School of BioSciences - Research Publications
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ItemGenetic sex test for the short-beaked echidna (Tachyglossus aculeatus)(SPRINGER, 2022-04-30)Monotremes (echidnas and platypus) possess five X and four or five Y sex chromosomes, respectively, that evolved independently from the sex chromosomes found in therian mammals. While the platypus has obvious venomous spurs in the male, the short-beaked echidna (Tachyglossus aculeatus) lacks easily identifiable sexually dimorphic characteristics, making it difficult to sex adults out of the breeding season and almost impossible to sex juveniles or embryonic material. Here, we used restriction-site associated DNA sequencing (RADseq) to identify novel sex-specific markers in the short-beaked echidna. We identified and validated a subset of male-specific markers that can be used as a non-invasive genetic sex test for the short-beaked echidna. We also assessed how laboratory conditions, including DNA extraction protocol and number of PCR cycles, can influence the outcome of genetic sex tests. The combined use of these markers will provide a valuable toolkit for researchers, conservationists, and zoo-keepers to reliably and non-invasively determine sex in the short-beaked echidna.
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ItemThe Unique Penile Morphology of the Short-Beaked Echidna, Tachyglossus aculeatus(KARGER, 2021-09)Monotremes diverged from therian mammal ancestors approximately 184 million years ago and have a number of novel reproductive characteristics. One in particular is their penile morphology. There are differences between echidna and platypus phalluses, but both are somewhat similar in structure to the reptilian phallus. The echidna penis consists of 4 rosette glans, each of which contains a termination of the quadrifurcate urethra, but it appears that only 2 of the 4 glans become erect at any one time. Despite this, only a few historical references describe the structure of the echidna penis and none provides an explanation for the mechanisms of unilateral ejaculation. This study confirmed that the echidna penis contains many of the same overall structures and morphology as other mammalian penises and a number of features homologous with reptiles. The corpus cavernosum is well supplied with blood, extends up to the base of the glans penis and is primarily responsible for erection. However, the echidna possesses 2 distinct corpora spongiosa separated by a septum, each of which surround the urethra only distal to the initial urethral bifurcation in the glans penis. Together with the bifurcation of the main penile artery, this provides a mechanism by which blood flow could be directed to only one corpus spongiosum at a time to maintain an open urethra that supplies 2 of the 4 glans to facilitate unilateral ejaculation.
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ItemPlasma progesterone secretion during gestation of the captive short-beaked echidna(BIOSCIENTIFICA LTD, 2021-10)This study describes the progesterone profile during pregnancy in sexually mature female captive short-beaked echidnas (Tachyglossus aculeatus aculeatus). Echidnas were monitored daily by video surveillance to confirm key reproductive behaviour. Plasma samples were collected and pouch morphology was assessed three times a week. The pouch of the female echidna only develops during gestation and it was possible to create a four-stage grading system using the most distinguishable characteristics of pouch development. Maximum pouch development was associated with declining progesterone concentrations, with the pouch closing in a drawstring-like manner at oviposition. Control of pouch development in pregnant echidnas is not yet clear but later pouch development is associated with a decrease in progesterone and pouch closure and may be under mechanical influences of the egg or young in the pouch. The length of pregnancy was 16.7 ± 0.2 days with a 15.1 ± 1.0 days luteal phase followed by an incubation period in the pouch. Eggs could be detected in utero at least 4 days before oviposition. Plasma progesterone peaked at 10.5 ± 0.9 ng/mL within 12 days of mating but then declined to basal levels within 1 day of oviposition and remained basal throughout egg incubation, confirming that progesterone is elevated throughout pregnancy and that gestation does not extend beyond the luteal phase. After the loss of an egg or pouch young, most females entered a second oestrous cycle and ovulated, suggesting echidnas are seasonally polyoestrous. The duration of the luteal phase in the echidna corresponds with that observed in other mammals.
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ItemNo Preview AvailableComparing the potential for maternal-fetal signalling in oviparous and viviparous lizards(ROYAL SOC, 2022-12-05)The evolution of a placenta requires several steps including changing the timing of reproductive events, facilitating nutrient exchange, and the capacity for maternal-fetal communication. To understand the evolution of maternal-fetal communication, we used ligand-receptor gene expression as a proxy for the potential for cross-talk in a live-bearing lizard (Pseudemoia entrecasteauxii) and homologous tissues in a related egg-laying lizard (Lampropholis guichenoti). Approximately 70% of expressed ligand/receptor genes were shared by both species. Gene ontology (GO) analysis showed that there was no GO-enrichment in the fetal membranes of the egg-laying species, but live-bearing fetal tissues were significantly enriched for 50 GO-terms. Differences in enrichment suggest that the evolution of viviparity involved reinforcing specific signalling pathways, perhaps to support fetal control of placentation. One identified change was in transforming growth factor beta signalling. Using immunohistochemistry, we show the production of the signalling molecule inhibin beta B (INHBB) occurs in viviparous fetal membranes but was absent in closely related egg-laying tissues, suggesting that the evolution of viviparity may have involved changes to signalling via this pathway. We argue that maternal-fetal signalling evolved through co-opting expressed signalling molecules and recruiting new signalling molecules to support the complex developmental changes required to support a fetus in utero. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.
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ItemValidation of a non-invasive assessment technique for quantifying faecal glucocorticoid metabolite concentrations in the short-beaked echidna (Tachyglossus aculeatus)(ACADEMIC PRESS INC ELSEVIER SCIENCE, 2022-10-01)The monotreme adrenocortical response to stress may not rely as heavily on the hypothalamic-pituitaryadrenal (HPA) axis compared to other mammals. This study aimed to validate a technique in which glucocorticoid metabolites could be quantified non-invasively in short-beaked echidna faeces by examining the secretion of glucocorticoids (GC) using an adrenocorticotrophic hormone (ACTH) challenge on sexually mature captive echidnas. Echidnas were housed individually for 15 days, with the ACTH challenge occurring on day five. Blood samples were collected on day five during the challenge and faecal samples were collected each morning for the 15 days. Both sample types were analysed for glucocorticoids (GC) or its metabolites. Plasma corticosterone concentrations increased significantly after 30 min and 60 min relative to time 0, whilst plasma cortisol concentrations increased significantly after 60 min. The ACTH challenge also resulted in an increase in glucocorticoid metabolite concentration in faecal samples from four of the six echidnas detected one to two days post ACTH injection, thereby validating a non-invasive method to assess adrenal response in the echidna. These results confirm that echidnas respond to a synthetic ACTH challenge in a similar manner to that of eutherian species indicating that echidnas appear to use the HPA axis in their stress response.
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ItemProteomics and Deep Sequencing Comparison of Seasonally Active Venom Glands in the Platypus Reveals Novel Venom Peptides and Distinct Expression Profiles(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-11)The platypus is a venomous monotreme. Male platypuses possess a spur on their hind legs that is connected to glands in the pelvic region. They produce venom only during the breeding season, presumably to fight off conspecifics. We have taken advantage of this unique seasonal production of venom to compare the transcriptomes of in- and out-of-season venom glands, in conjunction with proteomic analysis, to identify previously undiscovered venom genes. Comparison of the venom glands revealed distinct gene expression profiles that are consistent with changes in venom gland morphology and venom volumes in and out of the breeding season. Venom proteins were identified through shot-gun sequenced venom proteomes of three animals using RNA-seq-derived transcripts for peptide-spectral matching. 5,157 genes were expressed in the venom glands, 1,821 genes were up-regulated in the in-season gland, and 10 proteins were identified in the venom. New classes of platypus-venom proteins identified included antimicrobials, amide oxidase, serpin protease inhibitor, proteins associated with the mammalian stress response pathway, cytokines, and other immune molecules. Five putative toxins have only been identified in platypus venom: growth differentiation factor 15, nucleobindin-2, CD55, a CXC-chemokine, and corticotropin-releasing factor-binding protein. These novel venom proteins have potential biomedical and therapeutic applications and provide insights into venom evolution.
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ItemOrientation pinwheels in primary visual cortex of a highly visual marsupial(AMER ASSOC ADVANCEMENT SCIENCE, 2022-09-30)Primary visual cortices in many mammalian species exhibit modular and periodic orientation preference maps arranged in pinwheel-like layouts. The role of inherited traits as opposed to environmental influences in determining this organization remains unclear. Here, we characterize the cortical organization of an Australian marsupial, revealing pinwheel organization resembling that of eutherian carnivores and primates but distinctly different from the simpler salt-and-pepper arrangement of eutherian rodents and rabbits. The divergence of marsupials from eutherians 160 million years ago and the later emergence of rodents and rabbits suggest that the salt-and-pepper structure is not the primitive ancestral form. Rather, the genetic code that enables complex pinwheel formation is likely widespread, perhaps extending back to the common therian ancestors of modern mammals.
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ItemReproductive behaviour before and after oestrus and oviposition in the captive short-beaked echidna (Tachyglossus aculeatus).(CSIRO Publishing, 2022-09)CONTEXT: Most of our current knowledge regarding echidna reproductive behaviour is based on qualitative measurements; therefore, it is unclear if specific behavioural cues could be utilised in their captive reproductive management. AIMS: This study aimed to identify quantitative changes in general and reproductive behaviour of echidna breeding pairs and pregnant females that might facilitate the detection of oestrus and impending oviposition and provide a summary of reproductive behaviour observed in a captive colony over a three-year observation period. METHODS: Three echidna breeding pairs and two trios were monitored daily for seven reproductive and eight general behaviours during the 2020 breeding season. After confirmed copulation, females were monitored for four egg-laying and eight general behaviours until egg incubation. General observations of reproductive behaviours during the 2018-2020 breeding seasons were recorded as part of routine husbandry. KEY RESULTS: For breeding pairs, there was a significant rate of change over time before and after copulation for the behaviours 'urogenital sniffing', 'rolling' and 'copulation attempt'. For pregnant females, time engaged in 'pacing' significantly increased while 'time eating' and the 'quantity of food eaten' significantly decreased on the day of oviposition. We were not able to identify oestrus from specific behaviours, but our observations suggest that the female echidna's period of receptivity is less than 24h. CONCLUSIONS: The frequency that males express 'urogenital sniffing', 'rolling' and 'copulation attempt' toward the female can be used to alert zookeepers that copulation has likely occurred. Increased pacing, reduced feeding time and quantity of food eaten can aid zookeepers to identify impending oviposition. IMPLICATIONS: This study demonstrates that there are quantifiable changes in specific echidna behaviours that can be incorporated into zoo husbandry practices to improve the reproductive management of this species.
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ItemPlacental imprinting of SLC22A3 in the IGF2R imprinted domain is conserved in therian mammals(BMC, 2022-08-27)BACKGROUND: The eutherian IGF2R imprinted domain is regulated by an antisense long non-coding RNA, Airn, which is expressed from a differentially methylated region (DMR) in mice. Airn silences two neighbouring genes, Solute carrier family 22 member 2 (Slc22a2) and Slc22a3, to establish the Igf2r imprinted domain in the mouse placenta. Marsupials also have an antisense non-coding RNA, ALID, expressed from a DMR, although the exact function of ALID is currently unknown. The eutherian IGF2R DMR is located in intron 2, while the marsupial IGF2R DMR is located in intron 12, but it is not yet known whether the adjacent genes SLC22A2 and/or SLC22A3 are also imprinted in the marsupial lineage. In this study, the imprinting status of marsupial SLC22A2 and SLC22A3 in the IGF2R imprinted domain in the chorio-vitelline placenta was examined in a marsupial, the tammar wallaby. RESULTS: In the tammar placenta, SLC22A3 but not SLC22A2 was imprinted. Tammar SLC22A3 imprinting was evident in placental tissues but not in the other tissues examined in this study. A putative promoter of SLC22A3 lacked DNA methylation, suggesting that this gene is not directly silenced by a DMR on its promoter as seen in the mouse. Based on immunofluorescence, we confirmed that the tammar SLC22A3 is localised in the endodermal cell layer of the tammar placenta where nutrient trafficking occurs. CONCLUSIONS: Since SLC22A3 is imprinted in the tammar placenta, we conclude that this placental imprinting of SLC22A3 has been positively selected after the marsupial and eutherian split because of the differences in the DMR location. Since SLC22A3 is known to act as a transporter molecule for nutrient transfer in the eutherian placenta, we suggest it was strongly selected to control the balance between supply and demand of nutrients in marsupial as it does in eutherian placentas.
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ItemEvolution of the Short Form of DNMT3A, DNMT3A2, Occurred in the Common Ancestor of Mammals(OXFORD UNIV PRESS, 2022-07-02)Genomic imprinting is found in marsupial and eutherian mammals, but not in monotremes. While the primary regulator of genomic imprinting in eutherians is differential DNA methylation between parental alleles, conserved imprinted genes in marsupials tend to lack DNA methylation at their promoters. DNA methylation at eutherian imprinted genes is mainly catalyzed by a DNA methyltransferase (DNMT) enzyme, DNMT3A. There are two isoforms of eutherian DNMT3A: DNMT3A and DNMT3A2. DNMT3A2 is the primary isoform for establishing DNA methylation at eutherian imprinted genes and is essential for eutherian genomic imprinting. In this study, we investigated whether DNMT3A2 is also present in the two other mammalian lineages, marsupials and monotremes. We identified DNMT3A2 in both marsupials and monotremes, although imprinting has not been identified in monotremes. By analyzing genomic sequences and transcriptome data across vertebrates, we concluded that the evolution of DNMT3A2 occurred in the common ancestor of mammals. In addition, DNMT3A/3A2 gene and protein expression during gametogenesis showed distinct sexual dimorphisms in a marsupial, the tammar wallaby, and this pattern coincided with the sex-specific DNA methylation reprogramming in this species as it does in mice. Our results show that DNMT3A2 is present in all mammalian groups and suggests that the basic DNMT3A/3A2-based DNA methylation mechanism is conserved at least in therian mammals.