 81 Operational Taxonomic Units (OTU). Only five OTUs could be identified to species level by comparing sequences against the BOLD database using BLAST, and four more to the genus level. Adults and larvae could be successfully associated in 18 cases across six families. The high diversity of Trichoptera in this area highlights their potential as bioindicators for water quality assessment. Conclusions: This study provides an example of how molecular approaches can benefit the exploration of hidden diversity in unexplored areas and can be a valuable tool to link life stages. However, our study also highlights the need to improve DNA barcode reference libraries of Trichoptera for the Oriental region.

The First Chromosome-level Genome Assembly of Cheumatopsyche charites Malicky and Chantaramongkol, 1997 (Trichoptera: Hydropsychidae) Reveals How It Responds to Pollution.

Trichoptera is a highly adapted group of freshwater insects. They are generally more sensitive to dissolved oxygen and water quality than most freshwater organisms, and this sensitivity allows them to be used as reliable biological indicators of water quality. At present, there exists no chromosome-level genome of a hydropsychid species. Cheumatopsyche charites Malicky & Chantaramongkol, 1997 can successfully survive and thrive in polluted streams where other caddisflies are infrequent, suggesting that they are tolerant to latent contamination. Here we report a high-quality chromosome-level genome assembly of C. charites generated combining PacBio long reads and Hi-C reads. We obtained a genome assembly of 223.23 Mb, containing 68 scaffolds with an N50 length of 13.97 Mb, and 155 contigs (99.67%) anchored into 16 pseudochromosomes. We identified 36.12 Mb (16.18%) of the genome as being composed of repetitive elements, identified 369 noncoding RNAs, and predicted 8,772 protein-coding genes (96.80% BUSCO completeness). Gene family evolution analyses identified 7,148 gene families, of which 41 experienced rapid evolution. The expanded gene families were shown to be involved in detoxification metabolism, digestive absorption, and resistance to viruses or bacteria. This high-quality genome provides a valuable genomic basis for the study of trichopteran evolution.

Morphological description and DNA-based association of the last instar larva of Erotesis schachti Malicky 1982 (Trichoptera: Leptoceridae), an endemic of the Iberian Peninsula.

In this paper we describe the main morphological characteristics that distinguish the full-grown larva of Erotesis schachti, an endemic of the Iberian Peninsula. The conspecificity of the larva and adult was confirmed by DNA analysis. Morphological features that easily discriminate it from the similar species Erotesis baltica are given.

Similar pattern, different paths: tracing the biogeographical history of Megaloptera (Insecta: Neuropterida) using mitochondrial phylogenomics.

The sequential breakup of the supercontinent Pangaea since the Middle Jurassic is one of the crucial factors that has driven the biogeographical patterns of terrestrial biotas. Despite decades of effort searching for concordant patterns between diversification and continental fragmentation among taxonomic groups, increasing evidence has revealed more complex and idiosyncratic scenarios resulting from a mixture of vicariance, dispersal and extinction. Aquatic insects with discreet ecological requirements, low vagility and disjunct distributions represent a valuable model for testing biogeographical hypotheses by reconstructing their distribution patterns and temporal divergences. Insects of the order Megaloptera have exclusively aquatic larvae, their adults have low vagility, and the group has a highly disjunct geographical distribution. Here we present a comprehensive phylogeny of Megaloptera based on a large-scale mitochondrial genome sequencing of 99 species representing >90% of the world genera from all major biogeographical regions. Molecular dating suggests that the deep divergence within Megaloptera pre-dates the breakup of Pangaea. Subsequently, the intergeneric divergences within Corydalinae (dobsonflies), Chauliodinae (fishflies) and Sialidae (alderflies) might have been driven by both vicariance and dispersal correlated with the shifting continent during the Cretaceous, but with strikingly different and incongruent biogeographical signals. The austral distribution of many corydalids appears to be a result of colonization from Eurasia through southward dispersal across Europe and Africa during the Cretaceous, whereas a nearly contemporaneous dispersal via northward rafting of Gondwanan landmasses may account for the colonization of extant Eurasian alderflies from the south.

Draft Genome Assemblies and Annotations of Agrypnia vestita Walker, and Hesperophylax magnus Banks Reveal Substantial Repetitive Element Expansion in Tube Case-Making Caddisflies (Insecta: Trichoptera).

Trichoptera (caddisflies) play an essential role in freshwater ecosystems; for instance, larvae process organic material from the water and are food for a variety of predators. Knowledge on the genomic diversity of caddisflies can facilitate comparative and phylogenetic studies thereby allowing scientists to better understand the evolutionary history of caddisflies. Although Trichoptera are the most diverse aquatic insect order, they remain poorly represented in terms of genomic resources. To date, all long-read based genomes have been sequenced from individuals in the retreat-making suborder, Annulipalpia, leaving ∼275 Ma of evolution without high-quality genomic resources. Here, we report the first long-read based de novo genome assemblies of two tube case-making Trichoptera from the suborder Integripalpia, Agrypnia vestita Walker and Hesperophylax magnus Banks. We find that these tube case-making caddisflies have genome sizes that are at least 3-fold larger than those of currently sequenced annulipalpian genomes and that this pattern is at least partly driven by major expansion of repetitive elements. In H. magnus, long interspersed nuclear elements alone exceed the entire genome size of some annulipalpian counterparts suggesting that caddisflies have high potential as a model for understanding genome size evolution in diverse insect lineages.

Molecular mechanisms underlying simplification of venation patterns in holometabolous insects.

How mechanisms of pattern formation evolve has remained a central research theme in the field of evolutionary and developmental biology. The mechanism of wing vein differentiation in Drosophila is a classic text-book example of pattern formation using a system of positional information, yet very little is known about how species with a different number of veins pattern their wings, and how insect venation patterns evolved. Here, we examine the expression pattern of genes previously implicated in vein differentiation in Drosophila in two butterfly species with more complex venation Bicyclus anynana and Pieris canidia We also test the function of some of these genes in B. anynana We identify both conserved as well as new domains of decapentaplegic, engrailed, invected, spalt, optix, wingless, armadillo, blistered and rhomboid gene expression in butterflies, and propose how the simplified venation in Drosophila might have evolved via loss of decapentaplegic, spalt and optix gene expression domains, via silencing of vein-inducing programs at Spalt-expression boundaries, and via changes in expression of vein maintenance genes.

Molecular identification of Stylops advarians (Strepsiptera: Stylopidae) in western Canada.

Strepsiptera are an enigmatic order of insects with extreme sexual dimorphism which makes it difficult to "match-up" free-living adult males with parasitic conspecific females of the Stylopidia, and free-living females of the Mengenillidae using morphological characters. Species identification is further complicated for the Stylopidia because adult females are endoparasitic and neotenic. Therefore, we used DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) to confirm the species identity of adult strepsipterans that were morphologically identified as Stylops advarians. These specimens, collected from Saskatoon (Saskatchewan, Canada), included one adult male, and eight females, the latter of which had been collected from solitary bees (Andrena milwaukeensis). Also included in the analyses were three pools of first-instar larvae that had emerged from three of the females. The results of the molecular analyses revealed that all specimens had an identical cox1 sequence, and belonged to a clade, with total statistical support (bootstrap value of 100%), that contained specimens of S. advarians from New York and Maine (USA). Hence, the results were consistent with the morphological identification of S. advarians. This study demonstrates the usefulness of a molecular approach for the identification of endoparasitic adult female and larval strepsipterans, life cycle stages that lack significant morphological characters for species identification.

Comparative genomic analysis of C-type lectin-domain genes in seven holometabolous insect species.

C-type lectins (CTLs) recognize various glycoconjugates through carbohydrate recognition domains (CRDs) and they play important roles in immune responses. In this study, comparative genomic analysis of CTLs were performed in 7 holometabolous species. CTL-S1 to S8 and CTL-X1 to X4 orthologous groups existed in the 7 species, while CTL-X5 group with dual-CRD, CTL-S11 group with triple-CRD, CTL-S9 group with a long C-terminus and Lepidopteran specific CTL-S10 group were not conserved. SliCTL-S12 to S14 cluster was only present in Spodoptera litura, and CTL-S genes were expanded on chromosomes 2 L and 2 R in Drosophila melanogaster. Most IMLs were clustered into three groups and the numbers of IMLs vary among species due to gene duplications. D. melanogaster specific CTLs and Lepidopteran IMLs within each of the three groups evolved more rapidly with higher dN/dS ratios. Two CRDs in IMLs clustered into two clades, with conserved Cys4-Cys5 and Cys1-Cys2 bonds in the first and second CRDs, respectively. The CTL-S and CTL-X family members in S. litura were mainly expressed in the fat body of 5th but not 6th instar larvae, and responded differently to S. litura nucleopolyhedrovirus (SpltNPV) and Nomuraea rileyi infection. The transcription levels of SliCTLs that expressed in fat body but not highly expressed in hemocytes were decreased at the middle and late stages of SpltNPV infection, and the mRNA levels of SliCTLs highly or specifically expressed in hemocytes were mainly decreased by SpltlNPV, N. rileyi and Bacillus thuringiensis infection. These results provide valuable information for further exploration of CTL functions in host-pathogen interaction.

An integrative phylogenomic approach to elucidate the evolutionary history and divergence times of Neuropterida (Insecta: Holometabola).

BACKGROUND: The latest advancements in DNA sequencing technologies have facilitated the resolution of the phylogeny of insects, yet parts of the tree of Holometabola remain unresolved. The phylogeny of Neuropterida has been extensively studied, but no strong consensus exists concerning the phylogenetic relationships within the order Neuroptera. Here, we assembled a novel transcriptomic dataset to address previously unresolved issues in the phylogeny of Neuropterida and to infer divergence times within the group. We tested the robustness of our phylogenetic estimates by comparing summary coalescent and concatenation-based phylogenetic approaches and by employing different quartet-based measures of phylogenomic incongruence, combined with data permutations. RESULTS: Our results suggest that the order Raphidioptera is sister to Neuroptera + Megaloptera. Coniopterygidae is inferred as sister to all remaining neuropteran families suggesting that larval cryptonephry could be a ground plan feature of Neuroptera. A clade that includes Nevrorthidae, Osmylidae, and Sisyridae (i.e. Osmyloidea) is inferred as sister to all other Neuroptera except Coniopterygidae, and Dilaridae is placed as sister to all remaining neuropteran families. Ithonidae is inferred as the sister group of monophyletic Myrmeleontiformia. The phylogenetic affinities of Chrysopidae and Hemerobiidae were dependent on the data type analyzed, and quartet-based analyses showed only weak support for the placement of Hemerobiidae as sister to Ithonidae + Myrmeleontiformia. Our molecular dating analyses suggest that most families of Neuropterida started to diversify in the Jurassic and our ancestral character state reconstructions suggest a primarily terrestrial environment of the larvae of Neuropterida and Neuroptera. CONCLUSION: Our extensive phylogenomic analyses consolidate several key aspects in the backbone phylogeny of Neuropterida, such as the basal placement of Coniopterygidae within Neuroptera and the monophyly of Osmyloidea. Furthermore, they provide new insights into the timing of diversification of Neuropterida. Despite the vast amount of analyzed molecular data, we found that certain nodes in the tree of Neuroptera are not robustly resolved. Therefore, we emphasize the importance of integrating the results of morphological analyses with those of sequence-based phylogenomics. We also suggest that comparative analyses of genomic meta-characters should be incorporated into future phylogenomic studies of Neuropterida.

Annotated Draft Genomes of Two Caddisfly Species Plectrocnemia conspersa CURTIS and Hydropsyche tenuis NAVAS (Insecta: Trichoptera).

Members of the speciose insect order Trichoptera (caddisflies) provide important ecosystem services, for example, nutrient cycling through breaking down of organic matter. They are also of industrial interest due to their larval silk secretions. These form the basis for their diverse case-making behavior that allows them to exploit a wide range of ecological niches. Only five genomes of this order have been published thus far, with variable qualities regarding contiguity and completeness. A low-cost sequencing strategy, that is, using a single Oxford Nanopore flow cell per individual along with Illumina sequence reads was successfully used to generate high-quality genomes of two Trichoptera species, Plectrocnemia conspersa and Hydropsyche tenuis. Of the de novo assembly methods compared, assembly of low coverage Nanopore reads (∼18×) and subsequent polishing with long reads followed by Illumina short reads (∼80-170× coverage) yielded the highest genome quality both in terms of contiguity and BUSCO completeness. The presented genomes are the shortest to date and extend our knowledge of genome size across caddisfly families. The genomic region that encodes for light (L)-chain fibroin, a protein component of larval caddisfly silk was identified and compared with existing L-fibroin gene clusters. The new genomic resources presented in this paper are among the highest quality Trichoptera genomes and will increase the knowledge of this important insect order by serving as the basis for phylogenomic and comparative genomic studies.

Preference of Polistes dominula wasps for trumpet creepers when infected by Xenos vesparum: A novel example of co-evolved traits between host and parasite.

The parasitic insect Xenos vesparum induces noticeable behavioral and physiological changes-e.g. castration-in its female host, the paper wasp Polistes dominula: parasitized putative workers avoid any colony task and desert the colony to survive in the nearby vegetation, like future queens and males do. In this long-term observational study, we describe the spectacular attraction of parasitized workers towards trumpet creeper bushes (Campsis radicans) in early-summer. Two thirds of all wasps that we sampled on these bushes were parasitized, whereas the parasite prevalence was much lower in our study area and most wasps sampled on other nearby flowering bushes were non-parasitized. First, we describe the occurrence and consistency of this phenomenon across different sites and years. Second, we evaluate the spatial behavior of parasitized wasps on C. radicans bushes, which includes site-fidelity, exploitation and defense of rich extra-floral nectaries on buds and calices. Third, we record two critical steps of the lifecycle of X. vesparum on C. radicans: the parasite's mating and a summer release of parasitic larvae, that can infect larval stages of the host if transported to the host's nest. In a nutshell, C. radicans bushes provide many benefits both to the parasite X. vesparum and to its host: they facilitate the parasite's mating and bivoltine lifecycle, a phenomenon never described before for this parasite, while, at the same time, they provide the wasp host with shelter inside trumpet flowers and extrafloral gland secretions, thus likely enhancing host survival and making it a suitable vector for the infection.

Signatures of DNA Methylation across Insects Suggest Reduced DNA Methylation Levels in Holometabola.

It has been experimentally shown that DNA methylation is involved in the regulation of gene expression and the silencing of transposable element activity in eukaryotes. The variable levels of DNA methylation among different insect species indicate an evolutionarily flexible role of DNA methylation in insects, which due to a lack of comparative data is not yet well-substantiated. Here, we use computational methods to trace signatures of DNA methylation across insects by analyzing transcriptomic and genomic sequence data from all currently recognized insect orders. We conclude that: 1) a functional methylation system relying exclusively on DNA methyltransferase 1 is widespread across insects. 2) DNA methylation has potentially been lost or extremely reduced in species belonging to springtails (Collembola), flies and relatives (Diptera), and twisted-winged parasites (Strepsiptera). 3) Holometabolous insects display signs of reduced DNA methylation levels in protein-coding sequences compared with hemimetabolous insects. 4) Evolutionarily conserved insect genes associated with housekeeping functions tend to display signs of heavier DNA methylation in comparison to the genomic/transcriptomic background. With this comparative study, we provide the much needed basis for experimental and detailed comparative analyses required to gain a deeper understanding on the evolution and function of DNA methylation in insects.

Phylogenetic relationships among tribes of the green lacewing subfamily Chrysopinae recovered based on mitochondrial phylogenomics.

Chrysopidae (green lacewings) is the second largest family in Neuroptera, and it includes medium-size lacewings largely recognized by the presence of golden-colored eyes, bright green bodies and delicate wings with dense venation patterns. The subfamily Chrysopinae includes 97% of the species diversity in the family and it is currently divided into four tribes: Ankylopterygini, Belonopterygini, Chrysopini and Leucochrysini. Here we sequenced and annotated the nearly complete mitochondrial genomes of four species of each these tribes: Abachrysa eureka, Italochrysa insignis, Leucochrysa pretiosa, Parankyloteryx sp. We then reconstructed the phylogenetic relationships with estimated divergence times among tribes of Chrysopinae based on the mt genomic data. Our results suggest that Chrysopinae sans Nothancyla verreauxi evolved as two reciprocally monophyletic lineages formed by stem members of the tribes Leucochrysini plus Belonopterygini on one hand, and the stem members of Ankylopterygini plus Chrysopini on the other. Our estimations of divergence times place the diversification of stem Chrysopinae into the extant tribes during the Middle Jurassic to Late Cretaceous. The relatively young ages previously estimated for the green lacewing divergences were probably underestimated due to false inferences of homology between non-sister taxa that are later correctly identified as homoplasy after more taxa are added.

Cytotaxonomy and molecular phylogeny of the genus Cerapanorpa Gao, Ma & Hua, 2016 (Mecoptera: Panorpidae).

The species of the genus Cerapanorpa Gao, Ma & Hua, 2016 (Mecoptera: Panorpidae) are characterized mainly by the presence of a finger-like anal horn on tergum VI of males and are distributed in the Oriental and eastern Palearctic regions. Herein, we investigated the pachytene banding patterns and reconstructed the Bayesian time-calibrated tree of some species of Cerapanorpa. All species examined display achiasmate meiosis and the same meiformula 2n = 42 + X0, reconfirming the monophyly of Cerapanorpa. The great variations in the size and number of heterochromatic bands suggest that they are reliable traits for species delimitation in Cerapanorpa. The existence of natural C-banding polymorphism indicates that chromosomal rearrangements likely have contributed to the diversification of chromosomal bands in Cerapanorpa. The closely related species of Cerapanorpa are reconfirmed to be evolutionarily independent entities by cytogenetic and molecular data. The divergence time estimated from the BEAST analysis shows that Cerapanorpa likely originated in the period from the Rupelian (30.7 Ma) to the Burdigalian (19.9 Ma), and most diversification occurred from the Burdigalian to the Piacenzian (17.4-2.8 Ma) in the Neogene. Our data suggest that chromosome rearrangements likely play a significant role in the speciation of Cerapanorpa.

A DNA barcode library for Germany's mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera).

Mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera) are prominent representatives of aquatic macroinvertebrates, commonly used as indicator organisms for water quality and ecosystem assessments. However, unambiguous morphological identification of EPT species, especially their immature life stages, is a challenging, yet fundamental task. A comprehensive DNA barcode library based upon taxonomically well-curated specimens is needed to overcome the problematic identification. Once available, this library will support the implementation of fast, cost-efficient and reliable DNA-based identifications and assessments of ecological status. This study represents a major step towards a DNA barcode reference library as it covers for two-thirds of Germany's EPT species including 2,613 individuals belonging to 363 identified species. As such, it provides coverage for 38 of 44 families (86%) and practically all major bioindicator species. DNA barcode compliant sequences (≥500 bp) were recovered from 98.74% of the analysed specimens. Whereas most species (325, i.e., 89.53%) were unambiguously assigned to a single Barcode Index Number (BIN) by its COI sequence, 38 species (18 Ephemeroptera, nine Plecoptera and 11 Trichoptera) were assigned to a total of 89 BINs. Most of these additional BINs formed nearest neighbour clusters, reflecting the discrimination of geographical subclades of a currently recognized species. BIN sharing was uncommon, involving only two species pairs of Ephemeroptera. Interestingly, both maximum pairwise and nearest neighbour distances were substantially higher for Ephemeroptera compared to Plecoptera and Trichoptera, possibly indicating older speciation events, stronger positive selection or faster rate of molecular evolution.

The mitogenome of Hydropsyche pellucidula (Hydropsychidae): first gene arrangement in the insect order Trichoptera.

We describe the mitochondrial genome of Hydropsyche pellucidula Curtis 1834, which is first described for the suborder Annulipalpia and the first in the order Trichoptera to show a non-canonical gene order. The mitogenome was obtained by de novo assembly of shotgun sequenced total genomic DNA using Illumina Miseq technology, which produced an average coverage of 115× and a minimum coverage of 48×. The mitochondrial genome includes 13 protein-coding genes, 2 rRNAs and 22 tRNAs. The genome is characterized by a rearrangement in the relative position of protein-coding and ribosomal genes. This mitogenome sequence will be useful for studying the family Hydropsychidae, which is commonly used for freshwater pollution biomonitoring.

Global map of oxytocin/vasopressin-like neuropeptide signalling in insects.

Oxytocin and vasopressin mediate a range of physiological functions that are important for osmoregulation, reproduction, social behaviour, memory and learning. The origin of this signalling system is thought to date back ~600 million years. Oxytocin/vasopressin-like peptides have been identified in several invertebrate species and they appear to be functionally related across the entire animal kingdom. There is little information available about the biology of this peptide G protein-coupled receptor signalling system in insects. Recently over 200 insect genome/transcriptome datasets were released allowing investigation of the molecular structure and phylogenetic distribution of the insect oxytocin/vasopressin orthologue - inotocin peptides and their receptors. The signalling system is present in early arthropods and representatives of some early-diverging lineages. However, Trichoptera, Lepidoptera, Siphonaptera, Mecoptera and Diptera, lack the presence of inotocin genes, which suggests the peptide-receptor system was probably lost in their common ancestor ~280 million-years-ago. In addition we detected several losses of the inotocin signalling system in Hemiptera (white flies, scale insects and aphids), and the complete absence in spiders (Chelicerata). This unique insight into evolutionarily patterns and sequence diversity of neuroendocrine hormones will provide opportunities to elucidate the physiology of the inotocin signalling system in one of the largest group of animals.

The complete mitochondrial genome of the Neochauliodes fraternus (Megaloptera: Corydalidae).

The mitochondrial genome of Neochauliodes fraternus (Megaloptera: Corydalidae) is a circular molecule of 15,768 bp in length, containing 37 typical mitochondrial genes: 13 protein-coding genes (PCGs), 2 ribosomal RNAs, 22 transfer RNAs and a non-coding AT-rich region. Its gene order and arrangement are identical to the common type found in most insect mitogenomes. All PCGs start with a typical ATN codon except for the ND1 which uses TTG as its start codon; all PCGs terminate in the common stop codon TAA or TAG, except for the COI, COIII, ND3, ND5, ND4 and CYTB which use single T as their stop codons. The non-coding AT-rich region is 1031 bp long, located between rrnS and tRNA(lle) genes. It contains some structures of repeated motifs and microsatellite-like elements characteristic of the megaloptera.