Atrazine disorganises laminin formation and reduces cell numbers in the tammar testis during early differentiation.

In brief: Atrazine, like oestrogen, disorganises laminin formation and reduces the number of germ cells and Sertoli cells in the developing testes of the tammar wallaby. This study suggests that interfering with the balance of androgen and oestrogen affects the integrity of laminin structure and testis differentiation. Abstract: The herbicide atrazine was banned in Europe in 2003 due to its endocrine disrupting activity but remains widely used. The integrity of the laminin structure in fetal testis cords requires oestrogen signalling but overexposure to xenoestrogens in the adult can cause testicular dysgenesis. However, whether xenoestrogens affect laminin formation in developing testes has not been investigated. Here we examined the effects of atrazine in the marsupial tammar wallaby during early development and compare it with the effects of the anti-androgen flutamide, oestrogen, and the oestrogen degrader fulvestrant. The tammar, like all marsupials, gives birth to altricial young, allowing direct treatment of the developing young during the male programming window (day 20-40 post partum (pp)). Male pouch young were treated orally with atrazine (5 mg/kg), flutamide (10 mg/kg), 17ß-oestradiol (2.5 mg/kg) and fulvestrant (1 mg/kg) daily from day 20 to 40 pp. Distribution of laminin, vimentin, SOX9 and DDX4, cell proliferation and mRNA expression of SRY, SOX9, AMH, and SF1 were examined in testes at day 50 post partum after the treatment. Direct exposure to atrazine, flutamide, 17ß-oestradiol, and fulvestrant all disorganised laminin but had no effect on vimentin distribution in testes. Atrazine reduced the number of germ cells and Sertoli cells when examined at day 40-50 pp and day 20 to 40 pp, respectively. Both flutamide and fulvestrant reduced the number of germ cells and Sertoli cells. Atrazine also downregulated SRY expression and impaired SOX9 nuclear translocation. Our results demonstrate that atrazine can compromise normal testicular differentiation during the critical male programming window.

Fused in sarcoma (FUS) inhibits milk production efficiency in mammals.

Efficient milk production in mammals confers evolutionary advantages by facilitating the transmission of energy from mother to offspring. However, the regulatory mechanism responsible for the gradual establishment of milk production efficiency in mammals, from marsupials to eutherians, remains elusive. Here, we find that mammary gland of the marsupial sugar glider contained milk components during adolescence, and that mammary gland development is less dynamically cyclic compared to that in placental mammals. Furthermore, fused in sarcoma (FUS) is found to be partially responsible for this establishment of low efficiency. In mouse model, FUS inhibit mammary epithelial cell differentiation through the cyclin-dependent kinase inhibitor p57Kip2, leading to lactation failure and pup starvation. Clinically, FUS levels are negatively correlated with milk production in lactating women. Overall, our results shed light on FUS as a negative regulator of milk production, providing a potential mechanism for the establishment of milk production from marsupial to eutherian mammals.

SURGICAL MANAGEMENT OF A TARSAL LUXATION IN A RED KANGAROO (OSPHRANTER RUFUS).

Joint luxations commonly occur in animals secondary to traumatic injury. Because of the unique hind-limb anatomy of macropods, surgical stabilization of orthopedic injuries is considered challenging, and reports of successful management are limited. A 4-yr-old male neutered red kangaroo (Osphranter rufus) presented with a dorsolateral luxation of the left tibiotarsal joint. Surgical reduction and tarsal arthrodesis were performed. Although the full range of motion of the tarsal joint was limited, this kangaroo was still able to ambulate normally at slow speeds following surgery and recovery. The aim of this report was to describe the surgical and postoperative management of a tibiotarsal luxation in a kangaroo. There were significant postoperative complications in this kangaroo, and antibiotic regional limb perfusion was used to treat wound and implant infection.

Metagenomic and Molecular Detection of Novel Fecal Viruses in Free-Ranging Agile Wallabies.

The agile wallaby (Notamacropus agilis) is one of the most abundant marsupial species in northern Queensland and a competent host for the zoonotic Ross River virus. Despite their increased proximity and interactions with humans, little is known about the viruses carried by these animals, and whether any are of conservation or zoonotic importance. Metagenomics and molecular techniques were used in a complementary manner to identify and characterize novel viruses in the fecal samples of free-ranging agile wallabies. We detected a variety of novel marsupial-related viral species including agile wallaby atadenovirus 1, agile wallaby chaphamaparvovirus 1-2, agile wallaby polyomavirus 1-2, agile wallaby associated picobirnavirus 1-9, and a known macropod gammaherpesvirus 3. Phylogenetic analyses indicate that most of these novel viruses would have co-evolved with their hosts (agile wallabies). Additionally, non-marsupial viruses that infect bacteria (phages), plants, insects, and other eukaryotes were identified. This study highlighted the utility of non-invasive sampling as well as the integration of broad-based molecular assays (consensus PCR and next generation sequencing) for monitoring the emergence of potential pathogenic viruses in wildlife species. Furthermore, the novel marsupial viruses identified in this study will enrich the diversity of knowledge about marsupial viruses, and may be useful for developing diagnostics and vaccines.

Intracranial inflammatory polyp with cerebellopontine compression and leptomeningitis secondary to chronic otitis in a red kangaroo.

CNS lesions associated with chronic otitis have not been reported in red kangaroos (Macropus rufus), to our knowledge. Here we describe an intracranial inflammatory polyp secondary to chronic otitis in a 6-y-old female red kangaroo with right auricular discharge, loss of balance, and head tilt. Autopsy highlighted a pale-yellow, firm, intracranial polypoid growth that extended from the right tympanic cavity through the internal acoustic meatus and intracranially, with compression of the right cerebellopontine angle. Anaerobic bacterial culture yielded Bacteroides pyogenes from fresh brain and a right external ear swab. Histologically, the tympanic cavity was effaced by neutrophils and macrophages surrounded by lymphocytes and plasma cells, as well as edematous fibrovascular tissue. The epithelial lining of the mucoperiosteum was hyperplastic, with epithelial pseudoglands surrounded by fibrovascular tissue. Areas of temporal bone lysis and remodeling were associated with the inflammatory changes, which occasionally surrounded adjacent nerves. Fibrovascular tissue and inflammatory cells extended from the tympanic cavity through the internal acoustic meatus and into the intracranial cavity, forming the polypoid growth observed grossly; the polyp consisted of a dense core of fibrovascular tissue with scattered clusters of neutrophils and foamy macrophages. Lymphocytes and plasma cells surrounded the leptomeningeal perivascular spaces in the brainstem, cerebellum, and occipital lobe.

HYPERLIPIDEMIA AND XANTHOMATOSIS IN YELLOW-FOOTED ROCK WALLABIES (PETROGALE XANTHOPUS) UNDER MANAGED CARE.

Xanthomas are localized lipid deposits in organs with associated granulomatous inflammation. Xanthomatosis is a rare condition in both human and veterinary medicine and is often linked to inherited or acquired dyslipidemias. Three female yellow-footed rock wallabies (Petrogale xanthopus) at a single institution were diagnosed via biopsy with cutaneous xanthomas secondary to hypertriglyceridemia and hypercholesterolemia, and an additional two female yellow-footed rock wallabies were diagnosed with xanthomas at a second institution. All cases presented with cutaneous masses at the haired skin and paw pad junctions of the extremities, and/or mucocutaneous junctions of the face or urogenital tract. The clinically affected individuals were overconditioned or obese, had lipemic serum, and had elevations in blood cholesterol and triglyceride levels. When full lipid panels were performed, inverse high- and low-density lipoprotein fractions were observed. Six other individuals at the first institution had identical husbandry but were of more appropriate body condition, were normolipidemic, and had no xanthomas. One of the affected animals was also concurrently diagnosed with hepatic lipidosis via liver biopsy. Pedigree review and evaluation for underlying endocrine diseases such as hypothyroidism were performed. Because all affected animals were found to be related, a genetic predisposition is possible but requires further investigation. Consideration for the predisposition of some individuals for obesity, hyperlipidemia, and subsequent xanthoma formation should be factored in the husbandry and medical management of this species.

Anaplastic thyroid carcinoma in a brush-tailed rock-wallaby (Petrogale penicillata).

BACKGROUND: Neoplasia is considered to be rare in macropods. Anaplastic thyroid carcinoma (ATC) also known as undifferentiated or giant cell carcinoma, is a rare but aggressive and lethal solid tumour reported to affect humans, dogs, cats, racoons and birds. It is derived from poorly differentiated follicular cells and lacks the characteristic architectural pattern of arrangement of tumour cells. ATC has not previously been reported in macropods. CASE REPORT: A brush-tailed rock-wallaby (Petrogale penicillata) was presented for a mass on the ventral neck. A clinical diagnosis of thyroid carcinoma was suspected based on radiology, ultrasound and cytology. Other than palliative care, treatment was declined. Four months later the wallaby was found dead and submitted for necropsy. Gross examination and histopathology demonstrated a unilateral ATC with vascular neoplastic emboli and distant metastases to the heart, lungs and liver. CONCLUSION: The clinical signs associated with thyroid tumours may be the result of localised growth and expansion of the thyroid, metastatic disease or a combination of these effects. Most thyroid tumours are nonfunctional. Based on the lack of typical clinical signs associated with functional thyroid tumours, in this case, we conclude that the thyroid tumour in this wallaby was likely to be nonfunctional. The cause of death in this wallaby was likely due to the heavy tumour burden with compromised cardiorespiratory function exacerbated by dorsoventral compression of the larynx. More study is needed to better understand thyroid neoplasia in macropods.

Successful bilateral phacoemulsification and vitrectomy in a Bennett's wallaby (Macropus rufogriseus).

CASE DESCRIPTION: A 7-month-old hand-reared female Bennett's wallaby (Macropus rufogriseus) was evaluated for bilateral ocular opacity of 3 months' duration. CLINICAL FINDINGS: On physical examination, the wallaby was in good overall condition. An ophthalmic examination revealed mature cataracts in both eyes (OU). The cataracts were suspected to have a nutritional origin as it has been described in hand-reared macropods. Results of tonometry were normal OU. Results of CBC and serum biochemistry were unremarkable. The wallaby was premedicated with medetomidine and ketamine hydrochloride. Ocular ultrasonography performed while the wallaby was anesthetized revealed a thickened and opaque lens, mildly heterogeneous vitreous humor, and no sign of retinal detachment OU. An electroretinogram confirmed functional retinae OU. TREATMENT AND OUTCOME: Surgery was considered necessary for welfare reasons. Phacoemulsification and vitrectomy were performed without intraoperative complication. The wallaby recovered uneventfully, and systemic NSAID, topical corticosteroid, and systemic and topical antimicrobial therapy were prescribed. One year later, the animal had vision OU. Physical examination did not show any vision-threatening postoperative complications. CLINICAL RELEVANCE: The etiology of nutritional cataracts is not fully understood in macropods, but this condition is rather common in these species. To our knowledge, this is the first detailed report of a successful bilateral phacoemulsification and vitrectomy in a Bennett's wallaby.

A WIDESPREAD NOVEL GAMMAHERPESVIRUS IN APPARENTLY HEALTHY WILD QUOKKAS (SETONIX BRACHYURUS): A THREATENED AND ENDEMIC WALLABY OF WESTERN AUSTRALIA.

Five genetically distinct macropodid marsupial herpesviruses have been reported [Macropodid alphaherpesviruses 1 and 2 (MaHV-1 and -2); Macropodid herpesviruses 3 to 5 (MaHV-3 to -5)]. MaHV-2 was originally isolated from an outbreak of fatal disease in captive quokkas (Setonix brachyurus) that were in contact with other macropodid species. This warranted a survey of the presence of herpesviruses in this threatened and endemic Western Australian (WA) wallaby. Blood samples from 142 apparently healthy quokkas were tested for exposure to MaHV-1 and -2 by serology. Of these 142, 121 [Rottnest Island (RI), n = 93; mainland WA, n = 28] were tested for herpesvirus infection by polymerase chain reaction (PCR). Antibodies to MaHV-1 and -2 were detected in one individual [prevalence, 0.7%; 95% confidence interval (CI), 0.1%-3.2%] from the mainland and none from RI. However, a novel gammaherpesvirus [designated Macropodid herpesvirus 6 (MaHV-6)] was detected by PCR in the blood of 13 of 121 individuals (11%; 95% CI, 6.2-17.2). Infection with MaHV-6 was significantly more prevalent on the mainland (7/28; i.e., 25%) compared with RI (6/93; i.e., 6.45%; difference in sample proportions, 95% CI, 6%-32%; P = 0.015). There was no association (P > 0.05) between infection with MaHV-6 and differences in hematology, blood chemistry, peripheral blood cell morphologies, or on clinical status. There was a significant association between infection with MaHV-6 and the presence of Theileria spp. in blood [odds ratio (OR) = 11.0; 95% CI, 2.31-52.3; P = 0.001] and yeast in the nasal lining (OR = 7.0; 95% CI, 1.54-31.8; P = 0.021), suggesting that quokkas may be more susceptible to infection with these microorganisms if also infected with MaHV-6. MaHV-6 infection may be a catalyst for vulnerability to disease with other infectious agents and may pose a significant threat to other macropods. These findings have implications for in situ and ex situ management programs of quokkas.

Respiratory characteristics of the tammar wallaby pouch young and functional limitations in a newborn with skin gas exchange.

A short gestation, low birth weight and presence of cutaneous exchange of O2 and CO2 comprise altricial features of newborn marsupials and that collectively implies a highly immature respiratory system. In the present study, we investigated various respiratory characteristics of the neonatal/postnatal tammar wallaby, a species of marsupial in which > 30% of the newborn's total O2 demands are supported by cutaneous rather than pulmonary gas exchange. The ventilatory response (HVR) to acute hypoxia (10% inspired O2) was absent in the newborn (1 day old) pouch young; a hypoxic hypometabolism contributed entirely to the hyperventilation (increased pulmonary convection requirement). A high (compared to older animals) resting metabolic cost to breathe and an inefficient respiratory system suggest the lack of a HVR might be due to an energetic constraint that impinges on their ability to sustain an increase in ventilation. The latter was supported by the inability of the newborn to tolerate metabolic-ventilatory stimulation following administration of the metabolic uncoupler, 2,4-dinitrophenol (2,4-DNP). At 1 week of age, the cost of breathing was reduced, which coincided with the expression of a significant ventilatory response to hypoxia, a more energetically efficient respiratory system, and tolerance to 2,4-DNP. These data suggest this species of marsupial is born with major respiratory insufficiency, and that their pronounced dependence on the skin for metabolic gas exchange is of critical importance for survival.

The non-human glycan, N-glycolylneuraminic acid (Neu5Gc), is not expressed in all organs and skeletal muscles of nine animal species.

Red meat-derived sialic acid (Sia), N-glycolylneuraminic acid (Neu5Gc), promotes the risk of carcinoma and inflammation. Expression in skeletal muscle and organs across animal species remains unknown. We measured Neu5Gc in skeletal muscle and organ tissues from nine species using UHPLC and found that: (1) neu5Gc concentration in skeletal muscle was highest in goats (166 ± 48.7 µg/g protein), followed by cattle, pig, sheep, horse, cat and deer: >75% was conjugated. No Neu5Gc was detected in kangaroo and dog muscles; (2) total Neu5Gc in organ meats was generally about 2-54% higher than in muscle. Surprisingly Neu5Gc was absent in seven organs of female deer; (3) nine commercial ovine meat cuts contained similar Neu5Gc levels. Thus, red meat Neu5Gc concentration is tissue and species-specific and absent in muscle and organ tissue of some species. Our study provides guidelines for animal meat preferences for consumers and sheds light on the functionality of Neu5Gc.

Trypanosoma cruzi Genotype I and Toxoplasma gondii Co-infection in a Red-Necked Wallaby.

While the health effects of trypanosomes in Australian mammals in their native range are not fully understood, there is evidence of an impact in those species introduced to other geographical regions. Here we report the pathological and molecular features of concurrent fatal trypanosomiasis and toxoplasmosis in an adult female captive red-necked wallaby (syn. Bennett's wallaby; Macropus rufogriseus) from Bee County, Texas, USA. The animal exhibited no clinical signs prior to sudden death. On necropsy, the main findings were generalized organ congestion and bilateral renal petechiation. Microscopically, the main finding was lymphohistiocytic and necrotizing pancarditis with intrasarcoplasmic protozoal pseudocysts containing amastigotes and occasional intrahistiocytic amastigotes, morphologically compatible with Trypanosoma cruzi, as well as rare intrasarcoplasmic protozoal tissue cysts with zoites morphologically compatible with Toxoplasma gondii. Other lesions included acute centrilobular to panlobular necrotizing hepatitis with intrahepatocellular T. gondii cysts, necrotizing splenitis, pulmonary oedema with fibrin, histiocytosis and rare fibrin microthrombi, and acute renal tubular degeneration with proteinosis and pigmented casts suggestive of haemoglobinuria or myoglobinuria. Immunohistochemical labelling confirmed intralesional T. gondii cysts and molecular analyses identified T. cruzi genotype I and T. gondii. This is a unique case that, to the best of our knowledge, represents the first description of T. cruzi and T. gondii co-infection, as well as the first record of naturally occurring infection T. cruzi genotype I infection in macropodids. This case adds to the epidemiological knowledge on Chagas disease in the USA, particularly in Texas where there is a high prevalence of human and canine trypanosomiasis.

Induction of synchronous oestrus but not ovulation after pre-treatment with the GnRH agonist, Lucrin® Depot, in the tammar wallaby.

We investigated the capacity for pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to induce synchronous oestrus and ovulation in the tammar wallaby (Notamacropus eugenii) after follicular suppression with Lucrin® Depot, a one-month GnRH agonist. On Day 0 pouch young were removed (RPY) to reactivate a normal oestrous cycle and then two groups were treated with vehicle (Control; n = 5 and Superovulation (SOvn); n = 5) and two groups were treated with 7.5 mg of Lucrin Depot (Lucrin; n = 6; Lucrin+SOvn, n = 6). On Day 20 RPY the SOvn and Lucrin+SOvn Group received 20 IU of PMSG, which was followed on Day 23 RPY with 500 IU of hCG. The Lucrin+SOvn females underwent a more synchronous oestrus with 5 of 6 mating on Day 26 RPY while the SOvn (n = 5/5) and Control (n = 5/5) Groups copulated over two days, between Day 25-27 RPY and Day 27-29 RPY respectively. Mating plugs were not detected in any of the females in the Lucrin Group by Day 31 RPY. Autopsy on Day 31 RPY confirmed all females in each treatment group had undergone a reactivated cycle as evidenced by the presence of a large corpus luteum (CL) in one ovary. At autopsy the females in the Lucrin+SOvn Group had highly stimulated reproductive tracts, and their ovaries contained many follicles >3 mm; 14 ± 2.1 and 15.3 ± 2.1 follicles >3 mm in the CL-bearing ovary and contralateral ovary respectively. Similarly, females in the SOvn Group had 11.4 ± 2.4 and 17.4 ± 1.9 follicles >3 mm in each respective ovary. Uterine flushing and ovarian histology confirmed that females in Lucrin+SOvn and SOvn Groups had not ovulated, but normal oocytes were present in the follicles. By comparison, the Control Group had ovulated with a single embryo being recovered from the uterus of 4 of 5 females. In contrast to all groups, females in the Lucrin Group showed follicular suppression (all follicles <1.5 mm) and an unstimulated reproductive tract. We conclude that a suppression plus stimulation regimen using Lucrin Depot followed by PMSG and hCG has the capacity to synchronise oestrus, and that 20 IU of PMSG stimulates the development of antral follicles >3 mm in both ovaries. However, a single 500 IU treatment of hCG on Day 23 RPY was not able to induce ovulation in the tammar wallaby.

Hormonal and Molecular Regulation of Phallus Differentiation in a Marsupial Tammar Wallaby.

Congenital anomalies in phalluses caused by endocrine disruptors have gained a great deal of attention due to its annual increasing rate in males. However, the endocrine-driven molecular regulatory mechanism of abnormal phallus development is complex and remains largely unknown. Here, we review the direct effect of androgen and oestrogen on molecular regulation in phalluses using the marsupial tammar wallaby, whose phallus differentiation occurs after birth. We summarize and discuss the molecular mechanisms underlying phallus differentiation mediated by sonic hedgehog (SHH) at day 50 pp and phallus elongation mediated by insulin-like growth factor 1 (IGF1) and insulin-like growth factor binding protein 3 (IGFBP3), as well as multiple phallus-regulating genes expressed after day 50 pp. We also identify hormone-responsive long non-coding RNAs (lncRNAs) that are co-expressed with their neighboring coding genes. We show that the activation of SHH and IGF1, mediated by balanced androgen receptor (AR) and estrogen receptor 1 (ESR1) signalling, initiates a complex regulatory network in males to constrain the timing of phallus differentiation and to activate the downstream genes that maintain urethral closure and phallus elongation at later stages.

Bilateral radial and ulnar fractures in a red kangaroo (Macropus rufus).

CASE DESCRIPTION: An 11-month-old sexually intact male red kangaroo (Macropus rufus) was examined because of bilateral radial and ulnar fractures. CLINICAL FINDINGS: Radiography of the forelimbs revealed bilateral, short oblique fractures in the proximal to mid diaphyses of the radii and ulnae. Fractures were overriding and moderately displaced. Wider than expected gaps were evident in the humeroulnar and humeroradial joints bilaterally. Although several physes remained open, no proximal radial physis was radiographically evident. TREATMENT AND OUTCOME: Dual bone fixation was performed bilaterally, and dynamic luxation of the left radial head was identified and stabilized intraoperatively. Although satisfactory function of both forelimbs was evident at 8 weeks and 26 months after surgery, a persistent gait abnormality affecting the right forelimb was noted. Twenty-six months after surgery, radiography revealed bilateral proximal radial physes and resolution of the abnormally wide gaps in the humeroradial and humeroulnar joints. Despite dual bone fixation, synostoses formed bilaterally and may have contributed to the persistent lameness in the kangaroo's right forelimb. CLINICAL RELEVANCE: Veterinarians treating kangaroos should be aware of difficulties in determining skeletal maturity and planning fracture stabilization because of potential differences in skeletal growth and fracture healing, compared with other species. We described critical issues observed in the treatment and outcome of the kangaroo of the present report and provided lessons learned as well as potential explanations of these issues to facilitate future treatment of kangaroos with forelimb fractures.

Metastatic Osteolytic Angioleiomyosarcoma Induced by a Foreign Body in a Kangaroo (Macropus giganteus).

An adult female Eastern Grey kangaroo (Macropus giganteus) was examined for lameness due to a mass located at the right tibiotarsal joint. Radiographs revealed a metal arrowhead-shaped foreign body within the mass alongside a pathologic fracture. Grossly, at necropsy, there was a multilobulated and haemorrhagic mass with intralesional metal fragments, associated osteolysis of the tibiotarsus and pulmonary metastatic nodules. Histologically the neoplasm was composed of plump elongated and fusiform cells that often formed concentrically around small vessels. The pulmonary nodules were composed of similar neoplastic cells, necrosis and haemorrhage. Neoplastic cells were positive immunohistochemically for vimentin and smooth muscle actin. The histological features, immunohistochemical profiles and behaviour of this tumour support a diagnosis of a primary angioleiomyosarcoma with lung metastasis associated with a metal foreign body.

Activity budget and spatial distribution of Bennett's wallabies (Macropus rufogriseus) in open versus closed exhibit designs.

Although many studies investigating the impacts of zoo exhibit designs on captive animals exist, none have been performed on how they influence the behavior and welfare of captive Bennett's wallabies (Macropus rufogriseus). Here, we assess the impact of exhibit design on the activity budget and spatial distribution of Bennett's wallabies. We compared animal behavior in two open exhibits (i.e. physical interaction between animals and visitors permitted) to two closed exhibits (i.e. physical interaction between animals and visitors prohibited). Behavioral data were collected using focal sampling, and spatial distribution was recorded on exhibit maps at regular time intervals. We found a significant increase in feeding and interactive behaviors in closed exhibits in comparison to open exhibits. However, other behaviors such as resting, locomotion, and vigilance did not vary with design. Functional use of space was similar between both designs; however, the effect of habituation may be relevant to consider in future studies. Although some support for visitor effects were present, our study provided no evidence for strong impacts of exhibit design on Bennett's wallaby welfare. Our study emphasizes the need for additional research into the impacts of how zoo environments affect Bennett's wallaby behavior and welfare.

Transcriptomic Analysis of MAP3K1 and MAP3K4 in the Developing Marsupial Gonad.

MAPKs affect gonadal differentiation in mice and humans, but whether this applies to all mammals is as yet unknown. Thus, we investigated MAPK expression during gonadal differentiation and after treatment with oestrogen in a distantly related mammal, the marsupial tammar wallaby, using our model of oestrogen-induced gonadal sex reversal. High-throughput RNA-sequencing was carried out on gonads collected from developing tammar 2 days before birth to 8 days after birth to characterise MAPK and key sexual differentiation markers. Day 25 foetal testes were cultured for 120 h in control medium or medium supplemented with exogenous oestrogen and processed for RNA-seq to identify changes in gene expression in response to oestrogen. MAPK pathway genes in the tammar were highly conserved at the sequence and amino acid level with those of mice and humans. Marsupial MAP3K1 and MAP3K4 clustered together in a separate branch from eutherian mammals. There was a marked decrease in the expression of male-determining genes SOX9 and AMH and increase in the female marker FOXL2 in oestrogen-treated male gonads. Only MAP3K1 expression increased in male gonads in response to oestrogen while other MAPK genes remained unaffected. This study suggests that MAP3K1 can be influenced by exogenous oestrogens during gonadal differentiation in this marsupial.

Effects of androgen and oestrogen on IGF pathways controlling phallus growth.

The development of the mammalian phallus involves hormone-dependent mesenchymal-epithelial signalling mechanisms that contribute to urethral closure and regulation of phallus elongation and growth. In marsupials, most differentiation and growth of the phallus occurs post-natally, making them amenable to direct hormone treatment. Expression of IGFs, FGFs, EFNB2, MAFB, DLX5 and AP-1 mRNAs in the phallus at day 50 post-partum (pp) were altered after treatment of tammar wallaby young from day 20 to 40 pp with androgen, oestrogen or after castration at day 25 pp. However, the most interesting changes occurred in the IGF pathway genes. Androgen treatment upregulated IGF1 in female phalluses and oestrogen treatment upregulated IGF1 in male phalluses, but it was downregulated by castration. IGFBP3 was higher in female phalluses and downregulated by androgen. IGF1 expression was higher in all untreated male than in female phalluses from day 50 to 150 pp, but IGFBP3 had the reverse pattern. At day 90 pp, when urethral closure in males is progressing and male phallus growth is accelerating. IGF1 and PCNA protein were only detected in the male urorectal septum, suggesting for the first time that closure and elongation may involve IGF1 activation of cell proliferation specifically in male phalluses. These effects of sex steroids on gene expression and on the IGF1 signalling pathway in particular, suggest that the developing phallus may be especially susceptible to perturbation by exogenous hormones.

Rapid Pliocene adaptive radiation of modern kangaroos.

Differentiating between ancient and younger, more rapidly evolved clades is important for determining paleoenvironmental drivers of diversification. Australia possesses many aridity-adapted lineages, the origins of which have been closely linked to late Miocene continental aridification. Using dental macrowear and molar crown height measurements, spanning the past 25 million years, we show that the most iconic Australian terrestrial mammals, "true" kangaroos (Macropodini), adaptively radiated in response to mid-Pliocene grassland expansion rather than Miocene aridity. In contrast, low-crowned, short-faced kangaroos radiated into predominantly browsing niches as the late Cenozoic became more arid, contradicting the view that this was an interval of global browser decline. Our results implicate warm-to-cool climatic oscillations as a trigger for adaptive radiation and refute arguments attributing Pleistocene megafaunal extinction to aridity-forced dietary change.