Biology subjects

Ord, S.

Publications and source records attributed to Ord, S..

8 recordsLinked to original sources

bioRxiv2026

Optimization of High Molecular Weight DNA Extractions from Dried, Museum-Grade Insects Enables Long-Read Sequencing, Phylogenetics, and Methylation Profiling

Developing an effective DNA extraction method that meets requirements for long-read sequencing of poorly preserved samples, such as museum specimens or ancient material, offers new opportunities for genomic analysis of endangered or extinct species for which samples are rare. However, these samples often yield degraded and highly fragmented DNA, rendering long-read sequencing infeasible for many specimens residing in museum collections. Herein, we demonstrate a protocol for successfully extracting DNA of sufficient quality for sequencing on the Oxford Nanopore Technologies long-read sequencing PromethION platform from a desiccated, museum-grade blue carpenter bee specimen (Xylocopa caerulea). We find the protocol is reproducible across specimens and yields high levels of long, endogenous X. caerulea-derived DNA, highlighting the utility of our method for enabling genomic studies of historical collections. From a single flow cell, we assembled the full-length mitochondrial genome and used this assembly to perform a phylogenetic analysis, accurately placing our X. caerulea specimen among related Xylocopa species, thus demonstrating the phylogenetic utility of long-read museomics. Using these long-read data, we analyzed native CpG methylation, finding endogenous methylation signals that correlate with genic and exonic sequences. This method expands the feasibility of genomic and epigenomic analyses from challenging samples, enhancing our ability to investigate the genomes of endangered and extinct species through archival resources.

genomics↗

bioRxiv2025

Getting a head start: Craniofacial heterochrony in marsupials involves dynamic changes to molecular and cellular mechanisms underlying neural crest development

The neural crest is a vertebrate innovation central to craniofacial development and evolution. While the gene regulatory networks guiding neural crest development are well characterized, the mechanisms generating species-specific craniofacial diversity remain poorly understood. Marsupials provide a unique model for studying neural crest plasticity, having evolved accelerated patterns of craniofacial development during embryogenesis. This adaptation arises in response to marsupials being born altricial after a short gestation yet require well-developed mouthparts to attach to a teat and continue development in the pouch. However, how marsupials achieve this heterochronic shift in neural crest development is largely unknown. In this study, we investigate the cellular and molecular mechanisms underlying their distinct heterochrony, revealing that marsupials produce dense pre-migratory aggregates of neural crest cells which undergo collective migration as epithelial-like sheets, potentially facilitating rapid establishment of the facial prominences. These cellular behaviours are unique amongst amniotes but resemble patterns in anamniotes which similarly exhibit accelerated craniofacial development to support early feeding. Marsupials appear to have evolved a similar mechanism of neural crest migration to facilitate their developmental heterochrony. These findings suggest that vertebrate neural crest migration may be shaped by the pace of craniofacial development during embryogenesis rather than phylogeny, providing new perspectives on neural crest plasticity and the developmental mechanisms driving craniofacial diversity across vertebrates.

developmental biology↗

bioRxiv2025

High-Yield Induced Ovulation in Adult Fat-Tailed Dunnarts by PMSG Treatment Combined with Estrus Synchronization

The fat-tailed dunnart, Sminthopsis crassicaudata, is an emerging laboratory-based marsupial model for research on comparative biology, reproduction and conservation. In females, the reproductive cycle lasts 31 days and approximately 10 oocytes are ovulated per cycle. Assisted reproductive technologies (ART) play a crucial role in marsupial conservation, but developing protocols to harvest large numbers of oocytes remains a key challenge. Specifically, producing sufficient mature metaphase II (MII) oocytes in dunnarts continues to be difficult. Ovarian follicle stimulation is common practice to achieve superovulation in many species and typically requires treatment of prepubertal female animals to avoid the impacts of endogenous hormone cycling. Alternatively, adult females can be stimulated during the intermediate or follicular phase. In this study, we aimed to develop a high-yield induced-ovulation protocol to collect higher number of MII oocytes from adult, cycling, female dunnarts. We first synchronized the female dunnart reproductive cycles using luteinizing hormone-releasing hormone (LHRH). The reproductive cycles were monitored by examining cytology of vaginal lavage samples. After administering four LHRH injections given at three-day intervals, 88.9% (n=36) of the adult female dunnarts responded to the treatment, with their estrous cycles synchronized at the diestrous stage. We then induced ovarian follicle development through two PMSG injections over 6 days, followed by hCG administration to trigger ovulation. By combining estrous cycle synchronization with PMSG stimulation, we achieved 77.8% (n=36) ovulation success and obtained an average of 20.1{+/-}9.1 (n=28) MII oocytes per adult dunnart. These data demonstrated that estrous cycle synchronization followed by the PMSG-hCG treatment yields consistent, highly efficient induced-ovulation in adult dunnarts. This approach of combining estrus synchronization and follicle stimulation to produce sufficient MII oocytes for ART purpose could be applied to other valuable marsupial species to support conservation efforts. Summary SentenceSuperovulation and robust production of mature MII oocytes can be induced by 10-day estrus synchronization using LHRH followed by 6-day PMSG stimulation of follicle growth in adult fat-tailed dunnarts, a marsupial species. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/671172v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@14c6685org.highwire.dtl.DTLVardef@decf75org.highwire.dtl.DTLVardef@64330org.highwire.dtl.DTLVardef@12538a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

On the ancestry and evolution of the extinct dire wolf

Dire wolves (Aenocyon dirus) are extinct predators of Pleistocene North America. Although phenotypically similar to living wolves (Canis lupus), dire wolves have yet to be placed confidently in the canid family tree. We generated 3.4x and 12.8x paleogenomes from two well-preserved dire wolves dating to > 13,000 and > 72,000 years ago, and estimated consensus species trees for these and 10 canid species. Our results revealed that [~]2/3 of dire wolf ancestry is derived from a lineage sister to the clade comprising the gray wolf, coyote, and dhole, and the remaining [~]1/3 from a lineage near the base of Canini diversity. We identified 80 genes evolving under diversifying selection in dire wolves. Our results underscore the power of paleogenomes to resolve long-standing taxonomic questions and contribute to growing evidence of the role of post-speciation gene flow as an evolutionary force.

evolutionary biology↗

bioRxiv2025

Generation and Assessment of High-Quality Fat-Tailed Dunnart Oocytes Following Superovulation in Prepubertal Animals

The fat-tailed dunnart, Sminthopsis crassicaudata, is a mouse-sized, polyovular, solitary dasyurid marsupial found in central and southern Australia. With the establishment of a chromosome-scale genome assembly, induced pluripotent stem cells, and targeted genetic editing, the dunnart is emerging as the laboratory marsupial model for comparative developmental, reproductive and conservation biology. The development of assisted reproductive technologies (ART) are critical to achieving these goals in this species. ART requires a large number of mature oocytes which are typically collected through stimulated and synchronised female reproductive cycles. While protocols for induced-ovulation or superovulation are standard in many placental mammals, there are no methods to date designed for marsupials. In the present study, prepubertal dunnarts were stimulated with pregnant mare serum gonadotrophin and human chronic gonadotrophin across 6 different treatment regimens. Our best regimen resulted in over 70% of prepubertal dunnarts ovulating with 82% normal oocytes. When the primed females were mated with stud males, 4-cell stage embryos were collected 48 h post-hCG administration. At around 96 h post-hCG, 50% (n=8) and 78% (n=9) of the embryos developed to blastocysts. Our results demonstrated successful stimulation of ovulation and mature oocyte collection in prepubertal dunnarts. Furthermore, we confirmed developmental competence of the induced ovulated oocytes through to at least the blastocyst stage. These findings represent the first robust hormonal regimen for predictable oocyte generation in any marsupial and will significantly contribute to the use of the dunnart in developmental and conservation biology.

developmental biology↗

bioRxiv2024

Marsupial limb patterning redefines the necessity of lateral plate mesoderm subdivision for limb formation

The tetrapod limb has long served as a model for elucidating molecular and cellular mechanisms driving tissue patterning, development and evolution. While significant advances have been made in understanding the drivers of limb initiation, outgrowth, and patterning, the early morphogenetic processes that transform the lateral plate mesoderm (LPM) into limb fields remain less resolved. Marsupial mammals provide a unique opportunity to investigate these foundational processes due to their accelerated forelimb development, driven by the functional demands of altricial neonates to crawl into the pouch at birth. Heterochronic formation of the forelimbs occurs prior to development of other surrounding structures, offering unparalleled insights into the plasticity of limb field specification. Here, we reveal that marsupial limb initiation and outgrowth bypasses physical subdivision of the LPM, a process previously considered critical for tetrapod limb formation. Instead, limb development proceeds through early activation of LPM-associated genes and proliferation before coelom formation, demonstrating remarkable morphogenetic plasticity. This evolutionary adaptation enables heterochronic limb development, redefining conserved processes to meet extreme functional constraints. These findings challenge previous models of tetrapod limb specification, highlighting the evolutionary plasticity of limb patterning mechanisms and reshaping our understanding of how selective pressures influence foundational developmental events.

developmental biology↗

bioRxiv2024

Enrichment of spermatogonial stem cells and staging of the testis cycle in a dasyurid marsupial, the fat-tailed dunnart

There is increasing interest in use of marsupial models in research, for use in next-generation conservation by improving fitness through genetic modification, and in de-extinction efforts. Specifically this includes dasyurid marsupials such as the Thylacine, Tasmanian devil, quolls and the small rodent-like dunnarts. Technologies for generating genetically modified Australian marsupials remains to be established. Given the need to advance research in this space, the fat-tailed dunnart (Sminthopsis crassicaudata) is being established as a model for marsupial spermatogonial stem cell isolation, modification and testicular transplantation. This species is small (60-90mm body size), polyovulatory (8-12 pups per birth), and can breed in standard rodent facilities when housed in a 12:12 light cycle. To develop the fat tailed dunnart as a model for next-generation marsupial conservation, this study aimed to enrich dunnart spermatogonial stem cells from whole testis digestions using a fluorescent dye technology and fluorescence-activated cell sorting. This approach is not dependent on antibodies or genetic reporter animals that are limiting factors when performing cell sorting on species separated from human and mouse by large evolutionary timescales. This study also assessed development of spermatogonia and spermatogenesis in the fat-tailed dunnart, by making the first definition of the cycle of the seminiferous epithelium in any dasyurid. Overall, this is the first detailed study to assess the cycle of dasyurid spermatogenesis and provides a valuable method to enrich marsupial spermatogonial stem cells for cellular, functional and molecular analysis.

cell biology↗

bioRxiv2024

Genetically engineered resistance to bufotoxin in marsupial ATP1A1

The introduction of the bufotoxin-secreting cane toad (Rhinella marina) to Queensland in 1935 has had a devastating impact on wildlife in the Australian tropics. Having evolved for millions of years in the absence of cane toads or other bufotoxin-secreting organisms, many of the Australias native predators that include cane toads in their diet suffered large population declines following cane toad invasion to their habitat. One marsupial species, the northern quoll (Dasyurus hallucatus), is now classified as endangered (IUCN Red List) largely due to bufotoxin ingestion. This study aimed to introduce bufotoxin resistance into a marsupial cell line by editing part of the ATP1A1 gene encoding the extracellular H1-H2 domain - the binding target of bufotoxin. To this end, CRISPR prime editing was used to replace the part of the wildtype ATP1A1 gene encoding the H1-H2 domain in fibroblasts of a related marsupial model, the fat-tailed dunnart (Sminthopsis crassicaudata), with modifications known to be associated with bufotoxin resistance. The genetically modified cell population showed a >45-fold increase in resistance to bufalin (an active component of bufotoxin) compared to wild type. This study provides a proof of concept towards engineering genetic resistance in the northern quoll to halt or even reverse its current population decline.

bioengineering↗