otb: an Automated HiC/HiFi Pipeline Assembles the Prosapia bicincta Genome.

The implementation of a new genomic assembly pipeline named only the best (otb) has effectively addressed various challenges associated with data management during the development and storage of genome assemblies. otb, which incorporates a comprehensive pipeline involving a setup layer, quality checks, templating, and the integration of Nextflow and Singularity. The primary objective of otb is to streamline the process of creating a HiFi/HiC genome, aiming to minimize the manual intervention required in the genome assembly process. The Two-lined spittlebug, (Prosapia bicincta, Hemiptera: Cercopidae), a true bug insect herbivore, serves as a practical test case for evaluating otb. The two-lined spittlebug is both a crucial agricultural pest and a genomically understudied insect belonging to the order Hemiptera. This insect is a significant threat to grasslands and pastures, leading to plant wilting and phytotoxemia when infested. Its presence in tropical and subtropical regions around the world poses a long-term threat to the composition of plant communities in grassland landscapes, impacting rangelands, and posing a substantial risk to cattle production.

FAIR Header Reference genome: a TRUSTworthy standard.

The lack of interoperable data standards among reference genome data-sharing platforms inhibits cross-platform analysis while increasing the risk of data provenance loss. Here, we describe the FAIR bioHeaders Reference genome (FHR), a metadata standard guided by the principles of Findability, Accessibility, Interoperability and Reuse (FAIR) in addition to the principles of Transparency, Responsibility, User focus, Sustainability and Technology. The objective of FHR is to provide an extensive set of data serialisation methods and minimum data field requirements while still maintaining extensibility, flexibility and expressivity in an increasingly decentralised genomic data ecosystem. The effort needed to implement FHR is low; FHR's design philosophy ensures easy implementation while retaining the benefits gained from recording both machine and human-readable provenance.

Genome report: chromosome-scale genome assembly of the West Indian fruit fly Anastrepha obliqua (Diptera: Tephritidae).

The West Indian fruit fly, Anastrepha obliqua, is a major pest of mango in Central and South America and attacks more than 60 species of host fruits. To support current genetic and genomic research on A. obliqua, we sequenced the genome using high-fidelity long-read sequencing. This resulted in a highly contiguous contig assembly with 90% of the genome in 10 contigs. The contig assembly was placed in a chromosomal context using synteny with a closely related species, Anastrepha ludens, as both are members of the Anastrepha fraterculus group. The resulting assembly represents the five autosomes and the X chromosome which represents 95.9% of the genome, and 199 unplaced contigs representing the remaining 4.1%. Orthology analysis across the structural annotation sets of high quality tephritid genomes demonstrates the gene annotations are robust, and identified genes unique to Anastrepha species that may help define their pestiferous nature that can be used as a starting point for comparative genomics. This genome assembly represents the first of this species and will serve as a foundation for future genetic and genomic research in support of its management as an agricultural pest.

DATA RESOURCES AND ANALYSES FAIR Header Reference genome: A TRUSTworthy standard.

The lack of interoperable data standards among reference genome data-sharing platforms inhibits cross-platform analysis while increasing the risk of data provenance loss. Here, we describe the FAIR-bioHeaders Reference genome (FHR), a metadata standard guided by the principles of Findability, Accessibility, Interoperability, and Reuse (FAIR) in addition to the principles of Transparency, Responsibility, User focus, Sustainability, and Technology (TRUST). The objective of FHR is to provide an extensive set of data serialisation methods and minimum data field requirements while still maintaining extensibility, flexibility, and expressivity in an increasingly decentralised genomic data ecosystem. The effort needed to implement FHR is low; FHR's design philosophy ensures easy implementation while retaining the benefits gained from recording both machine and human-readable provenance.

Reference genome for the Mojave poppy bee (Perdita meconis), a specialist pollinator of conservation concern.

The Mojave poppy bee, Perdita meconis Griswold (Hymenoptera: Anthophila: Andrenidae), is a species of conservation concern that is restricted to the eastern Mojave Desert of North America. It is a specialist pollinator of two poppy genera, Arctomecon and Argemone (Papaveraceae), and is being considered for listing under the US Endangered Species Act along with one of its pollinator hosts, the Las Vegas bearpoppy (Arctomecon californica). Here, we present a near chromosome-level genome of the Mojave poppy bee to provide a genomic resource that will aid conservation efforts and future research. We isolated DNA from a single, small (<7 mm), male specimen collected using non-ideal preservation methods then performed whole-genome sequencing using PacBio HiFi technology. After quality and contaminant filtering, the final draft genome assembly is 327 Mb, with an N50 length of 17.5 Mb. Annotated repetitive elements compose 37.3% of the genome, although a large proportion (24.87%) of those are unclassified repeats. Additionally, we annotated 18,245 protein-coding genes and 19,433 transcripts. This genome represents one of only a few genomes from the large bee family Andrenidae and one of only a few genomes for pollinator specialists. We highlight both the potential of this genome as a resource for future research, and how high-quality genomes generated from small, non-ideal (in terms of preservation) specimens could facilitate biodiversity genomics.

Evolution of five environmentally responsive gene families in a pine-feeding sawfly, Neodiprion lecontei (Hymenoptera: Diprionidae).

A central goal in evolutionary biology is to determine the predictability of adaptive genetic changes. Despite many documented cases of convergent evolution at individual loci, little is known about the repeatability of gene family expansions and contractions. To address this void, we examined gene family evolution in the redheaded pine sawfly Neodiprion lecontei, a noneusocial hymenopteran and exemplar of a pine-specialized lineage evolved from angiosperm-feeding ancestors. After assembling and annotating a draft genome, we manually annotated multiple gene families with chemosensory, detoxification, or immunity functions before characterizing their genomic distributions and molecular evolution. We find evidence of recent expansions of bitter gustatory receptor, clan 3 cytochrome P450, olfactory receptor, and antimicrobial peptide subfamilies, with strong evidence of positive selection among paralogs in a clade of gustatory receptors possibly involved in the detection of bitter compounds. In contrast, these gene families had little evidence of recent contraction via pseudogenization. Overall, our results are consistent with the hypothesis that in response to novel selection pressures, gene families that mediate ecological interactions may expand and contract predictably. Testing this hypothesis will require the comparative analysis of high-quality annotation data from phylogenetically and ecologically diverse insect species and functionally diverse gene families. To this end, increasing sampling in under-sampled hymenopteran lineages and environmentally responsive gene families and standardizing manual annotation methods should be prioritized.

Phylogenomic analysis provides diagnostic tools for the identification of Anastrepha fraterculus (Diptera: Tephritidae) species complex.

Insect pests cause tremendous impact to agriculture worldwide. Species identification is crucial for implementing appropriate measures of pest control but can be challenging in closely related species. True fruit flies of the genus Anastrepha Schiner (Diptera: Tephritidae) include some of the most serious agricultural pests in the Americas, with the Anastrepha fraterculus (Wiedemann) complex being one of the most important due to its extreme polyphagy and wide distribution across most of the New World tropics and subtropics. The eight morphotypes described for this complex as well as other closely related species are classified in the fraterculus species group, whose evolutionary relationships are unresolved due to incomplete lineage sorting and introgression. We performed multifaceted phylogenomic approaches using thousands of genes to unravel the evolutionary relationships within the A. fraterculus complex to provide a baseline for molecular diagnosis of these pests. We used a methodology that accommodates variable sources of data (transcriptome, genome, and whole-genome shotgun sequencing) and developed a tool to align and filter orthologs, generating reliable datasets for phylogenetic studies. We inferred 3031 gene trees that displayed high levels of discordance. Nevertheless, the topologies of the inferred coalescent species trees were consistent across methods and datasets, except for one lineage in the A. fraterculus complex. Furthermore, network analysis indicated introgression across lineages in the fraterculus group. We present a robust phylogeny of the group that provides insights into the intricate patterns of evolution of the A. fraterculus complex supporting the hypothesis that this complex is an assemblage of closely related cryptic lineages that have evolved under interspecific gene flow. Despite this complex evolutionary scenario, our subsampling analysis revealed that a set of as few as 80 loci has a similar phylogenetic resolution as the genome-scale dataset, offering a foundation to develop more efficient diagnostic tools in this species group.

Gut bacterial population and community dynamics following adult emergence in pest tephritid fruit flies.

Gut microbiota are important contributors to insect success. Host-microbe interactions are dynamic and can change as hosts age and/or encounter different environments. A turning point in these relationships the transition from immature to adult life stages, particularly for holometabolous insects where there is radical restructuring of the gut. Improved knowledge of population and community dynamics of gut microbiomes upon adult emergence inform drivers of community assembly and physiological aspects of host-microbe interactions. Here, we evaluated the bacterial communities of the pest tephritid species melon fly (Zeugodacus cucurbitae) and Medditeranean fruit fly (medfly, Ceratitis capitata) associated with the pupae life stage and timepoints immediately following adult eclosion. We used a combination of culturing to determine cultivatable bacterial titers, qPCR to determine 16S-rRNA SSU copy numbers, and 16S V4 sequencing to determine changes in communities. Both culturing and qPCR revealed that fly bacterial populations declined upon adult emergence by 10 to 100-fold followed by recovery within 24 h following eclosion. Titers reached ~ 107 CFUs (~ 108 16S rRNA copies) within a week post-emergence. We also observed concurrent changes in amplicon sequence variance (ASVs), where the ASV composition differed overtime for both melon fly and medfly adults at different timepoints. Medfly, in particular, had different microbiome compositions at each timepoint, indicating greater levels of variation before stabilization. These results demonstrate that tephritid microbiomes experience a period of flux following adult emergence, where both biomass and the makeup of the community undergoes dramatic shifts. The host-microbe dynamics we document suggest plasticity in the community and that there may be specific periods where the tephritid gut microbiome may be pliable to introduce and establish new microbial strains in the host.

Interspecific gene flow obscures phylogenetic relationships in an important insect pest species complex.

As genomic data proliferates, the prevalence of post-speciation gene flow is making species boundaries and relationships increasingly ambiguous. Although current approaches inferring fully bifurcating phylogenies based on concatenated datasets provide simple and robust answers to many species relationships, they may be inaccurate because the models ignore inter-specific gene flow and incomplete lineage sorting. To examine the potential error resulting from ignoring gene flow, we generated both a RAD-seq and a 500 protein-coding loci highly multiplexed amplicon (HiMAP) dataset for a monophyletic group of 12 species defined as the Bactrocera dorsalis sensu lato clade. With some of the world's worst agricultural pests, the taxonomy of the B. dorsalis s.l. clade is important for trade and quarantines. However, taxonomic confusion confounds resolution due to intra- and interspecific phenotypic variation and convergence, mitochondrial introgression across half of the species, and viable hybrids. We compared the topological convergence of our datasets using concatenated phylogenetic and various multispecies coalescent approaches, some of which account for gene flow. All analyses agreed on species delimitation, but there was incongruence between species relationships. Under concatenation, both datasets suggest identical species relationships with mostly high statistical support. However, multispecies coalescent and multispecies network approaches suggest markedly different hypotheses and detected significant gene flow. We suggest that the network approaches are likely more accurate because gene flow violates the assumptions of the concatenated phylogenetic analyses, but the data-reductive requirements of network approaches resulted in reduced statistical support and could not unambiguously resolve gene flow directions. Our study highlights the importance of testing for gene flow, particularly with phylogenomic datasets, even when concatenated approaches receive high statistical support.

A chromosome scale assembly of the parasitoid wasp Venturia canescens provides insight into the process of virus domestication.

The parasitoid wasp Venturia canescens is an important biological control agent of stored products moth pests and serves as a model to study the function and evolution of domesticated endogenous viruses (DEVs). The DEVs discovered in V. canescens are known as virus-like particles (VcVLPs), which are produced using nudivirus-derived components and incorporate wasp-derived virulence proteins instead of packaged nucleic acids. Previous studies of virus-derived components in the V. canescens genome identified 53 nudivirus-like genes organized in six gene clusters and several viral pseudogenes, but how VcVLP genes are organized among wasp chromosomes following their integration in the ancestral wasp genome is largely unknown. Here, we present a chromosomal scale genome of V. canescens consisting of 11 chromosomes and 56 unplaced small scaffolds. The genome size is 290.8 Mbp with a N50 scaffold size of 24.99 Mbp. A high-quality gene set including 11,831 protein-coding genes were produced using RNA-Seq data as well as publicly available peptide sequences from related Hymenoptera. A manual annotation of genes of viral origin produced 61 intact and 19 pseudogenized nudivirus-derived genes. The genome assembly revealed that two previously identified clusters were joined into a single cluster and a total of 5 gene clusters comprising of 60 intact nudivirus-derived genes were located in three chromosomes. In contrast, pseudogenes are dispersed among 8 chromosomes with only 4 pseudogenes associated with nudivirus gene clusters. The architecture of genes encoding VcVLP components suggests it originates from a recent virus acquisition and there is a link between the processes of dispersal and pseudogenization. This high-quality genome assembly and annotation represents the first chromosome-scale assembly for parasitoid wasps associated with VLPs, and is publicly available in the National Center for Biotechnology Information Genome and RefSeq databases, providing a valuable resource for future studies of DEVs in parasitoid wasps.

Chromosome-scale genome assembly of the rusty patched bumble bee, Bombus affinis (Cresson) (Hymenoptera: Apidae), an endangered North American pollinator.

The rusty patched bumble bee, Bombus affinis, is an important pollinator in North America and a federally listed endangered species. Due to habitat loss and large declines in population size, B. affinis is facing imminent extinction unless human intervention and recovery efforts are implemented. To better understand B. affinis biology and population genetic and genomic landscapes, we sequenced and assembled the B. affinis genome from a single haploid male. Whole genome HiFi sequencing on PacBio coupled with HiC sequencing resulted in a complete and highly contiguous contig assembly that was scaffolded into a chromosomal context, resolving 18 chromosomes distributed across the 365.1 Mb assembly. All material for both HiFi and HiC sequencing was derived from a single abdominal tissue segment from the single male. These assembly results, coupled with the minimal amount of tissue destructively sampled, demonstrate methods for generating contiguous and complete genomic resources for a rare and endangered species with limited material available and highlight the importance of sample preservation. Precise methods and applications of these methods are presented for potential applications in other species with similar limitations in specimen availability and curation considerations.

Whole-genome resequencing data support a single introduction of the invasive white pine sawfly, Diprion similis.

Biological introductions are unintended "natural experiments" that provide unique insights into evolutionary processes. Invasive phytophagous insects are of particular interest to evolutionary biologists studying adaptation, as introductions often require rapid adaptation to novel host plants. However, adaptive potential of invasive populations may be limited by reduced genetic diversity-a problem known as the "genetic paradox of invasions." One potential solution to this paradox is if there are multiple invasive waves that bolster genetic variation in invasive populations. Evaluating this hypothesis requires characterizing genetic variation and population structure in the invaded range. To this end, we assemble a reference genome and describe patterns of genetic variation in the introduced white pine sawfly, Diprion similis. This species was introduced to North America in 1914, where it has rapidly colonized the thin-needled eastern white pine (Pinus strobus), making it an ideal invasion system for studying adaptation to novel environments. To evaluate evidence of multiple introductions, we generated whole-genome resequencing data for 64 D. similis females sampled across the North American range. Both model-based and model-free clustering analyses supported a single population for North American D. similis. Within this population, we found evidence of isolation-by-distance and a pattern of declining heterozygosity with distance from the hypothesized introduction site. Together, these results support a single-introduction event. We consider implications of these findings for the genetic paradox of invasion and discuss priorities for future research in D. similis, a promising model system for invasion biology.

Chromosome-scale genome assembly of the pink bollworm, Pectinophora gossypiella, a global pest of cotton.

The pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), is a major global pest of cotton. Current management practices include chemical insecticides, cultural strategies, sterile insect releases, and transgenic cotton producing crystalline (Cry) protein toxins of the bacterium Bacillus thuringiensis (Bt). These strategies have contributed to the eradication of P. gossypiella from the cotton-growing areas of the United States and northern Mexico. However, this pest has evolved resistance to Bt cotton in Asia, where it remains a critical pest, and the benefits of using transgenic Bt crops have been lost. A complete annotated reference genome is needed to improve global Bt resistance management of the pink bollworm. We generated the first chromosome-level genome assembly for pink bollworm from a Bt-susceptible laboratory strain (APHIS-S) using PacBio continuous long reads for contig generation, Illumina Hi-C for scaffolding, and Illumina whole-genome re-sequencing for error correction. The pseudo-haploid assembly consists of 29 autosomes and the Z sex chromosome. The assembly exceeds the minimum Earth BioGenome Project quality standards, has a low error rate, is highly contiguous at both the contig and scaffold levels (L/N50 of 18/8.26 MB and 14/16.44 MB, respectively), and is complete, with 98.6% of lepidopteran single-copy orthologs represented without duplication. The genome was annotated with 50% repeat content and 14,107 protein-coding genes, further assigned to 41,666 functional annotations. This assembly represents the first publicly available complete annotated genome of pink bollworm and will serve as the foundation for advancing molecular genetics of this important pest species.

HiMAP2: Identifying phylogenetically informative genetic markers from diverse genomic resources.

Multiplexed amplicon sequencing offers a cost-effective and rapid solution for phylogenomic studies that include a large number of individuals. Selecting informative genetic markers is a critical initial step in designing such multiplexed amplicon panels, but screening various genomic data and selecting markers that are informative for the question at hand can be laborious. Here, we present a flexible and user-friendly tool, HiMAP2, for identifying, visualizing and filtering phylogenetically informative loci from diverse genomic and transcriptomic resources. This bioinformatics pipeline includes orthology prediction, exon extraction and filtering of aligned exon sequences according to user-defined specifications. Additionally, HiMAP2 facilitates exploration of the final filtered exons by incorporating phylogenetic inference of individual exon trees with raxml-ng as well as the estimation of a species tree using astral. Finally, results of the marker selection can be visualized and refined with an interactive Bokeh application that can be used to generate publication-quality figures. Source code and user instructions for HiMAP2 are available at https://github.com/popphylotools/HiMAP_v2.

Insight into weevil biology from a reference quality genome of the boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae).

The boll weevil, Anthonomus grandis grandis Boheman, is one of the most historically impactful insects due to its near destruction of the US cotton industry in the early 20th century. Contemporary efforts to manage this insect primarily use pheromone baited traps for detection and organophosphate insecticides for control, but this strategy is not sustainable due to financial and environmental costs. We present a high-quality boll weevil genome assembly, consisting of 306 scaffolds with approximately 24,000 annotated genes, as a first step in the identification of gene targets for novel pest control. Gene content and transposable element distribution are similar to those found in other Curculionidae genomes; however, this is the most contiguous and only assembly reported to date for a member in the species-rich genus Anthonomus. Transcriptome profiles across larval, pupal, and adult life stages led to identification of several genes and gene families that could present targets for novel control strategies.

A Chromosome-Scale Genome Assembly of a Helicoverpa zea Strain Resistant to Bacillus thuringiensis Cry1Ac Insecticidal Protein.

Helicoverpa zea (Lepidoptera: Noctuidae) is an insect pest of major cultivated crops in North and South America. The species has adapted to different host plants and developed resistance to several insecticidal agents, including Bacillus thuringiensis (Bt) insecticidal proteins in transgenic cotton and maize. Helicoverpa zea populations persist year-round in tropical and subtropical regions, but seasonal migrations into temperate zones increase the geographic range of associated crop damage. To better understand the genetic basis of these physiological and ecological characteristics, we generated a high-quality chromosome-level assembly for a single H. zea male from Bt-resistant strain, HzStark_Cry1AcR. Hi-C data were used to scaffold an initial 375.2 Mb contig assembly into 30 autosomes and the Z sex chromosome (scaffold N50 = 12.8 Mb and L50 = 14). The scaffolded assembly was error-corrected with a novel pipeline, polishCLR. The mitochondrial genome was assembled through an improved pipeline and annotated. Assessment of this genome assembly indicated 98.8% of the Lepidopteran Benchmark Universal Single-Copy Ortholog set were complete (98.5% as complete single copy). Repetitive elements comprised approximately 29.5% of the assembly with the plurality (11.2%) classified as retroelements. This chromosome-scale reference assembly for H. zea, ilHelZeax1.1, will facilitate future research to evaluate and enhance sustainable crop production practices.

A Unified Protocol for CRISPR/Cas9-Mediated Gene Knockout in Tephritid Fruit Flies Led to the Recreation of White Eye and White Puparium Phenotypes in the Melon Fly.

Tephritid fruit flies are among the most invasive and destructive agricultural pests worldwide. Over recent years, many studies have implemented the CRISPR/Cas9 genome-editing technology to dissect gene functions in tephritids and create new strains to facilitate their genetics, management, and control. This growing literature allows us to compare diverse strategies for delivering CRISPR/Cas9 components into tephritid embryos, optimize procedures, and advance the technology to systems outside the most thoroughly studied species within the family. Here, we revisit five years of CRISPR research in Tephritidae and propose a unified protocol for candidate gene knockout in fruit flies using CRISPR/Cas9. We demonstrated the efficiency of our protocol by disrupting the eye pigmentation gene white eye (we) in the melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae). High rates of somatic and germline mutagenesis were induced by microinjecting pre-assembled Cas9-sgRNA complexes through the chorion of embryos at early embryogenesis, leading to the rapid development of new mutant lines. We achieved comparable results when targeting the we orthologue in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), illustrating the reliability of our methods when transferred to other related species. Finally, we functionally validated the recently discovered white pupae (wp) loci in the melon fly, successfully recreating the white puparium phenotype used in suppression programs of this and other major economically important tephritids. This is the first demonstration of CRISPR-based genome-editing in the genus Zeugodacus, and we anticipate that the procedures described here will contribute to advancing genome-editing in other non-model tephritid fruit flies.

HiFiAdapterFilt, a memory efficient read processing pipeline, prevents occurrence of adapter sequence in PacBio HiFi reads and their negative impacts on genome assembly.

BACKGROUND: Pacific Biosciences HiFi read technology is currently the industry standard for high accuracy long-read sequencing that has been widely adopted by large sequencing and assembly initiatives for generation of de novo assemblies in non-model organisms. Though adapter contamination filtering is routine in traditional short-read analysis pipelines, it has not been widely adopted for HiFi workflows. RESULTS: Analysis of 55 publicly available HiFi datasets revealed that a read-sanitation step to remove sequence artifacts derived from PacBio library preparation from read pools is necessary as adapter sequences can be erroneously integrated into assemblies. CONCLUSIONS: Here we describe the nature of adapter contaminated reads, their consequences in assembly, and present HiFiAdapterFilt, a simple and memory efficient solution for removing adapter contaminated reads prior to assembly.

Identification of sex chromosomes and primary sex ratio in the small hive beetle, a worldwide parasite of honey bees.

BACKGROUND: The small hive beetle (SHB), Aethina tumida, has emerged as a worldwide threat to honey bees in the past two decades. These beetles harvest nest resources, feed on larval bees, and ultimately spoil nest resources with gelatinous slime together with the fungal symbiont Kodamaea ohmeri. RESULTS: Here, we present the first chromosome-level genome assembly for the SHB. With a 99.1% representation of conserved (BUSCO) arthropod genes, this resource enables the study of chemosensory, digestive, and detoxification traits critical for SHB success and possible control. We use this annotated assembly to characterize features of SHB sex chromosomes and a female-skewed primary sex ratio. We also found chromosome fusion and a lower recombination rate in sex chromosomes than in autosomes. CONCLUSIONS: Genome-enabled insights will clarify the traits that allowed this beetle to exploit hive resources successfully and will be critical for determining the causes of observed sex ratio asymmetries.