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The complete mitochondrial genome of the wallaroo (Macropus robustus) and the phylogenetic relationship among Monotremata, Marsupialia, and Eutheria.

The complete mitochondrial DNA (mtDNA) (16,896 nt) of the wallaroo (Macropus robustus) was sequenced. The concatenated amino acid sequences of 12 mitochondrial protein-coding genes of the wallaroo plus those of a number of other mammals were included in a phylogenetic study of early mammalian divergences. The analysis joined monotremes and marsupials (the Marsupionta hypothesis) to the exclusion of eutherians. The analysis rejected significantly the commonly acknowledged Theria hypothesis, according to which Marsupialia and Eutheria are grouped together to the exclusion of Monotremata. The region harboring the gene for lysine tRNA (tRNA-Lys) in the mtDNA of other vertebrates is in the wallaroo occupied by a sequence (tRNA-Lys) that lacks both an anticodon loop as well as the anticodon for the amino acid lysine. An alternative tRNA-Lys gene was not identified in any other region of the mtDNA of the wallaroo, suggesting that a tRNA-Lys of nuclear origin is imported into marsupial mitochondria. Previously described RNA editing of tRNA-Asp and rearrangement of some tRNA genes were reconfirmed in the mtDNA of the wallaroo. The divergence between Monotremata/Marsupialia and Eutheria was timed to approximately 130 million years before present (MYBP). The same calculations suggested that Monotremata and Marsupialia diverged approximately 115 MYBP and that Australian and American marsupials separated approximately 75 MYBP. The findings also show that many, probably most, extant eutherian orders had their origin in middle to late Cretaceous times, 115-65 MYBP.

Animals↗

Development of craniofacial musculature in Monodelphis domestica (Marsupialia, Didelphidae).

Development of craniofacial muscles of Monodelphis domestica (Marsupialia, Didelphidae) is described. In a period of 4-6 days all craniofacial muscles in M. domestica progress from myoblast condensation, to striated myofibers that are aligned in the direction of adult muscles and possess multiple, lateral nuclei. This process begins 1 to 2 days before birth and continues during the first few days after birth. Compared to other aspects of cranial development, muscle development in M. domestica is rapid. This rapid and more or less simultaneous emergence of craniofacial muscles differs from the previously described pattern of development of the cranial skeleton in marsupials, which displays a mosaic of acceleration and deceleration of regions and individual elements. Unlike the skeletal system, craniofacial muscles show no evidence of regional specialization during development. M. domestica resembles eutherian mammals in the relatively rapid and more or less simultaneous differentiation of all craniofacial muscles. It differs from eutherian taxa in that most stages of myogenesis occur postnatally, following the onset of function. The timing of the development of muscular and skeletal structures is compared and it is concluded that the relatively early development of muscle is not reflected by any particular acceleration of the differentiation or growth of skeletal structures. Finally, the difficulties in accounting for complex internal arrangements of muscles such as the tongue, given current models of myogenesis are summarized.

Aging↗

Responses of the digestive tract of the omnivorous northern brown bandicoot, Isoodon macrourus (Marsupialia: Peramelidae), to plant- and insect-containing diets.

The gastrointestinal tract of omnivores such as bandicoots (Marsupialia: Peramelidae) must be able to process foods as different as invertebrates, fungi and plant material. We studied the mechanisms involved in the utilisation by captive northern brown bandicoots (Isoodon macrourus) of insect larvae and milled lucerne (Medicago sativa) hay incorporated into a basal diet of a commercial small carnivore mix. Animals on the plant-basal mix digested less dry matter, energy, lipid, fibre and total nitrogen, but consumed 79% more dry matter than those on the insect-basal mix. Consequently intake of digestible energy (i.e. energy absorbed) was not significantly different between diets. Mean retention time (MRT, the mean time a marker remains in the tract) of a large particle marker was shorter on the plant-basal mix, reflecting its higher intake, but MRT of a solute marker was not significantly different between diets. Consequently the solute marker was retained longer than the particle marker on the plant-basal mix, indicating selective retention of solutes and very small particles in the caecum on this diet. This was confirmed by a higher proportion of small particles in the caecum than the distal colon of road-killed I. macrourus. Thus the main responses by I. macrourus to the plant-basal mix appeared to be an increase in gastrointestinal tract capacity (from radiographic evidence), selective retention of solutes and very small particles in the caecum, and facilitated passage of less tractable large particles through the colon. As a consequence, food intake was higher on the plant-basal mix, which compensated for its lower digestibility, and intake of digestible energy was similar to that on the insect-basal mix. This considerable flexibility of the morphologically rather simple digestive tract of northern brown bandicoots helps to explain their ability to cope with naturally variable diets consisting of mainly invertebrates in summer to much more plant and fungal material in winter, and to survive in nutritionally dynamic environments such as heathlands where there can be dramatic changes in food type and availability following periodic wildfires.

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Durikainema macropi gen. et sp. nov. (Muspiceoidea: Robertdollfusidae). A remarkable nematode from Macropodidae (Marsupialia).

Durikainema macropi gen. et sp. nov. (Muspiceoidea: Robertdollfusidae) is described from the mesenteric and hepatic portal veins of Macropus giganteus Shaw 1790, M. agilis (Gould 1842) and M. rufogriseus (Desmarest 1817) (Marsupialia: Macropodidae) from Queensland, Australia. It is also known from histological sections of hepatic portal veins of M. robustus Gould 1841, M. fuliginosus (Desmarest 1817) and Lagorchestes conspicillatus Gould 1842. The new genus resembles the Enoplina in cephalic and caudal characters and the Dorylaimina in other characters. Durikainema resembles Robertdollfusa Chabaud and Campana 1950 in its small form, absence of mouth oesophagus and anus, atrophied digestive tube, reduction of female genital apparatus to a uterine pouch, viviparity and cephalic cuticular inflation in larvae. It differs from this genus in its complex and well developed cephalic structures and its well developed body musculature in both sexes. Durikainema is tentatively placed in the Robertdollfusidae, Muspiceoidea. Larvae develop beyond firststage in the uterus of the female. They have been found in the non-peripheral blood of male and female M. giganteus, the lactating mammary gland of female M. agilis but not the non-lactating glands of the same female and in the deep capillaries of thigh skin of male M. agilis. Transmission of the parasite may be direct by a percutaneous or milk route, or indirect by a haemophagous arthropod.

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Sutarostrongylus Johnsoni sp. n. (Nematoda: Trichostrongylina, Heligmosomoidea) from the pademelon, Thylogale stigmatica (Marsupialia) from Queensland, Australia.

A new species of heligmosomoid nematode Sutarostrongylus johnsoni sp. n., belonging to the sub-family Herpetostrongylinae Skrjabin & Schultz, is described from the small intestine (duodenum) of the red-legged pademelon, Thylogale stigmatica (Gould, 1860) (Marsupialia: Macropodidae), from north-eastern Queensland, Australia. The only other species of the genus S. kirkpatricki Beveridge & Durelte-Desset, 1986 occurs in the related host, the red-necked pademelon, T. thetis (Lesson, 1827) in south-eastern Queensland. The new species differs in having longer spicules and asymmetrical spicule tips as well as the presence of a comârete which develops on the right ventral aspect of the mid-region of the body. The synlophe of the new species is unusual in that the inclination of the axis of orientation changes from being oblique in the anterior part of the body to being frontal in the posterior part. The same change may occur in some species of Austrostrongylus Chandler, 1924. Current data suggest that species of Sutarostrongylus are limited to a single genus of host, Thylogale Gray, 1837 and support the suggestion that both nematode species exhibit morphological features which are intermediate between those occurring in Herpetotostrongylinae in dasyurid marsupials and those occurring in macropodid marsupials.

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Milk composition in the eastern quoll, Dasyurus viverrinus (Marsupialia:Dasyuridae).

The milk constituents of Dasyurus viverrinus, a carnivorous marsupial, exhibited major quantitative and qualitative changes during the course of lactation. The milk produced in the early stages of lactation was dilute, about 13-16% (w/w) solids before 3 weeks with carbohydrate representing the major fraction. In the latter stages of lactation the milk was concentrated, around 30% solids, and lipid was the predominant fraction. Palmitic acid was the major fatty acid present in early-stage milk but oleic acid became predominant in milk after 10 weeks post-partum. The changes in milk composition in D. viverrinus were similar to those described for the milks of herbivorous marsupials which therefore suggests that this pattern may be uniform throughout the Marsupialia.

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Timing of mating, sperm dynamics, and ovulation in a wild population of agile Antechinus (Marsupialia: dasyuridae).

Timing of mating, sperm transport and storage, and ovulation were examined in a wild population of agile Antechinus (Marsupialia: Dasyuridae) in order to ascertain the validity of direct comparisons between captive and field-based mating studies in this species. Mating commenced in early August, and all females had ovulated by the 27th of the month. Fifty-five percent of the mated females caught that had not yet ovulated were captured on 19-20 August. This corresponded with a peak (67%) in the ovulation date determined for pregnant females. Approximately 25.9 x 10(3) spermatozoa per side were recovered from the reproductive tract of each mated female captured (range: 1.7 x 10(3)-75.5 x 10(3) spermatozoa per side). Spermatozoa were consistently found in greater numbers in the lower isthmus (19.7 x 10(3) +/- 19.9 x 10(3) spermatozoa per side) of the oviduct ( approximately 67% of all sperm found in the female tract; range 17-94%) than elsewhere in the reproductive tract. Few spermatozoa were found in the upper isthmus, and none were detected in the ampulla. Sperm number in the female reproductive tract supports the hypothesis that females will mate several times within the one estrus. At the conclusion of the rut, approximately 80.0 x 10(3) spermatozoa remained in each testis and approximately 630 x 10(3) spermatozoa in each epididymis. Most epididymal spermatozoa were restricted to the distal corpus/proximal cauda regions of the duct. This study shows that both field and laboratory reproductive data correlate well in the agile Antechinus and that successful breeding is indeed an exercise in reproductive brinkmanship.

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Early differentiation of the testis in the native cat, Dasyurus viverrinus (Marsupialia).

The development of the testis in the native cat Dasyurus viverrinus (Marsupialia) is described, from Day 3 to 2.5 months post partum. The gonad rudiment consists of a mass of undifferentiated blastema cells of mesenchymal origin. The primordial germ cells populate the rudiment around the neonatal period. On Day 3 two cell types autodifferentiate from the gonadal blastema (pre-Sertoli and stroma cells) and the tunica albuginea begins to form. Pre-Sertoli cells are large, pale, cells which encompass the primordial germ cells and come to form a zone just beneath the tunica albuginea. By the end of the second week the cell complex has become transformed into a series of sex cords which encircle the gonad rudiment peripherally and converge on the rete cord in the hilar region. The stroma cells are fibroblast-like cells and occupy the central portion of the rudiment; by the eighth week, Leydig cells are appearing among them. No evidence was found for mesothelial invaginations or mesonephric contributions to the gonad. The classically recognised regions of 'cortex' and 'medulla' cannot be recognised and the seminiferous tubules differ in arrangement from that in eutherians.

Aging↗

Lectin histochemical study of early postnatal vascularization of the developing pars distalis adenohypophysis of the gray short-tailed opossum (Monodelphis domestica, Marsupialia).

Individuals from 18 litters between 0.5 day and 13 days post partum, and two adult specimens of Monodelphis domestica (Marsupialia), were studied by TEM and lectin histochemistry. Positive reactions for PO-lectins in the developing pituitary were found in the vascular and/or perivascular elements; secretory cells did not react. Positive vascular reactions varied with both the type of lectin and the age of the developing animal. In the adults, the reactions were reversed: there were no vascular, but a variety of positive cellular reactions. It is concluded that the adenohypophyseal blood vascular system is very far from being complete and mature in the newborn M. domestica. According to specific data taken from the literature, the lectin histochemistry indicates a sequential appearance of several components of the extracellular matrix, suggesting also that the actively organ-invading capillaries may be a good model for the study of basement membrane development.

Aging↗

Changes in milk composition during lactation in the common brushtail possum, Trichosurus vulpecula (Marsupialia: Phalangeridae).

The milk constituents of Trichosurus vulpecula, a folivorous marsupial, showed marked quantitative and qualitative changes during the course of lactation. The milk produced in the early stages of lactation was dilute, with about 9-13% (w/w) solids during the first 3 weeks, comprising mostly carbohydrate and protein (35-40%). At 20 weeks, about three-quarters of the way through lactation, the milk was much more concentrated, about 28% solids, with lipid the predominant fraction (30-35%), after a marked decline in carbohydrate content (20-25%). Concentrations of the electrolytes sodium and potassium also underwent marked changes. The changes in milk composition of T. vulpecula during the first three-quarters of lactation were similar to those described for a range of herbivorous, insectivorous and carnivorous marsupials. In the last quarter of lactation, however, brushtail possum milk maintained a relatively stable composition, with higher levels of carbohydrate and lower levels of lipid than for other marsupials. There appears to be a uniform pattern of changes in milk composition throughout the Marsupialia over most of lactation, with family differences evident only in the latter stages.

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The parasympathetic nervous system and its influence on heart rate in torpid western pygmy possums, Cercatetus concinnus (Marsupialia: Burramyidae).

We investigated the effect of parasympathetic antagonism on the patterns of heart rate during torpor in the western pygmy possum Cercatetus concinnus (Marsupialia: Burramyidae). This is the first study to examine the influence of the autonomic nervous system on cardiac function in a metatherian hibernator. During torpor, antagonism of the parasympathetic nervous system eliminated the ventilatory tachycardia, variability in instantaneous heart rate, and increased the overall heart rate. These findings are consistent with previous studies on other mammalian heterotherms, which have shown that the parasympathetic nervous system is responsible for these patterns in heart rate. During extended bouts of torpor (2 to 3 days) the ventilatory tachycardia persisted throughout each bout, which indicates that the parasympathetic nervous system remained functional during that time. It has been suggested that the progressive removal of autonomic tone is characteristic of deep steady-state hibernation. There is no evidence to suggest that such a state was going to be reached in the possums in this study. To date there is little evidence that clearly demonstrates a physiological basis for the distinction between shallow, daily torpor and deep hibernation.

Journal Article↗

Mitochondrial DNA-like sequences in the nuclear genome of the opossum genus Didelphis (Marsupialia: Didelphidae).

This study reports the occurrence of mtDNA-like sequences in the nuclear genome of the opossum genus Didelphis (Didelphidae, Marsupialia). A specific primer pair designed to amplify a region encompassing a 3' terminal 118 bp region of the cytochrome b gene, the Thr and Pro tRNA genes, and a 489 bp region of the D-loop of the D. virginiana mtDNA, was used in highly stringent PCR reactions. These PCR reactions resulted in several fragments per individual varying in size from 259 bp to 1 kb. The sequencing of some of these fragments showed the occurrence of paralogous mtDNA-like sequences among the PCR amplified fragments. Analyses of qualitative aspects of these sequences, their transition/transversion ratios, and phylogenetic relationships were conclusive in showing the occurrence of mtDNA-like sequences in the nuclear genome of the genus Didelphis. Comparisons and phylogenetic analysis of orthologous mtDNA from the four Didelphis species and paralogous nuclear sequences suggested that mtDNA migration to the nuclear genome occurred more than once in Didelphis evolution.

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Antiserum to the egg coats of the fat-tailed dunnart (Marsupialia, Dasyuridae) cross-reacts with egg coats of other marsupial and eutherian species.

We are examining the extracellular coats of the brush-tailed possum as a possible target for an immunocontraceptive vaccine for biocontrol of this pest species in New Zealand. In this study we have compared the composition of the extracellular coats of the fat-tailed and stripe-faced dunnarts, brush-tailed possum, domestic rabbit, and laboratory mouse using histochemistry, immunocytochemistry, and immunofluorescence. The histochemistry of the luminal epithelium of the oviduct and mucoid coats of the marsupials and rabbit indicated that they contain acidic glycoproteins. Immunofluorescence showed that polyclonal antiserum raised against the extracellular coats of the oocyte and early embryo of the fat-tailed dunnart, cross-reacted with the extracellular coats of the oocytes of all five species. These results suggest that there are common epitopes on the extracellular coats of oocytes and early embryos of distinctly related therian species. Further work to characterise these proteins is required to determine whether there is close homology between the oviductal glycoproteins of these species.

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Ultrastructural evidence for loss of the trophoblastic layer in the chorioallantoic placenta of Australian bandicoots (Marsupialia: Peramelidae).

In most marsupials, placentation involves only the yolk sac; however, in the bandicoot family, Peramelidae, a functional chorioallantoic placentation develops in addition (Hill, 1895, 1897, 1900; Flynn, '22, '23). This duality is viewed as having evolutionary significance because most eutheria have both placentae. Furthermore, the bandicoot trophoblast was reported to vanish from the chorioallantoic site in late gestation (Hill, 1897; Flynn, '23); whereas, the eutherian trophoblast is identifiable throughout later pregnancy and may act as an immunological barrier between maternal and fetal genotypes (Kirby '68). Thus we have re-examined this singular chorioallantoic placenta of the bandicoot in plastic sections with light and electron microscopy. A distinctive feature of the bandicoot placentation is the transformation of the uterine simple columnar luminal epithelium into a highly vascular lining composed almost entirely of discrete syncytial masses (homokaryons). Endometrial blood vessels penetrate among the homokaryons to create a rich network of large diameter capillaries at extremely superficial locations near the maternal surface. In the chorioallantoic placenta (7 mm to 10-11 mm crown-rump embryos) the microvillous surface of the maternal homokaryons interdigitates with the microvillous border of the fetal trophoblast with desmosomal interaction. This trophoblast consists of a single layer of tall columnar undifferentiated cells rich in ribosomes-polysomes, poor in cytoplasmic membranes, and with large nuclei that have distinct clumps of heterochromatin and conspicuous nucleoli. It is thus remarkable that these undifferentiated cells disappear as a recognizable layer later in gestation (12 mm crown-rump embryos). Flynn's hypothesis that the trophoblastic cells disappear by fusing with maternal syncytia gains support from the existence of two populations of nuclei in the syncytial masses only at the chorioallantoic site. One population is comparable to that occurring in the homokaryons pf the yolk sac placenta, i.e., pale staining nuclei with little heterochromatin and small peripheral nucleoli. However, the other nuclei resemble those of the trophoblast cells. Since the trophoblastic cells before their disappearance as a layer possess properties associated with potential for further differentiation, the possibility of fusion between the maternal homokaryons and fetal trophoblastic cells to form heterokaryons composed of two genotypes merits fur

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Morphology and histochemistry of the mandibular gland of the Australian brush-tail possum Trichosurus vulpecula (Marsupialia).

A histological, ultrastructural and histochemical study of the mandibular gland of the Australian possum Trichosurus vulpecula revealed essentially similar features as those described earlier for the mandibular gland of the taxonomically relatively unrelated American opossum Didelphis. The secretory endpieces consist of a branched tubular part, composed of serous cells whose secretion granules contain neutral glycoproteins, and terminal acini, consisting of seromucous cells containing small amounts of sialomucins. Relatively short intercalated ducts lead to striated ducts of variable ultrastructural appearance. The striated ducts run in bundles in the center of each sublobule of the gland. The possible functional significance of the abundance and variability in ultrastructure of the striated ducts is discussed.

Adrenergic Fibers↗

Comparative postnatal ontogeny of the skull in the australidelphian metatherian Dasyurus albopunctatus (Marsupialia: Dasyuromorpha: Dasyuridae).

We describe the cranial ontogeny of an australidelphian marsupial, Dasyurus albopunctatus, using a combination of qualitative and quantitative approaches. We examined in detail qualitative morphological changes of just-weaned individuals as compared to old adults; specifically, changes in 31 morphological structures (e.g., processes, foramina) and 38 changes in cranial joints. We also interpreted growth-invariant structures in terms of their functional relevance. We performed a multivariate allometry analysis based on 14 cranial measurements taken from 31 specimens encompassing the entire postweaning period. Three variables (height of occipital plate, breadth of braincase, and height of mandible) showed the same allometric trends in D. albopunctatus and the three marsupial species studied previously in the same framework (Didelphis albiventris, Lutreolina crassicaudata, and Dromiciops gliroides). In addition, D. albopunctatus shared allometric trends in two variables (length of the upper postcanine row and length of the orbit) with the microbiotheriid D. gliroides. Most of the growth trends observed are interpreted as linked to the predominantly carnivorous dietary habit of adult D. albopunctatus. Because dasyuromorphians are most likely basal to the major Australasian radiation of marsupials, knowledge of ontogenetic changes in D. albopunctatus may shed light on the evolution of ontogeny in the highly diverse Australasian marsupial fauna.

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Sperm morphology and storage in the female reproductive tract of the fat-tailed dunnart, Sminthopsis crassicaudata (Marsupialia: Dasyuridae).

A study of spermatozoa in the isthmus of the oviduct and of the surrounding epithelial cells in the dasyurid marsupial, Sminthopsis crassicaudata, was carried out. At least 10% of the ejaculated spermatozoa probably populate the isthmus region, where many come to reside in crypts until around the time of ovulation. Ultrastructural observations of spermatozoa in this region indicated that they had intact acrosomes and were identical in their morphology with those in the cauda epididymidis. After ovulation spermatozoa rapidly disappeared, some of which may be phagocytosed by the cells lining the crypts. These epithelial cells were also found to have many large, electron-dense granules at the time of sperm storage, but the contents did not appear to be released until the zygotes passed along the tract. The secretory activity of these cells may thus relate more to the production of the shell membrane that comes to surround the zygote than to the cells performing a nutritive or protective function for the spermatozoa during their period of storage within the female reproductive tract.

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