Tammar Research Articles

Inhibiting p38α and β MAPK affects testis development in the marsupial tammar wallaby.

The MAPK genes are critical for gonadal differentiation in eutherian mammals but their role in marsupial mammals is unknown. Characterisation and phylogenetic analyses of the tammar wallaby MAPK genes shows these genes are highly conserved with their orthologues in mammalian and non-mammalian species. We cultured sexually indifferent tammar gonads in the presence of p38α and β MAPK inhibitor, SB202190. SB202190 downregulated SOX9 and AMH levels in XY treated gonads when compared to controls, similar to the effects of oestrogen on the MAPK pathway in males. In contrast, XX gonads treated with the SB202190 inhibitor showed no change in mRNA expression between the control and treated gonads for any of the markers tested. This study confirms that components of the MAPK pathway drive testis differentiation via the key downstream genes SOX9 and AMH in marsupials as is observed in eutherian mammals.

The extension of mammalian pregnancy required taming inflammation: Independent evolution of extended placentation in the tammar wallaby.

In the first live-bearing mammals, pregnancy was likely short and ended with a brief period of inflammatory maternal-fetal interaction. This mode of reproduction has been retained in many marsupials. While inflammation is key to successful implantation in eutherians, a key innovation in eutherians is the ability to switch off this inflammation after it has been initiated. This extended period, in which inflammation is suppressed, likely allowed for an extended period of placentation. Extended placentation has evolved independently in one lineage of marsupials, the macropodids (wallabies and kangaroos), with placentation lasting beyond the 2 to 4 d seen in other marsupial taxa, which allows us to investigate the role of inflammation response after attachment in the extension of placentation in mammals. By comparing gene expression changes at attachment in three marsupial species, the tammar wallaby, opossum, and fat-tailed dunnart, we show that inflammatory attachment is an ancestral feature of marsupial implantation. In contrast to eutherians, where attachment-related (quasi-) inflammatory reaction is even involved in epitheliochorial placentation (e.g., pig), this study found no evidence of a distinct attachment-related reaction in wallabies. Instead, only a small number of inflammatory genes are expressed at distinct points of gestation, including IL6 before attachment, LIF throughout placentation, and prostaglandins before birth. During parturition, a more distinct inflammatory reaction is detectable, likely involved in precipitating the parturition cascade similar to eutherians. We suggest that in wallaby, extended gestation became possible by avoiding an inflammatory attachment reaction, which is a different strategy than seen in eutherians.

PRKACB is a novel imprinted gene in marsupials

BackgroundGenomic imprinting results in parent-of-origin-specific gene expression and, among vertebrates, is found only in therian mammals: marsupials and eutherians. A differentially methylated region (DMR), in which the methylation status of CpG dinucleotides differs between the two alleles, can mark the parental identity of imprinted genes. We developed a computational pipeline that detected CpG islands (CGIs) marked by both methylated and unmethylated signals in whole genome bisulfite sequencing data. This approach identified candidate marsupial DMRs in a publicly available koala methylome. One of these candidate DMRs was associated with PRKACB, a gene encoding the protein kinase A catalytic subunit beta. Nothing is known about the imprinting status of PRKACB in eutherian mammals although mutations of this gene are associated with endocrine neoplasia and other developmental disorders.ResultsIn the tammar wallaby and brushtail possum there was parent-of-origin-specific DNA methylation in the PRKACB DMR in which the maternal allele was methylated and the paternal allele was unmethylated. There were multiple RNAs transcribed from this locus. Allele-specific expression analysis identified paternal expression of a PRKACB lncRNA and an mRNA isoform. Comparison of the PRKACB gene start site between marsupials and eutherians demonstrated that the CGI is longer in marsupials. The PRKACB gene product functions in the same signalling pathway as the guanine nucleotide-binding protein alpha subunit encoded at the GNAS locus, a known eutherian imprinted gene. In a mouse methylome Gnas had three differentially methylated CGIs, while in the koala methylome the GNAS locus had two unmethylated CGIs.ConclusionsWe conclude that PRKACB is a novel, DMR-associated marsupial imprinted gene. Imprinting of PRKACB in marsupials and GNAS in eutherians may indicate a conserved selection pressure for imprinting of the protein kinase A signalling pathway in therians with the two lineages adapting by imprinting different genes.

Imprinted X chromosome inactivation in marsupials: The paternal X arrives at the egg with a silent DNA methylation profile

X chromosome inactivation (XCI) is an epigenetic process that results in the transcriptional silencing of one X chromosome in the somatic cells of females. This phenomenon is common to both eutherian and marsupial mammals, but there are fundamental differences. In eutherians, the X chosen for silencing is random. DNA methylation on the eutherian inactive X is high at transcription start sites (TSSs) and their flanking regions, resulting in universally high DNA methylation. This contrasts XCI in marsupials where the paternally derived X is always silenced, and in which DNA methylation is low at TSSs and flanking regions. Here, we examined the DNA methylation status of the tammar wallaby X chromosome during spermatogenesis to determine the DNA methylation profile of the paternal X prior to and at fertilization. Whole genome enzymatic methylation sequencing was carried out on enriched flow-sorted populations of premeiotic, meiotic, and postmeiotic cells. We observed that the X displayed a pattern of DNA methylation from spermatogonia to mature sperm that reflected the inactive X in female somatic tissue. Therefore, the paternal X chromosome arrives at the egg with a DNA methylation profile that reflects the transcriptionally silent X in adult female somatic tissue. We present this epigenetic signature as a candidate for the long sought-after imprint for paternal XCI in marsupials.

The structure of the TH/INS locus and the parental allele expressed are not conserved between mammals

Parent-of-origin-specific expression of imprinted genes is critical for successful mammalian growth and development. Insulin, coded by the INS gene, is an important growth factor expressed from the paternal allele in the yolk sac placenta of therian mammals. The tyrosine hydroxylase gene TH encodes an enzyme involved in dopamine synthesis. TH and INS are closely associated in most vertebrates, but the mouse orthologues, Th and Ins2, are separated by repeated DNA. In mice, Th is expressed from the maternal allele, but the parental origin of expression is not known for any other mammal so it is unclear whether the maternal expression observed in the mouse represents an evolutionary divergence or an ancestral condition. We compared the length of the DNA segment between TH and INS across species and show that separation of these genes occurred in the rodent lineage with an accumulation of repeated DNA. We found that the region containing TH and INS in the tammar wallaby produces at least five distinct RNA transcripts: TH, TH-INS1, TH-INS2, lncINS and INS. Using allele-specific expression analysis, we show that the TH/INS locus is expressed from the paternal allele in pre- and postnatal tammar wallaby tissues. Determining the imprinting pattern of TH/INS in other mammals might clarify if paternal expression is the ancestral condition which has been flipped to maternal expression in rodents by the accumulation of repeat sequences.

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

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. 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), 17β-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, 17β-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.

Characterization of the marsupial endogenous retrovirus walb with a focus on satellite DNA formation

The walbRep megasatellite DNA found in the red-necked wallaby was formed from the walb endogenous retrovirus. Our previous PCR experiments suggested the presence of walb and absence of walbRep in the genome of the tammar wallaby, which diverged from the red-necked wallaby 2-3 Mya. The results failed to exclude the possibility that certain walbRep sequences might have remained undetected owing to variation in the primer-annealing regions; therefore, the aforementioned suggestion was not confirmed. To obtain conclusive evidence, we analyzed the structure of walb sequences drawn from the tammar wallaby genome database recently updated to a chromosome-level assembly. All walb copies existed as separate DNA segments, not constituting tandem repeats. We concluded that walbRep was formed in the red-necked wallaby lineage after its divergence from the tammar wallaby. We also confirm the presence of a walb copy with an anomalistic, complex structure and propose a simple model for its generation mechanism.

Characterization of extracellular spike waveforms recorded in wallaby primary visual cortex.

Extracellular recordings were made from 642 units in the primary visual cortex (V1) of a highly visual marsupial, the Tammar wallaby. The receptive field (RF) characteristics of the cells were objectively estimated using the non-linear input model (NIM), and these were correlated with spike shapes. We found that wallaby cortical units had 68% regular spiking (RS), 12% fast spiking (FS), 4% triphasic spiking (TS), 5% compound spiking (CS) and 11% positive spiking (PS). RS waveforms are most often associated with recordings from pyramidal or spiny stellate cell bodies, suggesting that recordings from these cell types dominate in the wallaby cortex. In wallaby, 70-80% of FS and RS cells had orientation selective RFs and had evenly distributed linear and nonlinear RFs. We found that 47% of wallaby PS units were non-orientation selective and they were dominated by linear RFs. Previous studies suggest that the PS units represent recordings from the axon terminals of non-orientation selective cells originating in the lateral geniculate nucleus (LGN). If this is also true in wallaby, as strongly suggested by their low response latencies and bursty spiking properties, the results suggest that significantly more neurons in wallaby LGN are already orientation selective. In wallaby, less than 10% of recorded spikes had triphasic (TS) or sluggish compound spiking (CS) waveforms. These units had a mixture of orientation selective and non-oriented properties, and their cellular origins remain difficult to classify.

The alternate pathway of androgen metabolism and window of sensitivity.

Since the discovery in 1968 that dihydrotestosterone (DHT) is a major mediator of androgen action, a convincing body of evidence has accumulated to indicate that the major pathway of DHT formation is the 5α-reduction of circulating testosterone in androgen target tissues. However, we now know that DHT can also be formed in peripheral tissues by the oxidation of 5α-androstane-3α,17β-diol (adiol). This pathway is responsible for the formation of the male phenotype. We discuss the serendipitous discovery in the tammar wallaby of an alternate pathway by which adiol is formed in the testes, secreted into plasma and converted in peripheral tissues to DHT. This alternate pathway is responsible for virilisation of the urogenital system in this species and is present in the testes at the onset of male puberty of all mammals studied so far. This is the first clear-cut function for steroid 5α-reductase 1 in males. Unexpectedly, the discovery of this pathway in this Australian marsupial has had a major impact in understanding the pathophysiology of aberrant virilisation in female newborns. Overactivity of the alternate pathway appears to explain virilisation in congenital adrenal hyperplasia CAH, in X-linked 46,XY disorders of sex development. It also appears to be important in polycystic ovarian syndrome (PCOS) since PCOS ovaries have enhanced the expression of genes and proteins of the alternate pathway. It is now clear that normal male development in marsupials, rodents and humans requires the action of both the classic and the alternate (backdoor) pathways.

Development of the ethmoid in a wallaby and implications for the homology of turbinal elements in marsupials.

The homologies of the turbinals (scroll bones) of the ethmoid are not well understood, including the potential implication for understanding mammalian phylogeny. Here we examine the postnatal development of this anatomical system in a marsupial mammal because previous work has shown that the adult pattern of five endoturbinals (ethmoturbinals) and two ectoturbinals (frontoturbinals) is conserved. Furthermore, marsupial phylogeny is fairly well resolved and provides a solid evolutionary framework for examining turbinal homologies. In this study, we documented the development of the ethmoid of the tammar wallaby, Notamacropus eugenii, using histology and computed tomography imagery of a growth series of pouch young. The pattern of development of the turbinal elements in the wallaby was compared to that in didelphids, as described in previous work. We found that four ethmoturbinals initially develop, followed later in development by an interturbinal; these five elements then develop into the bony endoturbinals found in adults. These data support the idea that endoturbinal III, derived from an interturbinal, has a distinctive development pattern from the other endoturbinals. This is consistent with what is seen in the didelphid marsupials, Caluromys philander and Monodelphis domestica, suggesting this is a common developmental pattern for marsupials. This article is part of the theme issue 'The mammalian skull: development, structure and function'.

Divergent patterns of meiotic double strand breaks and synapsis initiation dynamics suggest an evolutionary shift in the meiosis program between American and Australian marsupials.

In eutherian mammals, hundreds of programmed DNA double-strand breaks (DSBs) are generated at the onset of meiosis. The DNA damage response is then triggered. Although the dynamics of this response is well studied in eutherian mammals, recent findings have revealed different patterns of DNA damage signaling and repair in marsupial mammals. To better characterize these differences, here we analyzed synapsis and the chromosomal distribution of meiotic DSBs markers in three different marsupial species (Thylamys elegans, Dromiciops gliorides, and Macropus eugenii) that represent South American and Australian Orders. Our results revealed inter-specific differences in the chromosomal distribution of DNA damage and repair proteins, which were associated with differing synapsis patterns. In the American species T. elegans and D. gliroides, chromosomal ends were conspicuously polarized in a bouquet configuration and synapsis progressed exclusively from the telomeres towards interstitial regions. This was accompanied by sparse H2AX phosphorylation, mainly accumulating at chromosomal ends. Accordingly, RAD51 and RPA were mainly localized at chromosomal ends throughout prophase I in both American marsupials, likely resulting in reduced recombination rates at interstitial positions. In sharp contrast, synapsis initiated at both interstitial and distal chromosomal regions in the Australian representative M. eugenii, the bouquet polarization was incomplete and ephemeral, γH2AX had a broad nuclear distribution, and RAD51 and RPA foci displayed an even chromosomal distribution. Given the basal evolutionary position of T. elegans, it is likely that the meiotic features reported in this species represent an ancestral pattern in marsupials and that a shift in the meiotic program occurred after the split of D. gliroides and the Australian marsupial clade. Our results open intriguing questions about the regulation and homeostasis of meiotic DSBs in marsupials. The low recombination rates observed at the interstitial chromosomal regions in American marsupials can result in the formation of large linkage groups, thus having an impact in the evolution of their genomes.

Conserved H3K27me3-associated chromatin remodelling allows STRA8 but not MEIOSIN expression in mammalian germ cells.

Apart from mice, meiosis initiation factors and their transcriptional regulation mechanisms are largely unknown in mammals. This study suggests that STRA8 and MEIOSIN are both meiosis initiation factors in mammals, but their transcription is epigenetically regulated differently from each other. In the mouse, the timing of meiosis onset differs between sexes due to the sex-specific regulation of the meiosis initiation factors, STRA8 and MEIOSIN. Before the initiation of meiotic prophase I, the Stra8 promoter loses suppressive histone-3-lysine-27 trimethylation (H3K27me3) in both sexes, suggesting that H3K27me3-associated chromatin remodelling may be responsible for activating STRA8 and its co-factor MEIOSIN. Here we examined MEIOSIN and STRA8 expression in a eutherian (the mouse), two marsupials (the grey short-tailed opossum and the tammar wallaby) and two monotremes (the platypus and the short-beaked echidna) to ask whether this pathway is conserved between all mammals. The conserved expression of both genes in all three mammalian groups and of MEIOSIN and STRA8 protein in therian mammals suggests that they are the meiosis initiation factors in all mammals. Analyses of published DNase-seq and chromatin-immunoprecipitation sequencing (ChIP-seq) data sets confirmed that H3K27me3-associated chromatin remodelling occurred at the STRA8, but not the MEIOSIN, promoter in therian mammals. Furthermore, culturing tammar ovaries with an inhibitor of H3K27me3 demethylation before meiotic prophase I affected STRA8 but not MEIOSIN transcriptional levels. Our data suggest that H3K27me3-associated chromatin remodelling is an ancestral mechanism that allows STRA8 expression in mammalian pre-meiotic germ cells.

Marsupial genome analysis suggests that satellite DNA formation from walb endogenous retrovirus is an event specific to the red-necked wallaby.

We recently identified walbRep, a satellite DNA residing in the genome of the red-necked wallaby Notamacropus rufogriseus. It originates from the walb endogenous retrovirus and is organized in a manner in which the provirus structure is retained. The walbRep repeat units feature an average pairwise nucleotide identity as high as 99.5%, raising the possibility of a recent origin. The tammar wallaby N. eugenii is a species estimated to have diverged from the red-necked wallaby 2-3 million years ago. In PCR analyses of these two and other related species, walbRep-specific fragment amplification was observed only in the red-necked wallaby. Sequence database searches for the tammar wallaby resulted in sequence alignment lists that were sufficiently powerful to exclude the possibility of walbRep existence. These results suggested that the walbRep formation occurred in the red-necked wallaby lineage after its divergence from the tammar wallaby lineage, thus in a time span of maximum 3 million years.

Evolution of the Short Form of DNMT3A, DNMT3A2, Occurred in the Common Ancestor of Mammals.

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.

Comparative Study of Brain Size Ontogeny: Marsupials and Placental Mammals.

Simple SummaryThis study examined brain/body ontogenetic growth in marsupials and compared it with placental mammals. While marsupials display morphology and cerebral organization diverse from placentals, their neocortical arrangement and cellular composition is unclear. Unfortunately, knowledge of marsupial ontogenetic brain/body size allometry is limited. Since marsupial brain structure and volume differ when compared with those of placentals, marsupials are considered to possess simple behavioural patterns. This is misleading, since even at a basic observation, Australian marsupials display many of the same mental capacities as other mammals. Consequently, the study findings support further investigation into the intellectual abilities of marsupials.There exists a negative allometry between vertebrate brain size and body size. It has been well studied among placental mammals but less is known regarding marsupials. Consequently, this study explores brain/body ontogenetic growth in marsupials and compares it with placental mammals. Pouch young samples of 43 koalas (Phascolarctos cinereus), 28 possums (Trichosurus vulpecula), and 36 tammar wallabies (Macropus eugenii) preserved in a solution of 10% buffered formalin, as well as fresh juveniles and adults of 43 koalas and 40 possums, were studied. Their brain size/body size allometry was compared to that among humans, rhesus monkeys, dogs, cats, rats, guinea pigs, rabbits, wild pigs, and mice. Two patterns of allometric curves were found: a logarithmic one (marsupials, rabbits, wild pigs, and guinea pigs) and a logistic one (the rest of mammals).

From Embryo to Adult: The Complete Development and Unusual Replacement of the Dentition of the Tammar Wallaby (Macropus eugenii)

From Embryo to Adult: The Complete Development and Unusual Replacement of the Dentition of the Tammar Wallaby (Macropus eugenii)

Strategies for meiotic sex chromosome dynamics and telomeric elongation in Marsupials.

During meiotic prophase I, homologous chromosomes pair, synapse and recombine in a tightly regulated process that ensures the generation of genetically variable haploid gametes. Although the mechanisms underlying meiotic cell division have been well studied in model species, our understanding of the dynamics of meiotic prophase I in non-traditional model mammals remains in its infancy. Here, we reveal key meiotic features in previously uncharacterised marsupial species (the tammar wallaby and the fat-tailed dunnart), plus the fat-tailed mouse opossum, with a focus on sex chromosome pairing strategies, recombination and meiotic telomere homeostasis. We uncovered differences between phylogroups with important functional and evolutionary implications. First, sex chromosomes, which lack a pseudo-autosomal region in marsupials, had species specific pairing and silencing strategies, with implications for sex chromosome evolution. Second, we detected two waves of γH2AX accumulation during prophase I. The first wave was accompanied by low γH2AX levels on autosomes, which correlated with the low recombination rates that distinguish marsupials from eutherian mammals. In the second wave, γH2AX was restricted to sex chromosomes in all three species, which correlated with transcription from the X in tammar wallaby. This suggests non-canonical functions of γH2AX on meiotic sex chromosomes. Finally, we uncover evidence for telomere elongation in primary spermatocytes of the fat-tailed dunnart, a unique strategy within mammals. Our results provide new insights into meiotic progression and telomere homeostasis in marsupials, highlighting the importance of capturing the diversity of meiotic strategies within mammals.

Does a third intermediate model for the vomeronasal processing of information exist? Insights from the macropodid neuroanatomy

The study of the α-subunit of Gi2 and Go proteins in the accessory olfactory bulb (AOB) was crucial for the identification of the two main families of vomeronasal receptors, V1R and V2R. Both families are expressed in the rodent and lagomorph AOBs, according to a segregated model characterized by topographical anteroposterior zonation. Many mammal species have suffered from the deterioration of the Gαo pathway and are categorized as belonging to the uniform model. This scenario has been complicated by characterization of the AOB in the tammar wallaby, Notamacropus eugenii, which appears to follow a third model of vomeronasal organization featuring exclusive Gαo protein expression, referred to as the intermediate model, which has not yet been replicated in any other species. Our morphofunctional study of the vomeronasal system (VNS) in Bennett’s wallaby, Notamacropus rufogriseus, provides further information regarding this third model of vomeronasal transduction. A comprehensive histological, lectin, and immunohistochemical study of the Bennett’s wallaby VNS was performed. Anti-Gαo and anti-Gαi2 antibodies were particularly useful because they labeled the transduction cascade of V2R and V1R receptors, respectively. Both G proteins showed canonical immunohistochemical labeling in the vomeronasal organ and the AOB, consistent with the anterior–posterior zonation of the segregated model. The lectin Ulex europaeus agglutinin selectively labeled the anterior AOB, providing additional evidence for the segregation of vomeronasal information in the wallaby. Overall, the VNS of the Bennett’s wallaby shows a degree of differentiation and histochemical and neurochemical diversity comparable to species with greater VNS development. The existence of the third intermediate type in vomeronasal information processing reported in Notamacropus eugenii is not supported by our lectin-histochemical and immunohistochemical findings in Notamacropus rufogriseus.

Ancient viral integrations in marsupials: a potential antiviral defence.

Marsupial viruses are understudied compared to their eutherian mammal counterparts, although they may pose severe threats to vulnerable marsupial populations. Genomic viral integrations, termed ‘endogenous viral elements’ (EVEs), could protect the host from infection. It is widely known past viral infections and EVEs play an active role in antiviral defence in invertebrates and plants. This study aimed to characterise actively transcribed EVEs in Australian marsupial species, because they may play an integral role in cellular defence against viruses. This study screened publicly available RNA sequencing data sets (n = 35) and characterised 200 viral transcripts from thirteen Australian marsupial species. Of the 200 transcripts, 188 originated from either Bornaviridae, Filoviridae, or Parvoviridae EVEs. The other twelve transcripts were from putative active infections from members of the Herpesviridae and Anelloviridae, and Hepadnaviridae. EVE transcripts (n = 188) were mapped to marsupial genomes (where available, n = 5/13) to identify the genomic insertion sites. Of the 188 transcripts, 117 mapped to 39 EVEs within the koala, bare-nosed wombat, tammar wallaby, brushtail possum, and Tasmanian devil genomes. The remaining eight animals had no available genome (transcripts n = 71). Every marsupial has Bornaviridae, Filoviridae, and Parvoviridae EVEs, a trend widely observed in eutherian mammals. Whilst eutherian bornavirus EVEs are predominantly nucleoprotein-derived, marsupial bornavirus EVEs demonstrate a surprising replicase gene bias. We predicted these widely distributed EVEs were conserved within marsupials from ancient germline integrations, as many were over 65 million years old. One bornavirus replicase EVE, present in six marsupial genomes, was estimated to be 160 million years old, predating the American–Australian marsupial split. We considered transcription of these EVEs through small non-coding RNA as an ancient viral defence. Consistent with this, in koala small RNA sequence data sets, we detected Bornaviridae replicase and Filoviridae nucleoprotein produced small RNA. These were enriched in testis tissue, suggesting they could protect marsupials from vertically transmitted viral integrations.

Mortality trends for five species of macropods from a single institution from 1995 to 2016.

Macropods are commonly kept and exhibited species worldwide. Mortality records were reviewed for five species of macropods housed at Cleveland Metroparks Zoo from 1995 to 2016. One hundred sixty-seven deaths occurred including Western grey kangaroo (Macropus fuliginosus) (n = 22), Bennett's wallaby (M. rufogriseus) (n = 64), red kangaroo (M. rufus) (n = 56), wallaroo (M. robustus) (n = 9), and Tammar wallaby (M. eugenii) (n = 16). The leading cause of death was trauma, followed by bronchopneumonia for all species. Gastric dilatation was identified as a concern for red kangaroo. Females lived longer than males across species and were overrepresented by the data set. Oral necrobacillosis and osteomyelitis were health concerns for macropods in this review. Mortality reviews are important as they identifying collection trends and influencing long-term care.