Tammar Research Articles

Insights from Contrast‐Enhanced MicroCT into the Formation of the Marsupial Hindlimb

As the sister clade to placental mammals, marsupials offer insight into a completely separate experiment in mammalian evolution. As with many placental groups (e.g., ungulates, rodents, and lagomorphs), several marsupial groups like kangaroos and bandicoots evolved elongate limbs with reduced digit numbers for locomoting in open, arid habitats. However, unlike most placental lineages which specialized their limbs from a cursorial ancestor, marsupial lineages evolved their specialized hindlimbs from a grasp-climbing ancestor that was morphologically similar to extant didelphid marsupials. To examine the developmental changes involved in marsupial limb diversification we used contrast-enhanced microcomputed tomography (CE microCT) to characterise postnatal limb development in the grey short-tailed opossum and the tammar wallaby. We compared phosphotungstic acid (PTA) and Ruthenium Red (RR) as contrast agents for differentiating bone and cartilage in microCT scans. PTA staining was effective at contrasting soft tissues but it was difficult to differentiate bone and cartilage tissues without time-consuming manual segmentation. Preliminary results show that RR stains soft-tissues much more slowly than PTA and skin removal is required for stain penetration of postnatal specimens. Comparison of limb segment proportions between the opossum and tammar wallaby provides evidence that macropods undergo a pronounced late postnatal elongation of the distal hindlimb elements, especially the calcaneus, metatarsals and phalanges. Digital segmentation of the hindfoot of an early postnatal PTA-stained specimen of the tammar wallaby provides evidence that a cartilaginous condensation anterior to the reduced second and third toe is morphologically more similar to a prehallax than a first toe. Potential implications for the evolvability of tetrapod digit number and the ancestral constraints of arboreality on limb adaptation are considered.

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 (1day 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 1week 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.

Host reproductive cycle influences the pouch microbiota of wild southern hairy-nosed wombats (Lasiorhinus latifrons)

BackgroundMarsupials are born much earlier than placental mammals, with most crawling from the birth canal to the protective marsupium (pouch) to further their development. However, little is known about the microbiology of the pouch and how it changes throughout a marsupial’s reproductive cycle. Here, using stringent controls, we characterized the microbial composition of multiple body sites from 26 wild Southern Hairy-nosed Wombats (SHNWs), including pouch samples from animals at different reproductive stages.ResultsUsing qPCR of the 16S rRNA gene we detected a microbial community in the SHNW pouch. We observed significant differences in microbial composition and diversity between the body sites tested, as well as between pouch samples from different reproductive stages. The pouches of reproductively active females had drastically lower microbial diversity (mean ASV richness 19 ± 8) compared to reproductively inactive females (mean ASV richness 941 ± 393) and were dominated by gram positive bacteria from the Actinobacteriota phylum (81.7–90.6%), with the dominant families classified as Brevibacteriaceae, Corynebacteriaceae, Microbacteriaceae, and Dietziaceae. Three of the five most abundant sequences identified in reproductively active pouches had closest matches to microbes previously isolated from tammar wallaby pouches.ConclusionsThis study represents the first contamination-controlled investigation into the marsupial pouch microbiota, and sets a rigorous framework for future pouch microbiota studies. Our results indicate that SHNW pouches contain communities of microorganisms that are substantially altered by the host reproductive cycle. We recommend further investigation into the roles that pouch microorganisms may play in marsupial reproductive health and joey survival.

Male germline development in the tammar wallaby, Macropus eugenii.

Male germ cells undergo two consecutive processes - pre-spermatogenesis and spermatogenesis - to generate mature sperm. In eutherian mammals, epigenetic information such as DNA methylation is dynamically reprogrammed during pre-spermatogenesis, before and during mitotic arrest. In mice, by the time germ cells resume mitosis, the majority of DNA methylation is reprogrammed. The tammar wallaby has a similar pattern of germ cell global DNA methylation reprogramming to that of the mouse during early pre-spermatogenesis. However, early male germline development in the tammar or in any marsupial has not been described previously, so it is unknown whether this is a general feature regulating male germline development or a more recent phenomenon in mammalian evolutionary history. To answer this, we examined germ cell nuclear morphology and mitotic arrest during male germline development in the tammar wallaby (Macropus eugenii), a marsupial that diverged from mice and humans around 160 million years ago. Tammar pro-spermatogonia proliferated after birth and entered mitotic arrest after day 30 postpartum (pp). At this time, they began moving towards the periphery of the testis cords and their nuclear size increased. Germ cells increased in number after day 100 pp which is the time that DNA methylation is known to be re-established in the tammar. This is similar to the pattern observed in the mouse, suggesting that resumption of germ cell mitosis and the timing of DNA methylation reprogramming are correlated and conserved across mammals and over long evolutionary timescales.

Primordial germ cell expression of SSEA1 and DDX4 (VASA) in female Trichosurus vulpecula (Marsupialia) reveals conserved and unique molecular patterns during marsupial germ cell development.

Development of primordial germ cells (PGCs: precursors to adult gametes) is a key process in vertebrate sexual differentiation. Marsupials are ideal to investigate this phenomenon because much of PGC migration and development unusually occurs postnatally in pouch young. However, investigation of the molecular dynamics underpinning PGC development is restricted to one marsupial model species: the tammar wallaby (Macropus eugenii). Given the reproductive diversity among clades, marsupial PGCs likely exhibit diversity in molecular patterns that could help uncover their developmental dynamics. Here we characterise PGC marker expression (SSEA1 and DDX4) in developing ovaries of the brushtail possum, Trichosurus vulpecula. Female germ cells expressed DDX4 from 6 days postpartum (dpp) and almost all germ cells expressed DDX4 by meiosis (40 dpp), consistent with M. eugenii and eutherian mammals. In contrast, PGCs and oogonia expressed SSEA1 from 12 dpp, throughout proliferation and until entry into meiosis (40-63 dpp). SSEA1 expression was temporally distinct from that of M. eugenii, in which SSEA1 expression persists only until 14 dpp, indicating differential expression between marsupial species at equivalent stages of germ cell development. Hence, the molecular characteristics of M. eugenii germ cells cannot be assumed for all marsupials, as at least one key molecule exhibits species-specific expression.

Human chorionic gonadotrophin does not induce ovulation in the tammar wallaby

We investigated whether human chorionic gonadotrophin (hCG), rather than porcine luteinising hormone (pLH), could induce ovulation in the tammar wallaby (Notamacropus eugenii) after priming with pregnant mare serum gonadotrophin (PMSG). pLH is effective, but is more expensive and supply is problematic. On Day 0 pouch young were removed (RPY) and 5mgkg−1 of bromocriptine mesylate was administered to all non-pregnant wallabies (n=12) to reactivate an oestrous cycle. On Day 20 RPY each female received 20IU PMSG i.m. and three days later (Day 23 RPY), the Single-Dose group (n=6) received 500IU of hCG suspended in oil at 0900 hours, and the Multi-Dose group (n=6) received 500IU of hCG at 0900, 1230 and 1630 hours. From Day 25 to Day 28 RPY copulatory plugs were observed in the Single-Dose (n=4 of 6; Day 25.3 to 28.3 RPY) and Multi-Dose (n=5 of 6; Day 25 to 28 RPY) groups. Autopsy on Day 31 RPY revealed that all females had reactivated, and despite all having large preovulatory follicles (4–6mm) in both ovaries, no ovulations or embryos were observed. We conclude that hCG is not an effective source of exogenous LH for the induction of ovulation in the tammar wallaby.

A role for Msx genes in mammalian embryonic diapause.

Mammalian embryonic diapause is a reproductive phenomenon defined by the reversible arrest in blastocyst development and metabolic activity within the uterus which synchronously becomes quiescent to implantation. This natural strategy, evident in over 130 species across eight orders, can temporally uncouple conception from delivery until conditions are favorable for the survival of the mother and newborn. While the maternal endocrine milieu has been shown to be important for this process, the local molecular mechanisms by which the uterus and embryo achieve quiescence, maintain blastocyst survival and then resumes blastocyst activation with subsequent implantation in response to endocrine cues remains unclear. Here we review the first evidence that the proximal molecular control of embryonic diapause is conserved in three unrelated mammalian species which employ different endocrine programs to initiate diapause. In particular, uterine expression of muscle segment homeobox (Msx) genes Msx1 or Msx2 persists during diapause, followed by downregulation with blastocyst reactivation and implantation. Mice (Mus musculus) with conditional inactivation of Msx1 and Msx2 in the uterus fail to achieve diapause and reactivation. Remarkably, the mink (Neovison vison) and tammar wallaby (Macropus eugenii) share this pattern of MSX1 or MSX2 expression as in mice during delay - it persists during diapause and is rapidly downregulated upon implantation. Therefore, these findings were the first to provide evidence that there are common conserved molecular regulators in the uterus of unrelated mammals during embryonic diapause.

Conserved mechanisms for putting pregnancy on hold in the mouse, mink and tammar wallaby

Conserved mechanisms for putting pregnancy on hold in the mouse, mink and tammar wallaby

Sequence comparisons of cytochrome P450 aromatases from Australian animals predict differences in enzymatic activity and/or efficiency†.

Aromatase (P450arom, CYP19A1) is the terminal enzyme in the synthesis of the steroid hormone family of estrogens. Not surprisingly, this enzyme has structural similarities between the limited number of species studied thus far. This study examined the structure of aromatases from four diverse Australian species including a marsupial (tammar wallaby; Macropus eugenii), monotreme (platypus; Ornithorhynchus anatinus), ratite (emu; Dromaius novaehollandiae) and lizard (bearded dragon; Pogona vitticeps). We successfully built homology models for each species, using the only crystallographically determined structure available, human aromatase. The amino acid sequences showed high amino acid sequence identity to the human aromatase: wallaby 81%, platypus 73%, emu 75% and bearded dragon at 74%. The overall structure was highly conserved among the five species, although there were non-secondary structures (loops and bends) that were variable and flexible that may result in some differences in catalytic activity. At the N-terminal regions, there were deletions and variations that suggest that functional distinctions may be found. We found that the active sites of all these proteins were identical, except for a slight variation in the emu. The electrostatic potential across the surfaces of these aromatases highlighted likely variations to the protein-protein interactions of these enzymes with both redox partner cytochrome P450 reductase and possibly homodimerization in the case of the platypus, which has been postulated for the human aromatase enzyme. Given the high natural selection pressures on reproductive strategies, the relatively high degree of conservation of aromatase sequence and structure across species suggests that there is biochemically very little scope for changes to have evolved without the loss of enzyme activity.

Discrete Hedgehog Factor Expression and Action in the Developing Phallus.

Hypospadias is a failure of urethral closure within the penis occurring in 1 in 125 boys at birth and is increasing in frequency. While paracrine hedgehog signalling is implicated in the process of urethral closure, how these factors act on a tissue level to execute closure itself is unknown. This study aimed to understand the role of different hedgehog signalling members in urethral closure. The tammar wallaby (Macropus eugenii) provides a unique system to understand urethral closure as it allows direct treatment of developing offspring because mothers give birth to young before urethral closure begins. Wallaby pouch young were treated with vehicle or oestradiol (known to induce hypospadias in males) and samples subjected to RNAseq for differential expression and gene ontology analyses. Localisation of Sonic Hedgehog (SHH) and Indian Hedgehog (IHH), as well as the transcription factor SOX9, were assessed in normal phallus tissue using immunofluorescence. Normal tissue culture explants were treated with SHH or IHH and analysed for AR, ESR1, PTCH1, GLI2, SOX9, IHH and SHH expression by qPCR. Gene ontology analysis showed enrichment for bone differentiation terms in male samples compared with either female samples or males treated with oestradiol. Expression of SHH and IHH localised to specific tissue areas during development, akin to their compartmentalised expression in developing bone. Treatment of phallus explants with SHH or IHH induced factor-specific expression of genes associated with bone differentiation. This reveals a potential developmental interaction involved in urethral closure that mimics bone differentiation and incorporates discrete hedgehog activity within the developing phallus and phallic urethra.

Uterine epithelial remodelling during pregnancy in the marsupial Monodelphis domestica (Didelphidae): Implications for mammalian placental evolution.

Mammalian pregnancy involves remodelling of the uterine epithelium to enable placentation. In marsupials, such remodelling has probably played a key role in the transition from ancestral invasive placentation to non-invasive placentation. Identifying uterine alterations that are unique to marsupials with non-invasive placentation can thus elucidate mechanisms of marsupial placental evolution. We identified apical alterations to uterine epithelial cells prior to implantation in Monodelphis domestica, a member of the least derived living marsupial clade (Didelphidae) with invasive (endotheliochorial) placentation. We then compared these traits with those of Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae), both with non-invasive placentation, to identify which alterations to the uterine epithelium are ancestral and which facilitate secondarily evolved non-invasive placentation. In M.domestica, remodelling of the uterine epithelium involves reduced cellular heterogeneity and development of uterodome-like cells, suggesting that similar alterations may also have occurred in the marsupial common ancestor. These alterations also overlap with those of both T.vulpecula and Ma.eugenii, suggesting that the placental shift from invasive to non-invasive placentation in marsupials involves essential, conserved characteristics, irrespective of placental mode. However, unique apical alterations of both T.vulpecula and Ma.eugenii, relative to M.domestica, imply that lineage-specific alterations underpin the evolutionary shift to non-invasive placentation in marsupials.

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.

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.

Long-term maternal exposure to atrazine in the drinking water reduces penis length in the tammar wallaby Macropus eugenii

Marsupials are experiencing devastating population declines across Australia. Exposure to environmental endocrine disruptors, through ingestion of contaminated resources in the environment, could be contributing to this decline. Atrazine (ATZ), a widely used herbicide in Australia, is an endocrine disruptor with the ability to cause reproductive abnormalities in a diverse range of vertebrates. We exposed adult female wallabies (Macropus eugenii) to drinking water containing ATZ (450 p.p.m) throughout pregnancy, parturition and lactation. We assessed the outcome of this exposure to the reproductive development of their young by assessing gonad and phallus development. Both these organs are especially sensitive to perturbations in the hormonal environment during development. Although no gross abnormalities were seen in gonad structure, exposure to ATZ did alter the expression of genes required for normal testis function. Furthermore, long-term exposure to ATZ resulted in a significant reduction in penis length. These results demonstrate that ATZ exposure during gestation and lactation can significantly affect the development of male young by affecting virilisation. Given the known vulnerability of macropodid marsupials to endocrine disruption, as well as their overlapping distribution with agricultural areas, these data raise major concerns for the use of pesticides in areas with fragile marsupial populations.

WOMEN IN REPRODUCTIVE SCIENCE: Reproduction down under.

Australia is home to a unique assembly of mammals - the marsupials and monotremes. Despite this uniqueness, they have been largely ignored by the biomedical scientific community, and yet study of marsupials has contributed to modern research on reproduction, development, evolution, conservation, molecular and comparative genomics. My lifetime passion for these long-neglected Australian fauna has led to unexpected discoveries and insights that challenged assumptions and opened up new areas of international research. I used a range of disciplinary expertise to pursue the study of these unique mammals. My main experimental species has been the tammar wallaby that I have used as a model species to investigate and understand not only biomedical problems but also to provide knowledge that is critical for the continued conservation and management of Australia's dwindling native mammals. This model provided more than a few surprises for me and my wonderful team of students, post-docs and collaborators about how hormones, genes and signalling molecules control reproductive biology and development in a wider context as well as how the interactions of the environment with mother and conceptus, with mother and fetus and mother and young ultimately control most aspects of successful reproduction in mammals.

Variation in Toxoplasma gondii seroprevalence: effects of site, sex, species and behaviour between insular and mainland macropods

Context Feral cats threaten wildlife conservation through a range of direct and indirect effects. However, most studies that have evaluated the impacts of feral cats on species of conservation significance have focussed on direct impacts such as predation; few studies have considered the indirect impacts of cat-borne disease. Toxoplasma gondii, a cat-borne parasite, causes both acute and latent disease in a range of wildlife species, and macropods are particularly susceptible. Kangaroo Island is Australia’s third largest island and supports a high density of feral cats and high seroprevalence of T. gondii in multiple species, relative to the mainland. This suggests that Kangaroo Island has a high environmental contamination with the parasite and a high risk of infection for other species. Aims We aimed to describe T. gondii seroprevalence in culled and road-killed macropods, so as to assess the effects of island versus mainland location, sex, species and behaviour. Methods Macropod sera were tested for T. gondii IgG antibodies using a commercially available modified agglutination test. Key results The seroprevalence of T. gondii in culled western grey kangaroos (Macropus fuliginosus) was significantly higher on the island (20%, 11/54 positive) than on the mainland (0%, 0/61 positive). There was no difference in T. gondii seroprevalence between culled and road-killed (21%, 21/102 positive) kangaroos from the island. The seroprevalence of T. gondii was significantly higher in female (32%, 12/38 positive) than in male (13%, 8/60 positive) kangaroos, but we observed no sex effect in tammar wallabies (Macropus eugenii), and no effect of species. Conclusions The higher T. gondii seroprevalence in insular macropods supports previous reports of higher T. gondii exposure in other Kangaroo Island fauna. The lack of difference in T. gondii seroprevalence between culled and road-killed kangaroos suggests that T. gondii-positive animals are not more vulnerable to road mortality, in contrast to that suggested previously. Implications Our findings suggest greater potential adverse conservation impacts owing to toxoplasmosis on the island than on the mainland. In light of a recent study demonstrating higher cat abundance on the island than on the mainland, the higher observed T. gondii seroprevalence in insular macropods is likely to be a consequence of higher cat density.

Effect of WAM‐1 transgenic Arabidopsis thaliana on the development and gut microbial composition of diamondback moth (Plutella xylostella) larvae (Lepidoptera: Plutellidae)

AbstractWAM‐1 is a cathelicidin antimicrobial peptide found on the Macropus eugenii (Tammar wallaby) genome. It possesses antibiotic activity against a broad spectrum of bacteria and fungi. In order to investigate the impact of this WAM‐1 antimicrobial peptide on development and midgut microbial composition of Plutella xylostella (DBM) larvae, the WAM‐1 was introduced into Arabidopsis thaliana using the Agrobacterium‐mediated floral‐dip transformation method. WAM‐1 expression was confirmed by reverse transcriptase PCR analysis. The larval growth and developmental variations and midgut microbial compositions were analysed after DBM larvae had fed on transgenic plants. Transgenic plant‐fed DBM larvae showed significantly higher mortality, a prolonged developmental period and less weight gain. Enterobacteriaceae was the highest dominating family in all samples including transgenic and non‐transgenic plants fed larvae. However, its abundance was substantially lower in the WAM‐1 Arabidopsis‐fed larvae. Furthermore, Moraxellaceae and Bacillaceae were the next dominating families in the WAM‐1 transgenic Arabidopsis‐fed larvae whereas Enterococcaceae and Xanthomonadaceae were dominant in the non‐transgenic Arabidopsis‐fed larvae. Abundance of Pseudomonadaceae was also relatively higher in WAM‐1 transgenic‐fed larvae whereas it was much lower in other samples. Therefore, our findings suggest that WAM‐1 has an impact on gut microbial composition but a minor effect on larval development.

New species and a redescription of species of Cloacina von Linstow, 1898 (Nematoda: Strongyloidea) parasitic in macropodid marsupials

ABSTRACTFour new species of strongyloid nematodes belonging to the genus Cloacina von Linstow, 1898 parasitic in macropodid marsupials are described and a poorly known species is redescribed from new material. Cloacina elegans Johnston & Mawson, 1938 is redescribed from the black-flanked rock wallaby, Petrogale lateralis Gould, from central Australia. Cloacina hylas sp. nov. from the stomach of the black-stripe wallaby, Notamacropus dorsalis (Gray), differs from congeners in lacking ornamented lobes surrounding the stoma, in the shape of the cephalic papillae, the presence of oesophageal bosses but the lack of oesophageal denticles. Cloacina idas sp. nov. from the stomach of Godman’s rock wallaby, Petrogale godmani Thomas, differs from congeners in possessing a very slender oesophagus, eight leaf crown elements and the deirid in the anterior oesophageal region. Cloacina schultzi sp. nov. from the stomach of the yellow-footed rock wallaby, Petrogale xanthopus Gray, differs in possessing a sub-cylindrical oesophagus, a shallow buccal capsule and lip-like inflations of the leaf crown elements while Cloacina zetes sp. nov. from the stomach of the Western Australian tammar wallaby, Notamacropus eugenii (Desmarest), differs from all congeners in the shape of the leaf crown elements.

Sexual conflict in action: An antagonistic relationship between maternal and paternal sex allocation in the tammar wallaby, Notamacropus eugenii.

Sex ratio biases are often inconsistent, both among and within species and populations. While some of these inconsistencies may be due to experimental design, much of the variation remains inexplicable. Recent research suggests that an exclusive focus on mothers may account for some of the inconsistency, with an increasing number of studies showing variation in sperm sex ratios and seminal fluids. Using fluorescent in‐situ hybridization, we show a significant population‐level Y‐chromosome bias in the spermatozoa of wild tammar wallabies, but with significant intraindividual variation between males. We also show a population‐level birth sex ratio trend in the same direction toward male offspring, but a weaning sex ratio that is significantly female‐biased, indicating that males are disproportionately lost during lactation. We hypothesize that sexual conflict between parents may cause mothers to adjust offspring sex ratios after birth, through abandonment of male pouch young and reactivation of diapaused embryos. Further research is required in a captive, controlled setting to understand what is driving and mechanistically controlling sperm sex ratio and offspring sex ratio biases and to understand the sexually antagonistic relationship between mothers and fathers over offspring sex. These results extend beyond sex allocation, as they question studies of population processes that assume equal input of sex chromosomes from fathers, and will also assist with future reproduction studies for management and conservation of marsupials.