Genome assembly of the southern pine beetle ( Dendroctonus frontalis Zimmerman) reveals the origins of gene content reduction in Dendroctonus.

Dendroctonus frontalis , also known as southern pine beetle (SPB), represents the most damaging forest pest in the southeastern United States. Strategies to predict, monitor and suppress SPB outbreaks have had limited success. Genomic data are critical to inform on pest biology and to identify molecular targets to develop improved management approaches. Here, we produced a chromosome-level genome assembly of SPB using long-read sequencing data. Synteny analyses confirmed the conservation of the core coleopteran Stevens elements and validated the bona fide SPB X chromosome. Transcriptomic data were used to obtain 39,588 transcripts corresponding to 13,354 putative protein-coding loci. Comparative analyses of gene content across 14 beetle and 3 other insects revealed several losses of conserved genes in the Dendroctonus clade and gene gains in SPB and Dendroctonus that were enriched for loci encoding membrane proteins and extracellular matrix proteins. While lineage-specific gene losses contributed to the gene content reduction observed in Dendroctonus , we also showed that widespread misannotation of transposable elements represents a major cause of the apparent gene expansion in several non- Dendroctonus species. Our findings uncovered distinctive features of the SPB gene complement and disentangled the role of biological and annotation-related factors contributing to gene content variation across beetles.

A reference quality genome assembly for the Jewel scarab Chrysina gloriosa.

The jewel scarab Chrysina gloriosa is one of the most charismatic beetles in the United States and is found from the mountains of West Texas to the Southeastern Arizona sky islands. This species is highly sought by professional and amateur collectors worldwide due to its gleaming metallic coloration. However, the impact of the large-scale collection of this beetle on its populations is unknown, and there is a limited amount of genetic information available to make informed decisions about its conservation. As a first step, we present the genome of C. gloriosa, which we reconstructed using a single female specimen sampled from our ongoing effort to document population connectivity and the demographic history of this beetle. Using a combination of long-read sequencing and Omni-C data, we reconstructed the C. gloriosa genome at a near-chromosome level. Our genome assembly consisted of 454 scaffolds spanning 642 MB, with the ten largest scaffolds capturing 98% of the genome. The scaffold N50 was 72 MB, and the BUSCO score was 95.5%. This genome assembly will be an essential tool to accelerate understanding C. gloriosa biology and help make informed decisions for the conservation of Chrysina and other species with similar distributions in this region. This genome assembly will further serve as a community resource for comparative genomic analysis.

A High-Quality Blue Whale Genome, Segmental Duplications, and Historical Demography.

The blue whale, Balaenoptera musculus, is the largest animal known to have ever existed, making it an important case study in longevity and resistance to cancer. To further this and other blue whale-related research, we report a reference-quality, long-read-based genome assembly of this fascinating species. We assembled the genome from PacBio long reads and utilized Illumina/10×, optical maps, and Hi-C data for scaffolding, polishing, and manual curation. We also provided long read RNA-seq data to facilitate the annotation of the assembly by NCBI and Ensembl. Additionally, we annotated both haplotypes using TOGA and measured the genome size by flow cytometry. We then compared the blue whale genome with other cetaceans and artiodactyls, including vaquita (Phocoena sinus), the world's smallest cetacean, to investigate blue whale's unique biological traits. We found a dramatic amplification of several genes in the blue whale genome resulting from a recent burst in segmental duplications, though the possible connection between this amplification and giant body size requires further study. We also discovered sites in the insulin-like growth factor-1 gene correlated with body size in cetaceans. Finally, using our assembly to examine the heterozygosity and historical demography of Pacific and Atlantic blue whale populations, we found that the genomes of both populations are highly heterozygous and that their genetic isolation dates to the last interglacial period. Taken together, these results indicate how a high-quality, annotated blue whale genome will serve as an important resource for biology, evolution, and conservation research.

The Genome of the Yellow Mealworm, Tenebrio molitor: It's Bigger Than You Think.

BACKGROUND: Insects are a sustainable source of protein for human food and animal feed. We present a genome assembly, CRISPR gene editing, and life stage-specific transcriptomes for the yellow mealworm, Tenebrio molitor, one of the most intensively farmed insects worldwide. METHODS: Long and short reads and long-range data were obtained from a T. molitor male pupa. Sequencing transcripts from 12 T. molitor life stages resulted in 279 million reads for gene prediction and genetic engineering. A unique plasmid delivery system containing guide RNAs targeting the eye color gene vermilion flanking the muscle actin gene promoter and EGFP marker was used in CRISPR/Cas9 transformation. RESULTS: The assembly is approximately 53% of the genome size of 756.8 ± 9.6 Mb, measured using flow cytometry. Assembly was complicated by a satellitome of at least 11 highly conserved satDNAs occupying 28% of the genome. The injection of the plasmid into embryos resulted in knock-out of Tm vermilion and knock-in of EGFP. CONCLUSIONS: The genome of T. molitor is longer than current assemblies (including ours) due to a substantial amount (26.5%) of only one highly abundant satellite DNA sequence. Genetic sequences and transformation tools for an insect important to the food and feed industries will promote the sustainable utilization of mealworms and other farmed insects.

A chromosome scale assembly of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), genome.

OBJECTIVE: The tarnished plant bug (TPB), Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), is a pest damaging many cultivated crops in North America. Although partial transcriptome data are available for this pest, a genome assembly was not available for this species. This assembly of a high-quality chromosome-length genome of TPB is aimed to develop the genetic resources that can provide the foundation required for advancing research on this species. RESULTS: The initial genome of TPB assembled with paired-end nucleotide sequences generated with Illumina technology was scaffolded with Illumina HiseqX reads generated from a proximity ligated (HiC) library to obtain a high-quality genome assembly. The final assembly contained 3963 scaffolds longer than 1 kbp to yield a genome of 599.96 Mbp. The N50 of the TPB genome assembly was 35.64 Mbp and 98.68% of the genome was assembled into 17 scaffolds larger than 1 Mbp. This megabase scaffold number is the same as the number of chromosomes observed in karyotyping of this insect. The TPB genome is known to have high repetitive DNA content, and the reduced assembled genome size compared to flowcytometric estimates of approximately 860 Mbp may be due to the collapsed assembly of highly similar regions.

Genome and Genetic Engineering of the House Cricket (Acheta domesticus): A Resource for Sustainable Agriculture.

Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics.

Differential Gene Expression of Innate Immune Response Genes Consequent to Solenopsis invicta Virus-3 Infection

The red imported fire ant Solenopsis invicta Buren (fire ant hereafter) is a global pest that inflicts billions of dollars in damages to the United States economy and poses a major threat on a global scale. Concerns with the broad-spectrum application of insecticides have facilitated the hunt for natural enemy-mediated controls. One of these, the virus Solenopsis invicta virus-3 (SINV-3 hereafter) is exceptionally virulent in laboratory settings. However, despite high mortality rates in the laboratory and documented widespread SINV-3 prevalence in the southern United States, the fire ant remains a major pest. To explore this paradox, we document the immune response elicited by the fire ant when infected with SINV-3. We sequence the fire ant transcriptome prior to and following infection with SINV-3, and identify and discuss in detail genes in immune response pathways differentially expressed following infection with SINV-3. This information provides insights into genes and pathways involved in the SINV-3 infection response in the fire ant and offers avenues to pursue, to suppress key immune response genes and force the fire ant to succumb to SINV-3 infection in the field.

Genomic diversification of the specialized parasite of the fungus-growing ant symbiosis.

Fungi shape the diversity of life. Characterizing the evolution of fungi is critical to understanding symbiotic associations across kingdoms. In this study, we investigate the genomic and metabolomic diversity of the genus Escovopsis, a specialized parasite of fungus-growing ant gardens. Based on 25 high-quality draft genomes, we show that Escovopsis forms a monophyletic group arising from a mycoparasitic fungal ancestor 61.82 million years ago (Mya). Across the evolutionary history of fungus-growing ants, the dates of origin of most clades of Escovopsis correspond to the dates of origin of the fungus-growing ants whose gardens they parasitize. We reveal that genome reduction, determined by both genomic sequencing and flow cytometry, is a consistent feature across the genus Escovopsis, largely occurring in coding regions, specifically in the form of gene loss and reductions in copy numbers of genes. All functional gene categories have reduced copy numbers, but resistance and virulence genes maintain functional diversity. Biosynthetic gene clusters (BGCs) contribute to phylogenetic differences among Escovopsis spp., and sister taxa in the Hypocreaceae. The phylogenetic patterns of co-diversification among BGCs are similarly exhibited across mass spectrometry analyses of the metabolomes of Escovopsis and their sister taxa. Taken together, our results indicate that Escovopsis spp. evolved unique genomic repertoires to specialize on the fungus-growing ant-microbe symbiosis.

The Genomic Basis of Evolutionary Novelties in a Leafhopper.

Evolutionary innovations generate phenotypic and species diversity. Elucidating the genomic processes underlying such innovations is central to understanding biodiversity. In this study, we addressed the genomic basis of evolutionary novelties in the glassy-winged sharpshooter (Homalodisca vitripennis, GWSS), an agricultural pest. Prominent evolutionary innovations in leafhoppers include brochosomes, proteinaceous structures that are excreted and used to coat the body, and obligate symbiotic associations with two bacterial types that reside within cytoplasm of distinctive cell types. Using PacBio long-read sequencing and Dovetail Omni-C technology, we generated a chromosome-level genome assembly for the GWSS and then validated the assembly using flow cytometry and karyotyping. Additional transcriptomic and proteomic data were used to identify novel genes that underlie brochosome production. We found that brochosome-associated genes include novel gene families that have diversified through tandem duplications. We also identified the locations of genes involved in interactions with bacterial symbionts. Ancestors of the GWSS acquired bacterial genes through horizontal gene transfer (HGT), and these genes appear to contribute to symbiont support. Using a phylogenomics approach, we inferred HGT sources and timing. We found that some HGT events date to the common ancestor of the hemipteran suborder Auchenorrhyncha, representing some of the oldest known examples of HGT in animals. Overall, we show that evolutionary novelties in leafhoppers are generated by the combination of acquiring novel genes, produced both de novo and through tandem duplication, acquiring new symbiotic associations that enable use of novel diets and niches, and recruiting foreign genes to support symbionts and enhance herbivory.

High-quality, chromosome-scale genome assemblies: comparisons of three Diaphorina citri (Asian citrus psyllid) geographic populations.

The Asian citrus psyllid, Diaphorina citri, is the insect vector of the causal agent of huanglongbing (HLB), a devastating bacterial disease of commercial citrus. Presently, few genomic resources exist for D. citri. In this study, we utilized PacBio HiFi and chromatin confirmation contact (Hi-C) sequencing to sequence, assemble, and compare three high-quality, chromosome-scale genome assemblies of D. citri collected from California, Taiwan, and Uruguay. Our assemblies had final sizes of 282.67 Mb (California), 282.89 Mb (Taiwan), and 266.67 Mb (Uruguay) assembled into 13 pseudomolecules-a reduction in assembly size of 41-45% compared with previous assemblies which we validated using flow cytometry. We identified the X chromosome in D. citri and annotated each assembly for repetitive elements, protein-coding genes, transfer RNAs, ribosomal RNAs, piwi-interacting RNA clusters, and endogenous viral elements. Between 19,083 and 20,357 protein-coding genes were predicted. Repetitive DNA accounts for 36.87-38.26% of each assembly. Comparative analyses and mitochondrial haplotype networks suggest that Taiwan and Uruguay D. citri are more closely related, while California D. citri are closely related to Florida D. citri. These high-quality, chromosome-scale assemblies provide new genomic resources to researchers to further D. citri and HLB research.

The Spruce Budworm Genome: Reconstructing the Evolutionary History of Antifreeze Proteins.

Insects have developed various adaptations to survive harsh winter conditions. Among freeze-intolerant species, some produce "antifreeze proteins" (AFPs) that bind to nascent ice crystals and inhibit further ice growth. Such is the case of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), a destructive North American conifer pest that can withstand temperatures below -30°C. Despite the potential importance of AFPs in the adaptive diversification of Choristoneura, genomic tools to explore their origins have until now been limited. Here we present a chromosome-scale genome assembly for C. fumiferana, which we used to conduct comparative genomic analyses aimed at reconstructing the evolutionary history of tortricid AFPs. The budworm genome features 16 genes homologous to previously reported C. fumiferana AFPs (CfAFPs), 15 of which map to a single region on chromosome 18. Fourteen of these were also detected in five congeneric species, indicating Choristoneura AFP diversification occurred before the speciation event that led to C. fumiferana. Although budworm AFPs were previously considered unique to the genus Choristoneura, a search for homologs targeting recently sequenced tortricid genomes identified seven CfAFP-like genes in the distantly related Notocelia uddmanniana. High structural similarity between Notocelia and Choristoneura AFPs suggests a common origin, despite the absence of homologs in three related tortricids. Interestingly, one Notocelia AFP formed the C-terminus of a "zonadhesin-like" protein, possibly representing the ancestral condition from which tortricid AFPs evolved. Future work should clarify the evolutionary path of AFPs between Notocelia and Choristoneura and assess the role of the "zonadhesin-like" protein as precursor of tortricid AFPs.

Spodoptera littoralis genome mining brings insights on the dynamic of expansion of gustatory receptors in polyphagous noctuidae.

The bitter taste, triggered via gustatory receptors, serves as an important natural defense against the ingestion of poisonous foods in animals, and the increased host breadth is usually linked to an increase in the number of gustatory receptor genes. This has been especially observed in polyphagous insect species, such as noctuid species from the Spodoptera genus. However, the dynamic and physical mechanisms leading to these gene expansions and the evolutionary pressures behind them remain elusive. Among major drivers of genome dynamics are the transposable elements but, surprisingly, their potential role in insect gustatory receptor expansion has not been considered yet. In this work, we hypothesized that transposable elements and possibly positive selection would be involved in the highly dynamic evolution of gustatory receptor in Spodoptera spp. We first sequenced de novo the full 465 Mb genome of S. littoralis, and manually annotated the main chemosensory genes, including a large repertoire of 373 gustatory receptor genes (including 19 pseudogenes). We also improved the completeness of S. frugiperda and S. litura gustatory receptor gene repertoires. Then, we annotated transposable elements and revealed that a particular category of class I retrotransposons, the SINE transposons, was significantly enriched in the vicinity of gustatory receptor gene clusters, suggesting a transposon-mediated mechanism for the formation of these clusters. Selection pressure analyses indicated that positive selection within the gustatory receptor gene family is cryptic, only 7 receptors being identified as positively selected. Altogether, our data provide a new good quality Spodoptera genome, pinpoint interesting gustatory receptor candidates for further functional studies and bring valuable genomic information on the mechanisms of gustatory receptor expansions in polyphagous insect species.

The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success.

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N50 of 53.6 Mb, and L50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research.

Special Issue: Genetic Basis of Phenotypic Variation in Drosophila and Other Insects.

Next-generation sequencing provides a nearly complete genomic sequence for model and non-model species alike; however, this wealth of sequence data includes no road map [...].

Population genomics and phylogeography of the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), in the United States, northern Mexico, and Argentina.

The boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), is an important pest of commercial cotton across the Americas. In the United States, eradication of this species is complicated by re-infestations of areas where eradication has been previously successful and by the existence of morphologically similar variants that can confound identification efforts. To date, no study has applied a high-throughput sequencing approach to better understand the population genetic structure of the boll weevil. Furthermore, only a single study has investigated genetic relationships between populations in North and South America. We used double digest restriction site-associated DNA sequencing (ddRADseq) to resolve the population genomic structure of the boll weevil in the southern United States, northern Mexico, and Argentina. Additionally, we assembled the first complete mitochondrial genome for this species and generated a preliminary whole genome assembly, both of which were used to improve the identification of informative loci. Downstream analyses revealed two main lineages-one consisting of populations found geographically west of the Sierra Madre Occidental mountain range and the second consisting of populations found to the east-were revealed, and both were sub-structured. Population geographic structure was consistent with the isolation by distance model, indicating that geogrpahic distance is likely a primary mechanism driving divergence in this species. Boll weevil populations from Argentina were found to be more closely related to the eastern lineage, suggesting a recent colonization of South America by the eastern lineage, but additional sampling across Mexico, Central America and South America is needed to further clarify their origin. Finally, we uncovered an instance of population turnover or replacement, highlighting the temporal instability of population structure.

Sequencing, assembly and annotation of the whole-insect genome of Lymantria dispar dispar, the European gypsy moth.

The European gypsy moth, Lymantria dispar dispar (LDD), is an invasive insect and a threat to urban trees, forests and forest-related industries in North America. For use as a comparator with a previously published genome based on the LD652 pupal ovary-derived cell line, as well as whole-insect genome sequences obtained from the Asian gypsy moth subspecies L. dispar asiatica and L. dispar japonica, the whole-insect LDD genome was sequenced, assembled and annotated. The resulting assembly was 998 Mb in size, with a contig N50 of 662 Kb and a GC content of 38.8%. Long interspersed nuclear elements constitute 25.4% of the whole-insect genome, and a total of 11,901 genes predicted by automated gene finding encoded proteins exhibiting homology with reference sequences in the NCBI NR and/or UniProtKB databases at the most stringent similarity cutoff level (i.e., the gold tier). These results will be especially useful in developing a better understanding of the biology and population genetics of L. dispar and the genetic features underlying Lepidoptera in general.

Comparison of long-read sequencing technologies in interrogating bacteria and fly genomes.

The newest generation of DNA sequencing technology is highlighted by the ability to generate sequence reads hundreds of kilobases in length. Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. We used whole-genome sequencing data produced by three PacBio protocols (Sequel II CLR, Sequel II HiFi, RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. In both organisms tested, Sequel II assemblies had the highest consensus accuracy, even after accounting for differences in sequencing throughput. ONT and PacBio CLR had the longest reads sequenced compared to PacBio RS II and HiFi, and genome contiguity was highest when assembling these datasets. ONT Rapid Sequencing libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assembly or polishing eukaryotic genome assemblies, and an ONT-Illumina hybrid approach would be more cost-effective for many users. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs. The ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

Whole-genome assembly of Culex tarsalis.

The mosquito, Culex tarsalis, is a key vector in the western United States due to its role in transmission of zoonotic arboviruses that affect human health. Extensive research has been conducted on Cx. tarsalis ecology, feeding behavior, vector competence, autogeny, diapause, genetics, and insecticide resistance. Population genetic analyses in the western U.S. have identified at least three genetic clusters that are geographically distinct. However, in-depth genetic studies have been hindered by the lack of a reference genome. In this study, we present the first whole-genome assembly of this mosquito species (CtarK1) based on PacBio HiFi reads from high-molecular-weight DNA extracted from a single male. The CtarK1 assembly is 790 Mb with an N50 of 58 kb, which is 27% larger than Culex quinquefasciatus (578 Mb). This difference appears to be mostly composed of transposable elements. To annotate CtarK1, we used a previously assembled Cx. tarsalis transcriptome and approximately 17,456 protein genes from Cx. quinquefasciatus (N = 17,456). Genome completeness was assessed using the Benchmarking Universal Single-Copy Orthologs (BUSCO) tool, which identified 84.8% of the 2799 Dipteran BUSCO genes. Using a Bayesian phylogeny based on mitochondrial genomes, we place Cx. tarsalis in the context of other mosquito species and estimate the divergence between Cx. tarsalis and Cx. quinquefasciatus to be between 15.8 and 22.2 million years ago (MYA). Important next steps from this work include characterizing the genetic basis of diapause and sex determination in Culex mosquitoes.

Lineage-specific patterns of chromosome evolution are the rule not the exception in Polyneoptera insects.

The structure of a genome can be described at its simplest by the number of chromosomes and the sex chromosome system it contains. Despite over a century of study, the evolution of genome structure on this scale remains recalcitrant to broad generalizations that can be applied across clades. To address this issue, we have assembled a dataset of 823 karyotypes from the insect group Polyneoptera. This group contains orders with a range of variations in chromosome number, and offer the opportunity to explore the possible causes of these differences. We have analysed these data using both phylogenetic and taxonomic approaches. Our analysis allows us to assess the importance of rates of evolution, phylogenetic history, sex chromosome systems, parthenogenesis and genome size on variation in chromosome number within clades. We find that fusions play a key role in the origin of new sex chromosomes, and that orders exhibit striking differences in rates of fusions, fissions and polyploidy. Our results suggest that the difficulty in finding consistent rules that govern evolution at this scale may be due to the presence of many interacting forces that can lead to variation among groups.