Sarah L. Whiteley

Climate warming can influence sex determination and embryonic growth in oviparous reptiles that rely on external temperatures during development. Warmer incubation temperatures can stimulate growth in ectotherms until oxygen supply can no longer keep pace with growth rate; at which point metabolically expensive organs like the brain suffer. We aimed to understand the impact of heat and sex on brain development in central bearded dragons, Pogona vitticeps (Agamidae), a uniquely thermosensitive species that exhibits sex-reversal at warm incubation temperatures. Three sexual phenotypes are possible in P. vitticeps : genetically driven ZW females and ZZ males, with the latter phenotype thermally overridden at incubation temperatures >32 °C to induce ZZ sex-reversed females. To visualise changes in brain anatomy, we used buffered Lugol’s iodine to perform contrast-enhanced micro computed tomography (µCT) and synchrotron propagation-based phase-contrast CT scanning. A total of 57 embryos were sampled across developmental stages and incubated at two temperature treatments: cool (28 °C, n = 26) and warm (36 °C, n = 21). Using high-resolution models of neuroanatomy, we found that the telencephalic region of the forebrain is significantly larger in individuals with ZZ genotypes (males and sex-reversed females) compared to concordant ZW females. Although incubation temperature significantly influenced growth rate and body size during development, overall brain size was not significantly different across temperature treatments. Our results indicate that the effects of genotypic sex on brain development are more important than thermal influences, and thus the brain may be somewhat buffered to the effects of warm incubation temperatures in this arid-adapted dragon.

Contrast-enhanced micro-computed tomography (µCT) has become a valuable tool for addressing neuroanatomical questions that previously required destructive histology. As a relatively new technique, it remains subject to ongoing technical refinement, and taxon- and tissue-specific protocol optimisation. We evaluated the performance of two common contrast-enhancing agents, buffered Lugol’s iodine and phosphotungstic acid (PTA) in aqueous solution, for µCT visualisation of the nervous system in the Jacky dragon lizard, Amphibolurus muricatus by comparing staining time, contrast specificity and brain tissue shrinkage evaluated at three concentrations (1.25%, 2.5% and 5%). Buffered Lugol’s iodine outperformed aqueous PTA by requiring shorter staining times (as short as four days) and providing better white matter-grey matter differentiation, whereas PTA required longer incubation periods of approximately three weeks and produced poorer white-grey matter differentiation. Importantly, buffered Lugol’s iodine produced higher brain-to-endocast occupancy estimates than aqueous PTA, suggesting substantially reduced tissue shrinkage. PTA-treated specimens exhibited an estimated 13.3% reduction in occupancy and approximately 20% greater brain tissue shrinkage than buffered Lugol’s-treated specimens. Together, these findings demonstrate that buffered Lugol’s iodine staining provides the high level of internal tissue contrast required to resolve fine-scale neuroanatomical organisation in three dimensions, preserves the spatial relationships among anatomical structures, and reduces artefacts, all of which result in a closer correspondence between the brain and endocranial cavity. From a practical perspective, the study provides guidance for contrast agent selection in future µCT studies and reveals that buffered Lugol’s iodine is better suited to fine-scale neuroanatomical, volumetric and geometric morphometric analyses, whereas PTA is advantageous for applications prioritising general soft-tissue or musculoskeletal visualisation. Graphical Buffered Lugol’s iodine improves µCT visualisation of dragon lizard brains through faster staining, greater neural contrast, reduced tissue shrinkage and closer correspondence between brain and endocast surfaces compared with phosphotungstic acid (PTA).

Temperature-induced sex reversal is a poorly understood trait in reptiles, described for only a small number of species. In the Eastern three-lined skink (Bassiana duperreyi), genotypic females exposed to cold temperatures reverse sex and develop as phenotypic males. We characterized embryonic development in B. duperreyi for the first time using eggs from wild communal nests. We observed variation in developmental stage at lay, suggesting females coordinate nesting despite becoming gravid at different times, potentially representing an early evolutionary shift towards viviparity. The development of sexual traits was like that of the other species with temperature-induced sex reversal (Pogona vitticeps). Ovotestes were observed in a small number of individuals regardless of genotypic sex and across all three incubation temperatures (18, 20 and 23°C). Notably, ovotestes were observed later in development in B. duperreyi, potentially representing differences in the influence of temperature on sexual development between the two species. Genotypic females also displayed temporary pseudohermaphroditism, which is characterized by the simultaneous presence of ovaries and bilobed hemipenes. These data represent foundational information for B. duperreyi required to support future research on the molecular underpinnings of sex reversal.

Although once only characterized by human hepatitis deltavirus (HDV), membership of the family Kolmioviridae has dramatically expanded in recent years. Despite this transformation in our understanding of the host range of kolmioviruses, the evolutionary history of this enigmatic group of RNA viruses is unclear. Kolmioviruses are characterized as small (∼1.7kb) satellite viruses that encode a single ∼200 amino acid delta antigen (DAg) and require unrelated helper viruses for replication. Here, we describe eight novel kolmioviruses from metatranscriptomic studies of the American alligator ( Alligator missippiensis ), red kangaroo ( Osphranter rufus ), and central bearded dragon ( Pogona vitticeps ), as well as avian kolmioviruses mined from the Sequence Read Archive (SRA). Although the novel kolmioviruses were often found in samples co-infected by other viruses, there was no evidence for the presence of hepatitis B virus as seen in HDV. By employing a range of sequence data sets, alignment methods, alignment trimming methods, and substitution models, we provide an evolutionary history of the Kolmioviridae that maximizes the extent of virus-host co-divergence and refines estimates of their evolutionary timescale. Although DAg amino acid sequences are more conserved than nucleotide sequences and hence might be expected to result in more accurate phylogenetic trees, we show that full genome nucleotide sequences likely provide the best representation of kolmiovirus evolution. More broadly, our results reveal that irrespective of the data set used, multiple distinct kolmiovirus lineages have co-circulated throughout vertebrate evolution over timescales spanning hundreds of millions of years, with the association between HDV and HBV appearing only recently. Significance Statement: Kolmioviruses are satellite RNA viruses, with hepatitis deltavirus (HDV) associated with human disease following co-infection with hepatitis B virus (HBV) the best characterized. Although a growing number of animal kolmioviruses have been identified in metagenomic studies and associated with a range of helper viruses, the evolutionary origins and history of this important and unique group of viruses is unknown. By identifying novel kolmioviruses in a range vertebrate hosts, including American alligators, we show that highly diverse lineages of kolmioviruses have co-circulated for the duration of vertebrate evolution with clear evidence of virus-host co-divergence, and are associated with a variety of potential helper viruses. Despite this antiquity, we present evidence that the HDV-HBV association only recently evolved in human populations.

Specialized chemically-coated paper cards, such as Flinders Technology Associates (FTA) cards, provide simple and reliable storage of nucleic acids by protecting DNA from degradation. Owed to their simplicity, FTA cards are widely used in clinical testing, forensic science and specimen archives. Originally developed for PCR-based applications that only require short DNA fragments, FTA cards are now being explored as an avenue for whole-genome and epigenetic sequencing applications. FTA cards and their corresponding DNA extraction protocols have not kept pace with advances in sequencing technologies. Because the initial protocols developed for FTA cards were geared towards applications using PCR amplification of short fragments, they typically yield low molecular weight DNA. This issue is particularly pronounced for FTA elute cards where heat-based elution at 95°C leads to DNA denaturation and fragmentation. Isolation of DNA from nucleated blood deposited onto FTA elute cards poses an additional challenge when compared to FTA classic cards, because hemoglobin is irreversibly bound to the card matrix, making the majority of DNA present in nucleated blood inaccessible. Here, we describe an easy, fast, and inexpensive protocol to extract high molecular weight DNA (>10 kb) of nucleated blood stored on FTA elute cards suitable for most genomic library preparations including those that interrogate DNA methylation. Our protocol yields a 14-fold increase in yield compared to numerous alternatives. Using our protocol, we demonstrate that high molecular weight DNA can still be extracted even after storage at ambient temperature for over a decade. Moreover, we show that DNA methylation marks are preserved on FTA elute cards, broadening the utility of FTA elute cards. This opens possibilities for (epi-)genomic studies using historical samples and enabling specimen collection where access to chemicals or cryogenic storage is limited – reducing project costs and extending collection opportunities into remote areas.

Developing in vitro protocols for non-model species poses challenges, and yet are essential for advancing molecular biology studies. This is particularly true for sex determination research that relies on being able to functionally demonstrate the role of genes in determining sex and guiding the process of sex differentiation. Reptile species are attractive models for sex determination research as many species display thermolabile sex systems, allowing for exploration of gene-environment interactions. The first in vitro gonad culture technique for a turtle was published almost 35 years ago in 1990, but these techniques have seen limited use. This is likely because of challenges inherent to the system, where cultures need to be maintained for prolonged periods for gonadal differentiation to occur. All published techniques for long-term cultures involve removing the gonad from the surrounding mesonephros, which causes issues for proper testes development. Here we present the first protocol developed in the reptile model, Pogona vitticeps, that allows the long-term culture of the whole urogenital system supporting differentiation from biopotential gonads to ovaries or testes. Gross morphology is well maintained, and the gonad can be dissected from the mesonephros even after a culture period of up to 19 days. The cultured gonads display sex specific gene expression and morphology. This protocol will facilitate research in sex determination by providing an effective and low-cost alternative to existing protocols and expand capacity for functional manipulation studies in non-model species.

Background: The central bearded dragon ( Pogona vitticeps) is widely distributed in central eastern Australia and adapts readily to captivity. Among other attributes, it is distinctive because it undergoes sex reversal from ZZ genotypic males to phenotypic females at high incubation temperatures. Here, we report an annotated telomere to telomere phased assembly of the genome of a female ZW central bearded dragon. Results: Genome assembly length is 1.75 Gbp with a scaffold N50 of 266.2 Mbp, N90 of 28.1 Mbp, 26 gaps and 42.2% GC content. Most (99.6%) of the reference assembly is scaffolded into 6 macrochromosomes and 10 microchromosomes, including the Z and W microchromosomes, corresponding to the karyotype. The genome assembly exceeds standard recommended by the Earth Biogenome Project (6CQ40): 0.003% collapsed sequence, 0.03% false expansions, 99.8% k-mer completeness, 97.9% complete single copy BUSCO genes and an average of 93.5% of transcriptome data mappable back to the genome assembly. The mitochondrial genome (16,731 bp) and the model rDNA repeat unit (length 9.5 Kbp) were assembled. Male vertebrate sex genes Amh and Amhr2 were discovered as copies in the small non-recombining region of the Z chromosome, absent from the W chromosome. This, coupled with the prior discovery of differential Z and W transcriptional isoform composition arising from pseudoautosomal sex gene Nr5a1 , suggests that complex interactions between these genes, their autosomal copies and their resultant transcription factors and intermediaries, determines sex in the bearded dragon. Conclusion: This high-quality assembly will serve as a resource to enable and accelerate research into the unusual reproductive attributes of this species and for comparative studies across the Agamidae and reptiles more generally. Species Taxonomy: Eukaryota; Animalia; Chordata; Reptilia; Squamata; Iguania; Agamidae; Amphibolurinae; Pogona ; Pogona vitticeps (Ahl, 1926) (NCBI:txid103695). Graphical

This protocol describes the primary culture of lung fibroblasts from Pogona vitticeps and their application in lentivirus transfection efficiency assays. The protocol has been optimized for Pogona vitticeps but can be adapted for other species. The cultured fibroblasts provide a stable and reproducible system for studying gene delivery efficiency, making them a valuable tool for downstream molecular applications. The method ensures high cell viability and proliferation capacity, supporting long-term culture and experimental manipulation. Additionally, the fibroblasts maintain their characteristic morphology and gene expression profiles, allowing for accurate assessment of transfection efficiency and viral vector performance in reptilian cells.

Background: The central bearded dragon (Pogona vitticeps) is widely distributed in central eastern Australia and adapts readily to captivity. Among other attributes, it is distinctive because it undergoes sex reversal from ZZ genotypic males to phenotypic females at high incubation temperatures. Here, we report an annotated near telomere-to-telomere phased assembly of the genome of a female ZW central bearded dragon. Results: Genome assembly length is 1.75 Gbp with a scaffold N50 of 266.2 Mbp, N90 of 28.1 Mbp, 26 gaps, and 42.2% GC content. Most (99.6%) of the reference assembly is scaffolded into 6 macrochromosomes and 10 microchromosomes, including the Z and W microchromosomes, corresponding to the karyotype. The genome assembly exceeds standard recommended by the Earth Biogenome Project (6CQ40): 0.003% collapsed sequence, 0.03% false expansions, 99.8% k-mer completeness, 97.9% complete single-copy BUSCO genes, and an average of 93.5% of transcriptome data mappable back to the genome assembly. The mitochondrial genome (16,731 bp) and the model ribosomal DNA repeat unit (length 9.5 Kbp) were assembled. Male vertebrate sex genes Amh and Amhr2 were discovered as copies in the small non-recombining region of the Z chromosome, absent from the W chromosome. This, coupled with the prior discovery of differential Z and W transcriptional isoform composition arising from pseudo-autosomal sex gene Nr5a1, suggests that complex interactions between these genes, their autosomal copies, and their resultant transcription factors and intermediaries determine sex in the bearded dragon. Conclusion: This high-quality assembly will serve as a resource to enable and accelerate research into the unusual reproductive attributes of this species and for comparative studies across the Agamidae and reptiles more generally.

This protocol is for the in vitro culture of intact embryonic urogenital systems using an agarose substrate. This protocol has been optimised for the model reptile species, Pogona vitticeps, but could be adapted for other species. This protocol supports gonad differentiation from bipotential to ovaries or testes that are distinguishable morphologically and by gene expression patterns. Gross morphology is well maintained even during the longest culture duration (19 days) such that the gonads can be dissect from the surrounding mesonephros, improving utility for downstream sequencing applications.