Biology subjects

Stewart, R. J.

Publications and source records attributed to Stewart, R. J..

8 recordsLinked to original sources

bioRxiv2022

Uncovering hidden genetic diversity: allelic resolution of insect and spider silk genes

Arthropod silk is vital to the evolutionary success of hundreds of thousands of species. The primary proteins in silks are often encoded by long, repetitive gene sequences. Until recently, sequencing and assembling these complex gene sequences has proven intractable given their repetitive structure. Here, using high-quality long-read sequencing, we show that there is extensive variation--both in terms of length and repeat motif order--between alleles of silk genes within individual arthropods. Further, this variation exists across two deep, independent origins of silk which diverged more than 500 million years ago--(1) the insect clade containing caddisflies and butterflies and (2) spiders. This remarkable convergence in previously overlooked patterns of allelic variation across multiple origins of silk suggests mechanisms for the generation and maintenance of structural protein-coding genes. Future genomic efforts to connect genotypes to phenotypes should account for such allelic variation.

genomics↗

bioRxiv2022

Structural diversity of silk heavy-chain fibroins across major caddisfly (Trichoptera) clades

BackgroundLarvae of Trichoptera produce silk to build various underwater structures allowing them to exploit a wide range of aquatic environments. The heavy chain fibroin (h-fibroin) gene encodes the primary protein component of their silk. Studies on this long (>20 kbp) and highly repetitive gene have been limited by difficulties in its sequence assembly. Recently, high-quality long-read sequencing techniques have been successfully applied to obtain the full-length h-fibroin sequence. ResultsWe used three new and five previously published genomes to identify eight full-length h-fibroin gene and protein sequences of h-fibroin across the order Trichoptera covering various endpoints of the diversity of ecological silk use in caddisflies. We analyze these together with four existing high-quality h-fibroin sequences. Across the order, we observed conserved patterns in h-fibroin (high similarity of amino(n)-/carboxyl(c)-termini, presence of characteristic repeating structural modules). However, the sequence, number, and arrangement of these repeating modules varied across clades with increasing structural complexity of h-fibroin in fixed retreat and tube-case builders compared to cocoon-builders. We also found a higher percentage of proline in fixed-retreat makers. ConclusionThis study provides characterizations of the primary structure of h-fibroin from a diverse set of caddisflies. The interplay of conserved termini and basic motif structure with high variation in repeating modules as well as the variation in the percentage of proline might be linked to differences in mechanical properties (i.e., tensile strength, toughness) related to the different silk usage. This sets a starting point for future studies to screen and correlate amino acid motifs and other sequence features with quantifiable silk properties.

genomics↗

bioRxiv2022

Realizing the promise of biodiversity genomics with highly accurate long reads

BackgroundGenerating 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 6,750 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. ResultsHiFi 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. ConclusionsHighly 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.

genomics↗

bioRxiv2022

Conservation of three-dimensional structure in Lepidoptera and Trichoptera L-fibroins for 290 million years

The divergence of sister orders, Trichoptera (caddisflies) and Lepidoptera (moths and butterflies), from a silk-spinning ancestor occurred around 290 million years ago. Trichoptera larvae are mainly aquatic and Lepidoptera larvae are almost entirely terrestrial, distinct habitats that required molecular adaptation of their silk for deployment in water versus air. The major protein components of their silks are Heavy chain and Light chain fibroins. In an effort to identify molecular changes in L-fibroins that may have contributed to the divergent use of silk in water versus air, we used the ColabFold implementation of AlphaFold2 to predict three-dimensional structures of L-fibroins from both orders. Comparison of the structures revealed that despite the ancient divergence, profoundly different habitats, and low sequence conservation, a 10-helix core structure was strongly conserved in L-fibroins from both orders. Previously known intra- and intermolecular disulfide linkages were accurately predicted. Structure variations observed outside of the core may represent molecular changes that contributed to the evolution of silks adapted to water or air. The distributions of electrostatic potential, for example, are not conserved and present distinct order-specific surfaces for potential interactions with or modulation by external factors. Also, the interactions of L-fibroins with the H-fibroin C-termini are different between the orders; lepidopteran L-fibroins have N-terminal insertions, not present in trichopteran L-fibroins, which form an unstructured ribbon in isolation but become part of an intermolecular {beta}-sheet when folded with their corresponding H-fibroin C-termini. The results may serve as another example of protein structure prediction from deep sequence data of understudied proteins made possible by AlphaFold2.

evolutionary biology↗

bioRxiv2022

Long-read HiFi Sequencing Correctly Assembles Repetitive heavy fibroin Silk Genes in New Moth and Caddisfly Genomes

Insect silk is an incredibly versatile biomaterial. Lepidoptera and their sister lineage, Trichoptera, display some of the most diverse uses of silk with varying strength, adhesive qualities and elastic properties. It is well known that silk fibroin genes are long (> 20 kb) and have many repetitive motifs. These features make these genes challenging to sequence. Most research thus far has focused on conserved N- and C-terminal regions of fibroin genes because a full comparison of repetitive regions across taxa has not been possible. Using the PacBio Sequel II system and SMRT sequencing, we generated high fidelity (HiFi) long-read genomic and transcriptomic sequences for the Indianmeal moth (Plodia interpunctella) and genomic sequences for the caddisfly, Eubasilissa regina. Both genomes were highly contiguous (N50 = 9.7 Mbp/32.4 Mbp, L50 = 13/11) and complete (BUSCO Complete = 99.3%/95.2%), with complete and contiguous recovery of silk heavy fibroin gene sequences. This study demonstrates that HiFi long-read sequencing can significantly help our understanding of genes with highly contiguous, repetitive regions.

genomics↗

bioRxiv2020

Draft Genome Assemblies and Annotations of Agrypnia vestita Walker, and Hesperophylaxmagnus 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. While 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 three-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 (LINEs) 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. SignificanceThere is a lack of genomic resources for aquatic insects. So far, only three high-quality genomes have been assembled, all from individuals in the retreat-making suborder Annulipalpia. In this article, we report the first high-quality genomes of two case-making species from the suborder Integripalpia, which are essential for studying genomic diversity across this ecologically diverse insect order. Our research reveals larger genome sizes in the tube case-makers (suborder Integripalpia, infraorder Phryganides), accompanied by a disproportionate increase of repetitive DNA. This suggests that genome size is at least partly driven by a major expansion of repetitive elements. Our work shows that caddisflies have high potential as a model for understanding how genomic diversity might be linked to functional diversification and forms the basis for detailed studies on genome size evolution in caddisflies. Data depositionThis project has been deposited at NCBI under the Bioproject ID: PRJNA668166

genomics↗

bioRxiv2018

POFUT2-mediated O-glycosylation of MIC2 is dispensable for Toxoplasma gondii tachyzoites

Toxoplasma gondii is a ubiquitous obligate intracellular eukaryotic parasite that causes congenital birth defects, disease of the immunocompromised and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune response of many eukaryotic parasites and is also of great relevance to vaccine design. Here, we demonstrate that MIC2, the motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat domains (TSR). At least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at O-glycosylation sites. We demonstrate that the addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homologue (TgPOFUT2) encoded by TGGT1_273550. While POFUT2 homologues are important for stabilizing motility associated adhesins and host infection in other apicomplexan parasites, in T. gondii loss of TgPOFUT2 has only a modest impact on MIC2 levels and the wider proteome. Consistent with this, both plaque formation and tachyzoite infectivity are broadly similar in the presence or absence of TgPOFUT2. These findings demonstrate that TgPOFUT2 O-glycosylates MIC2 and that this glycan is dispensable in T. gondii tachyzoites.

microbiology↗

bioRxiv2018

Protein Kinase A Negatively Regulates Ca2+ signalling in Toxoplasma gondii

The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating forms and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is not understood. Here we show that the cAMP-dependent Protein Kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that that PKAc1 is sequestered to the parasite periphery by dual acylation of its regulatory subunit PKAr1. Newly invaded PKAc1-deficient parasites exit host cells shortly thereafter in a perforin-like protein 1 (PLP-1)-dependent fashion. We demonstrate that loss of PKAc1 results in an inability to rapidly downregulate cytosolic Ca2+ levels shortly after invasion. Furthermore, we demonstrate that PKAc1 also specifically negatively regulates resting cytosolic Ca2+ in conditions that mimic intracellularity. We also show that cAMP and cGMP have opposing role in microneme secretion, further supporting evidence that cAMP signalling has a suppressive role during motility. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.

microbiology↗