A comparison of dissection and 3D approaches to estimate muscle physiological cross-sectional area, validated by in vivo bite forces.

Performance traits such as bite forces are crucial to fitness and relate to the niche and adaptation of species. However, for many insects it is not possible to directly measure bite forces because they are too small. Biomechanical models of bite forces are therefore relevant to test hypotheses of adaptation in insects and other small organisms. Although such models are based on classical mechanics, combining forces, material properties and laws of levers, it is currently unknown how various models relate to bite forces measured in vivo. One critical component of these models is the physiological cross-sectional area (PCSA) of muscles, which relates to the maximum amount of force they can produce. Here, using the grasshopper Schistocerca gregaria, we compare various ways to obtain PCSA values and use in vivo measurements of bite forces to validate the biomechanical models. We show that most approaches used to derive PCSA (dissection, 3D muscle convex hull volume, muscle attachment area) are consistent with the expected relationships between PCSA and bite force, as well as with the muscle stress values known for insects. The only exception to this are PCSA values estimated by direct 3D muscle volume computation, which could be explained by noisy variation produced by shrinkage. This method therefore produces PCSA values which are uncorrelated to in vivo bite forces. Furthermore, despite the fact that all other methods do not significantly differ from expectations, their derived PCSA values vary widely, suggesting a lack of comparability between studies relying on different methods.

Diversity, Form, and Postembryonic Development of Paleozoic Insects.

While Mesozoic, Paleogene, and Neogene insect faunas greatly resemble the modern one, the Paleozoic fauna provides unique insights into key innovations in insect evolution, such as the origin of wings and modifications of postembryonic development including holometaboly. Deep-divergence estimates suggest that the majority of contemporary insect orders originated in the Late Paleozoic, but these estimates reflect divergences between stem groups of each lineage rather than the later appearance of the crown groups. The fossil record shows the initial radiations of the extant hyperdiverse clades during the Early Permian, as well as the specialized fauna present before the End Permian mass extinction. This review summarizes the recent discoveries related to the documented diversity of Paleozoic hexapods, as well as current knowledge about what has actually been verified from fossil evidence as it relates to postembryonic development and the morphology of different body parts.

Highly accurate long reads are crucial for realizing the potential of biodiversity genomics.

BACKGROUND: Generating the most contiguous, accurate genome assemblies given available sequencing technologies is a long-standing challenge in genome science. With the rise of long-read sequencing, assembly challenges have shifted from merely increasing contiguity to correctly assembling complex, repetitive regions of interest, ideally in a phased manner. At present, researchers largely choose between two types of long read data: longer, but less accurate sequences, or highly accurate, but shorter reads (i.e., >Q20 or 99% accurate). To better understand how these types of long-read data as well as scale of data (i.e., mean length and sequencing depth) influence genome assembly outcomes, we compared genome assemblies for a caddisfly, Hesperophylax magnus, generated with longer, but less accurate, Oxford Nanopore (ONT) R9.4.1 and highly accurate PacBio HiFi (HiFi) data. Next, we expanded this comparison to consider the influence of highly accurate long-read sequence data on genome assemblies across 6750 plant and animal genomes. For this broader comparison, we used HiFi data as a surrogate for highly accurate long-reads broadly as we could identify when they were used from GenBank metadata. RESULTS: HiFi reads outperformed ONT reads in all assembly metrics tested for the caddisfly data set and allowed for accurate assembly of the repetitive ~ 20 Kb H-fibroin gene. Across plants and animals, genome assemblies that incorporated HiFi reads were also more contiguous. For plants, the average HiFi assembly was 501% more contiguous (mean contig N50 = 20.5 Mb) than those generated with any other long-read data (mean contig N50 = 4.1 Mb). For animals, HiFi assemblies were 226% more contiguous (mean contig N50 = 20.9 Mb) versus other long-read assemblies (mean contig N50 = 9.3 Mb). In plants, we also found limited evidence that HiFi may offer a unique solution for overcoming genomic complexity that scales with assembly size. CONCLUSIONS: Highly accurate long-reads generated with HiFi or analogous technologies represent a key tool for maximizing genome assembly quality for a wide swath of plants and animals. This finding is particularly important when resources only allow for one type of sequencing data to be generated. Ultimately, to realize the promise of biodiversity genomics, we call for greater uptake of highly accurate long-reads in future studies.

Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock.

Most organisms possess time-keeping devices called circadian clocks. At the molecular level, circadian clocks consist of transcription-translation feedback loops (TTFLs). Although some components of the negative TTFL are conserved across the animals, important differences exist between typical models, such as mouse and the fruit fly. In Drosophila, the key components are PERIOD (PER) and TIMELESS (TIM-d) proteins, whereas the mammalian clock relies on PER and CRYPTOCHROME (CRY-m). Importantly, how the clock has maintained functionality during evolutionary transitions between different states remains elusive. Therefore, we systematically described the circadian clock gene setup in major bilaterian lineages and identified marked lineage-specific differences in their clock constitution. Then we performed a thorough functional analysis of the linden bug Pyrrhocoris apterus, an insect species comprising features characteristic of both the Drosophila and the mammalian clocks. Unexpectedly, the knockout of timeless-d, a gene essential for the clock ticking in Drosophila, did not compromise rhythmicity in P. apterus, it only accelerated its pace. Furthermore, silencing timeless-m, the ancestral timeless type ubiquitously present across animals, resulted in a mild gradual loss of rhythmicity, supporting its possible participation in the linden bug clock, which is consistent with timeless-m role suggested by research on mammalian models. The dispensability of timeless-d in P. apterus allows drawing a scenario in which the clock has remained functional at each step of transition from an ancestral state to the TIM-d-independent PER + CRY-m system operating in extant vertebrates, including humans.

New insights into immune genes and other expanded gene families of the house fly, Musca domestica, from an improved whole genome sequence.

The house fly, Musca domestica, is a pest of livestock, transmits pathogens of human diseases, and is a model organism in multiple biological research areas. The first house fly genome assembly was published in 2014 and has been of tremendous use to the community of house fly biologists, but that genome is discontiguous and incomplete by contemporary standards. To improve the house fly reference genome, we sequenced, assembled, and annotated the house fly genome using improved techniques and technologies that were not available at the time of the original genome sequencing project. The new genome assembly is substantially more contiguous and complete than the previous genome. The new genome assembly has a scaffold N50 of 12.46 Mb, which is a 50-fold improvement over the previous assembly. In addition, the new genome assembly is within 1% of the estimated genome size based on flow cytometry, whereas the previous assembly was missing nearly one-third of the predicted genome sequence. The improved genome assembly has much more contiguous scaffolds containing large gene families. To provide an example of the benefit of the new genome, we used it to investigate tandemly arrayed immune gene families. The new contiguous assembly of these loci provides a clearer picture of the regulation of the expression of immune genes, and it leads to new insights into the selection pressures that shape their evolution.

Universal fluorescence in situ hybridization (FISH) protocol for mapping repetitive DNAs in insects and other arthropods.

Repetitive DNAs comprise large portion of eukaryote genomes. In genome projects, the assembly of repetitive DNAs is challenging due to the similarity between repeats, which generate ambiguities for alignment. Fluorescence in situ hybridization (FISH) is a powerful technique for the physical mapping of various sequences on chromosomes. This technique is thus very helpful in chromosome-based genome assemblies, providing information on the fine architecture of genomes and their evolution. However, various protocols are currently used for FISH mapping, most of which are relatively laborious and expensive, or work properly only with a specific type of probes or sequences, and there is a need for a universal and affordable FISH protocol. Here we tested a FISH protocol for mapping of different DNA repeats, such as multigene families (rDNAs, U snDNAs, histone genes), satellite DNAs, microsatellites, transposable elements, DOP-PCR products, and telomeric motif (TTAGG)n, on the chromosomes of various insects and other arthropods. Different cell types and stages obtained from diverse tissues were used. The FISH procedure proved high quality and reliable results in all experiments performed. We obtained data on the chromosomal distribution of DNA repeats in representatives of insects and other arthropods. Thus, our results allow us to conclude that the protocol is universal and requires only time adjustment for chromosome/DNA denaturation. The use of this FISH protocol will facilitate studies focused on understanding the evolution and role of repetitive DNA in arthropod genomes.

Filling in the gaps: A reevaluation of the Lygus hesperus peptidome using an expanded de novo assembled transcriptome and molecular cloning.

Peptides are the largest and most diverse class of molecules modulating physiology and behavior. Previously, we predicted a peptidome for the western tarnished plant bug, Lygus hesperus, using transcriptomic data produced from whole individuals. A potential limitation of that analysis was the masking of underrepresented genes, in particular tissue-specific transcripts. Here, we reassessed the L. hesperus peptidome using a more comprehensive dataset comprised of the previous transcriptomic data as well as tissue-specific reads produced from heads and accessory glands. This augmented assembly significantly improves coverage depth providing confirmatory transcripts for essentially all of the previously identified families and new transcripts encoding a number of new peptide precursors corresponding to 14 peptide families. Several families not targeted in our initial study were identified in the expanded assembly, including agatoxin-like peptide, CNMamide, neuropeptide-like precursor 1, and periviscerokinin. To increase confidence in the in silico data, open reading frames of a subset of the newly identified transcripts were amplified using RT-PCR and sequence validated. Further PCR-based profiling of the putative L. hesperus agatoxin-like peptide precursor revealed evidence of alternative splicing with near ubiquitous expression across L. hesperus development, suggesting the peptide serves functional roles beyond that of a toxin. The peptides predicted here, in combination with those identified in our earlier study, expand the L. hesperus peptidome to 42 family members and provide an improved platform for initiating molecular and physiological investigations into peptidergic functionality in this non-model agricultural pest.

Multi-faceted analysis provides little evidence for recurrent whole-genome duplications during hexapod evolution.

BACKGROUND: Gene duplication events play an important role in the evolution and adaptation of organisms. Duplicated genes can arise through different mechanisms, including whole-genome duplications (WGDs). Recently, WGD was suggested to be an important driver of evolution, also in hexapod animals. RESULTS: Here, we analyzed 20 high-quality hexapod genomes using whole-paranome distributions of estimated synonymous distances (KS), patterns of within-genome co-linearity, and phylogenomic gene tree-species tree reconciliation methods. We observe an abundance of gene duplicates in the majority of these hexapod genomes, yet we find little evidence for WGD. The majority of gene duplicates seem to have originated through small-scale gene duplication processes. We did detect segmental duplications in six genomes, but these lacked the within-genome co-linearity signature typically associated with WGD, and the age of these duplications did not coincide with particular peaks in KS distributions. Furthermore, statistical gene tree-species tree reconciliation failed to support all but one of the previously hypothesized WGDs. CONCLUSIONS: Our analyses therefore provide very limited evidence for WGD having played a significant role in the evolution of hexapods and suggest that alternative mechanisms drive gene duplication events in this group of animals. For instance, we propose that, along with small-scale gene duplication events, episodes of increased transposable element activity could have been an important source for gene duplicates in hexapods.

Structural colours in diverse Mesozoic insects.

Structural colours, nature's most pure and intense colours, originate when light is scattered via nanoscale modulations of the refractive index. Original colours in fossils illuminate the ecological interactions among extinct organisms and functional evolution of colours. Here, we report multiple examples of vivid metallic colours in diverse insects from mid-Cretaceous amber. Scanning and transmission electron microscopy revealed a smooth outer surface and five alternating electron-dense and electron-lucent layers in the epicuticle of a fossil wasp, suggesting that multilayer reflectors, the most common biophotonic nanostructure in animals and even plants, are responsible for the exceptional preservation of colour in amber fossils. Based on theoretical modelling of the reflectance spectra, a reflective peak of wavelength of 514 nm was calculated, corresponding to the bluish-green colour observed under white light. The green to blue structural colours in fossil wasps, beetles and a fly most likely functioned as camouflage, although other functions such as thermoregulation cannot be ruled out. This discovery not only provides critical evidence of evolution of structural colours in arthropods, but also sheds light on the preservation potential of nanostructures of ancient animals through geological time.

Unusual mechanism of emission of vibratory signals in pygmy grasshoppers Tetrix tenuicornis (Sahlberg, 1891) (Orthoptera: Tetrigidae).

Acoustic communication plays an important role in the life of insects and especially in representatives of the order Orthoptera. Their vibrational signalling, unlike signalling by sound, is poorly studied. The pygmy grasshoppers Tetrix tenuicornis (Sahlberg, 1891) belonging to the ancestral family Tetrigidae (Orthoptera) can produce several types of substrate-borne vibratory signals using their mid-legs. The emission of these signals is not accompanied by visible movements of any parts of the body. The goal of our study was to elucidate the mechanism of production of these vibrations. For this, we synchronously recorded the vibratory signals and the muscle activity in various regions of the legs and thorax in freely moving males. The obtained results revealed an unusual mechanism for the emission of acoustic signals. We found that the strongest muscle activity during the emission of the vibratory signals was recorded in the mesofemur and mesotibia. According to the position of the electrode, these muscles are the flexor and extensor of the tibia, levators and depressors of the tarsus, and probably pretarsus. The motor system employed during the emission of vibratory signals was most similar to that of the jump of locusts and probably is performed as a result of co-contraction of antagonistic muscles of the tibia, tarsus, and pretarsus. The data obtained make significant additions to the presentation of a variety of insect acoustic communication systems.

Gastric canthariasis caused by invasion of mealworm beetle larvae in weaned pigs in large-scale farming.

BACKGROUND: Mealworm beetle T. molitor (Coleoptera: Tenebrionidae) (Linnaeus, 1758) is one of the most important cosmopolitan primary storage pests, scavenging on a variety of post-harvest grains and affecting the quality and safety of food and feed. In addition to being an important factor in feed hygiene, the insect can also be an epidemiological factor of canthariasis. Livestock infestations with T. molitor are rarely reported. This article describes T. molitor-caused canthariasis in pigs in large scale closed-cycle farming. RESULTS: In the spring, we registered a significantly increased mortality among weaned pigs. In autopsy, live 3-6 mm long T. molitor larvae were found in their stomachs, especially in the non-glandular oesophageal region, on average 2-3 larvae per 10 cm2 of gastric mucosa. Corrective actions reduced the number of deaths back to basal levels. CONCLUSIONS: This is the first documented case of potentially lethal gastric canthariasis in weaned pigs, caused by invasion of T. molitor larvae. Although canthariasis caused by T. molitor has not been a significant problem in farm animals so far, our case indicates that the presence of mealworm beetles is a potential threat to animal welfare and health.

RNA virome screening in diverse but ecologically related citrus pests reveals potential virus-host interactions.

As an evergreen ecosystem, citrus orchards have specialized pest species and stable ecological homeostasis; thus, they provide an ideal model for investigating RNA viromes in diverse but ecologically related species. For this purpose, we collected specialized citrus pests from three classes of invertebrates, Insecta, Arachnida, and Gastropoda and we constructed two kinds of libraries (RNA and small RNA) for the pests by deep sequencing. In total, six virus-derived sequences were identified, including four Picornavirales, one Jingchuvirales and one Nidovirales. The picornavirus-derived small RNAs showed significant small RNA peaks and symmetric distribution patterns along the genome, which suggests these viruses infected the hosts and triggered host antiviral immunity RNA interference. Screening of virus-derived sequences in multiple species of citrus pests (n = 10 per species) showed that Eotetranychus kankitus picorna-like virus and Tetranychus urticae mivirus may be present in multiple pests. Our investigation in citrus pests confirmed that RNA viruses revealed by metagenomics could impact host immunity (e.g. RNAi). An approach with parallel deep sequencing of RNAs and small RNAs is useful not only for viral discoveries but also for understanding virus-host interactions of ecologically related but divergent pest species.

Multispecies coalescent analysis confirms standing phylogenetic instability in Hexapoda.

The multispecies coalescent (MSC) has been increasingly used in phylogenomic analyses due to the accommodation of gene tree topological heterogeneity by taking into account population-level processes, such as incomplete lineage sorting. In this sense, the phylogeny of insect species, which are characterized by their large effective population sizes, is suitable for a coalescent-based analysis. Furthermore, studies so far recovered short internal branches at early divergences of the insect tree of life, indicating fast evolutionary radiations that increase the probability of incomplete lineage sorting in deep time. Here, we investigated the performance of the MSC for a phylogenomic data set of hexapods compiled by Misof et al. (2014, Science 346:763). Our analysis recovered the monophyly of most insect orders, and major phylogenetic relationships were in agreement with current insect systematics. We identified, however, some evolutionary associations that were consistently problematic. Most noticeable, Hexapod monophyly was disrupted by the sister group relationship between the remiped crustacean and Insecta. Additionally, the interordinal relationships within Polyneoptera and Neuropteroidea were found to be phylogenetically unstable. We show that these controversial phylogenetic arrangements were also poorly supported by previous analyses, and therefore, we evaluated their robustness to stochastic errors from sampling sites and terminals, confirming standing problems in hexapod phylogeny in the genomics age.

Using genomic data to study insecticide resistance in the house fly, Musca domestica.

The house fly, Musca domestica, is a major pest at livestock facilities throughout the world. Insecticides have been the most common control strategy for flies, but many populations have evolved resistance. The speed by which we are able to identify the mutations responsible for resistance has been a major challenge for the development of high throughput resistance monitoring assays as new insecticides are introduced for control. This is particularly true for mutations that cause trans regulation of a gene, which then results in resistance. In this paper we take advantage of the conserved homology of dipteran chromosomes to assign 3069 genes to chromosomes. Of these, 234 were of toxicological interest (CYPs, esterases/hydrolases, glutathione S-transferases (GSTs) and target sites). The chromosomal location of genes known from linkage analysis studies matched the location predicted by homology mapping in ten out of ten cases, indicating a high reliability of our approach. The CYPs, esterases/hydrolases and GSTs were not randomly distributed throughout the genome. They clustered on chromosomes, but the pattern was different between the CYPs, esterases/hydrolases and GSTs. Examples are provided for how the availability of the house fly genome, combined with an ability to assign genes to chromosomes, will help to accelerate research in house flies.

Redefining the extinct orders Miomoptera and Hypoperlida as stem acercarian insects.

BACKGROUND: The systematic positions of the extinct insect orders Hypoperlida, Miomoptera and Permopsocida were enigmatic and unstable for nearly a century. The recent studies based on new material, especially from the Cenomanian Burmese amber, shed light on evolutionary history of Acercaria resolving Permopsocida as the stem group of Condylognatha. However, the knowledge of the remaining two orders differs significantly. RESULTS: In this study, we describe new specimens and evaluate morphology of various structures with emphasis on the mouthparts and wing venation. Our results are primary based on revisions of the type specimens with a proper delimitation of taxa Hypoperlida and Miomoptera followed by their significance for the evolutionary history of Acercaria. Three new genera as Belmomantis gen. nov., Elmomantis gen. nov., and Mazonopsocus gen. nov. are designated as members of Palaeomanteidae. The Pennsylvanian Mazonopsocus provides a minimum age for calibration, in accordance to the presence of crown acercarians during the late Carboniferous. CONCLUSIONS: This contribution demonstrates that Hypoperlida and Miomoptera are stem groups of Acercaria. The putative clade (Hypoperlida + Miomoptera) is appearing as potential sister group of (Psocodea + (Permopsocida + (Thripida + Hemiptera))).

The first report of gynandromorphy in termites (Isoptera; Kalotermitidae; Neotermes koshunensis).

This is the first report of gynandromorphy in Isoptera. An Asian dry-wood termite, Neotermes koshunensis (Shiraki) [Kalotermitidae], possessing both male and female phenotypic characteristics, was found on Okinawa Island, Japan. This deformed individual showed morphological and anatomical hermaphroditism in the abdomen. The right side of the seventh sternite was the female form and contained an ovary, while the left side was the male form and contained a testis. Genotypic analysis revealed that this individual was a genotypic bilateral chimera. These results suggested that the termite was a bilateral gynandromorph with a male left side and a female right side. As reported previously in other insects, double fertilization (by two sperms, one with an X and one with a Y chromosome) of a binucleate egg is the most likely mechanism that generated this genotypic bilateral chimera. N. koshunensis has the ability to reproduce through parthenogenesis, in which the secondary polar body is likely to be used for nuclear phase recovery. If the second polar body in this mechanism has high fertility and healthy embryogenic potential, like an egg nucleus, some of gynandromorphs might be produced by a side effect of parthenogenetic ability.

Ancient origin of high taxonomic richness among insects.

Insects are a hyper-diverse group, comprising nearly three-quarters of all named animal species on the Earth, but the environmental drivers of their richness and the roles of ecological interactions and evolutionary innovations remain unclear. Previous studies have argued that family-level insect richness increased continuously over the evolutionary history of the group, but inclusion of extant family records artificially inflated the relative richness of younger time intervals. Here we apply sampling-standardization methods to a species-level database of fossil insect occurrences, removing biases present in previous richness curves. We show that insect family-richness peaked 125 Ma and that Recent values are only 1.5-3 times as high as the Late Palaeozoic. Rarefied species-richness data also tentatively suggest little or no net increase in richness over the past 125 Myr. The Cretaceous peak in family richness was coincident with major radiations within extant groups but occurred prior to extinctions within more basal groups. Those extinctions may in part be linked to mid-Cretaceous floral turnover following the evolution of flowering plants. Negligible net richness change over the past 125 Myr implies that major radiations within extant groups were offset by reduced richness within groups that are now relict or extinct.

First protein and peptide characterization of the tarsal adhesive secretions in the desert locust, Schistocerca gregaria, and the Madagascar hissing cockroach, Gromphadorhina portentosa.

Peptides and proteins have been largely neglected in the analysis of insect tarsal adhesives. After extraction of the protein fraction of the tarsal secretion of the desert locust, Schistocerca gregaria, and Madagascar hissing cockroach, Gromphadorhina portentosa, we combined Fourier transform infrared spectroscopy (FTIR), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) analyses for protein mass detection. In both these insects, SDS-PAGE analysis revealed several protein bands ranging from 8-190 kDa in both the tarsal secretion and the tibia control sample. Two (S. gregaria) and one (G. portentosa) protein bands exclusively occurred in the tarsal secretion and can be considered to belong to peptides and proteins specific to this secretion. MALDI-TOF analyses revealed 83 different proteins/peptides of 1-7 kDa in S. gregaria, and 48 of 1-11 kDa in G. portentosa. 59 (S. gregaria) and 27 (G. portentosa) proteins exclusively occurred in the tarsal secretion. In G. portentosa, a characteristic series of signal peaks occurred in the range of c. 10-12 kDa, each peak being approximately 160 Da apart. Such a pattern is indicative of proteins modified by glycosylation. Our approach demonstrates that extensive sampling involving considerable time and manpower to sample the adhesive fluid directly from the tarsi opens up a perspective for extracting peptides and proteins in sufficient quantities. This makes them accessible to the field of proteomics and thus to elucidate their possible function in the adhesive process.

Rates and patterns of molecular evolution in freshwater versus terrestrial insects.

Insect lineages have crossed between terrestrial and aquatic habitats many times, for both immature and adult life stages. We explore patterns in molecular evolutionary rates between 42 sister pairs of related terrestrial and freshwater insect clades using publicly available protein-coding DNA sequence data from the orders Coleoptera, Diptera, Lepidoptera, Hemiptera, Mecoptera, Trichoptera, and Neuroptera. We furthermore test for habitat-associated convergent molecular evolution in the cytochrome c oxidase subunit I (COI) gene in general and at a particular amino acid site previously reported to exhibit habitat-linked convergence within an aquatic beetle group. While ratios of nonsynonymous-to-synonymous substitutions across available loci were higher in terrestrial than freshwater-associated taxa in 26 of 42 lineage pairs, a stronger trend was observed (20 of 31, pbinomial = 0.15, pWilcoxon = 0.017) when examining only terrestrial-aquatic pairs including fully aquatic taxa. We did not observe any widespread changes at particular amino acid sites in COI associated with habitat shifts, although there may be general differences in selection regime linked to habitat.