Monodelphis Domestica Research Articles

Seroprevalence ofToxoplasma gondiiin Wild Marsupials and Rodents from the Atlantic Forest of Pernambuco State, Northeastern Region, Brazil

Felids are important in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in their feces. Cats acquire T. gondii infection in nature by ingesting tissues of small mammals and birds. Serum samples of 223 feral marsupials and 174 feral rodents captured in 7 segments of the Atlantic Forest of the State of Pernambuco, northeastern region of Brazil, and in urban areas of the municipality of Recife were examined for antibodies to T. gondii by the modified agglutination test (MAT). Antibodies (MAT ≥ 25) were found in 6.7% (15 of 223) of the marsupials and 5.7% (10 of 174) of the rodents. No association was observed between seropositivity in marsupials or rodents and sex, age, or different areas of collection (P > 0.05). This is the first study on the seroprevalence of T. gondii in marsupials and rodents performed in the Atlantic Forest of the northeastern region of Brazil. The presence of antibodies to T. gondii are reported for the first time in long-furred woolly mouse opossum ( Micoureus demerarae ), murine mouse opossum ( Marmosa murina ), brown four-eyed opossum ( Metachirus nudicaudatus ), and gray short-tailed opossum ( Monodelphis domestica ).

Differential changes in the cellular composition of the developing marsupial brain.

Throughout development both the body and the brain change at remarkable rates. Specifically, the number of cells in the brain undergoes dramatic nonlinear changes, first exponentially increasing in cell number and then decreasing in cell number. Different cell types, such as neurons and glia, undergo these changes at different stages of development. The current investigation used the isotropic fractionator method to examine the changes in cellular composition at multiple developmental milestones in the short-tailed opossum, Monodelphis domestica. Here we report several novel findings concerning marsupial brain development and organization. First, during the later stages of neurogenesis (P18), neurons make up most of the cells in the neocortex, although the total number of neurons remains the same throughout the life span. In contrast, in the subcortical regions, the number of neurons decreases dramatically after P18, and a converse relationship is observed for nonneuronal cells. In the cerebellum, the total number of cells gradually increases until P180 and then remains constant, and then the number of neurons is consistent across the developmental ages examined. For the three major structures examined, neuronal density and the percentage of neurons within a structure are highest during neurogenesis and then decrease after this point. Finally, the total number of neurons in the opossum brain is relatively low compared with other small-brained mammals such as mice. The relatively low number of neurons and correspondingly high number of nonneurons suggests that in the marsupial brain nonneurons may play a significant role in signal processing.

Identification of a Novel PNMA-MS1 Gene in Marsupials Suggests the LTR Retrotransposon-Derived PNMA Genes Evolved Differently in Marsupials and Eutherians

Two major gene families derived from Ty3/Gypsy long terminal repeat (LTR) retrotransposons were recently identified in mammals. The sushi-ichi retrotransposon homologue (SIRH) family comprises 12 genes: 11 in eutherians including Peg10 and Peg11/Rtl1 that have essential roles in the eutherian placenta and 1 that is marsupial specific. Fifteen and 12 genes were reported in the second gene family, para-neoplastic antigen MA (PNMA), in humans and mice, respectively, although their biological functions and evolutionary history remain largely unknown. Here, we identified two novel candidate PNMA genes, PNMA-MS1 and -MS2 in marsupials. Like all eutherian-specific PNMA genes, they exhibit the highest homology to a Gypsy12_DR (DR, Danio rerio) Gag protein. PNMA-MS1 is conserved in both Australian and South American marsupial species, the tammar wallaby and grey short-tailed opossum. However, no PNMA-MS1 orthologue was found in eutherians, monotremes or non-mammalian vertebrates. PNMA-MS1 was expressed in the ovary, mammary gland and brain during development and growth in the tammar, suggesting that PNMA-MS1 may have acquired a marsupial-specific function. However, PNMA-MS2 seems to be a pseudogene. The absence of marsupial orthologues of eutherian PNMA genes suggests that the retrotransposition events of the Gypsy12_DR-related retrotransposons that gave rise to the PNMA family occurred after the divergence of marsupials and eutherians.

Expression patterns of Oct4, Cdx2, Tead4, and Yap1 proteins during blastocyst formation in embryos of the marsupial, Monodelphis domestica Wagner

The marsupial blastocyst forms in an entirely different manner from its eutherian counterpart, involving cell-zona rather than cell-cell adhesion during the 8- to-16-cell transition. While the eutherian blastocyst consists of a spherical trophoblast completely enveloping a pluripotent inner cell mass, or pluriblast, the marsupial blastocyst forms initially as a bowl-shaped monolayer of cells lining the zona pellucida at the embryonic pole (ep). This monolayer contains a small patch of centrally positioned pluriblast cells edged with trophoblast cells that later coalesce at the abembryonic pole. Using immunocytochemistry, we examined the localization of the proteins Oct4, Cdx2, Tead4, Sox2, and Yap1 in opossum embryos to determine if their temporal expression pattern differed from that in the mouse, given the important differences in cell behavior preceding blastocyst formation in these mammals. Our results indicate that these proteins are expressed in similar temporal patterns despite the topological differences between mouse and opossum cleavage-stage embryos and blastocysts. That the Hippo-pathway protein Yap1 localized specifically around the approximately 128-cell stage to opossum trophoblast nuclei but remained in the cytoplasm of pluriblast cells suggests that this transcriptional regulator participates in allocating cells to the trophoblast lineage, as it does in mouse. Interestingly, in both mouse and opossum embryos, expression of the pluripotency marker Oct4 persisted after Cdx2, which signals trophoblast specification, began to be expressed in trophoblast cells. This and the observation that Cdx2 is present in opossum embryos well before blastomere-zona adhesion even occurs suggests that the proteins studied may have other roles in early mammalian embryonic development.

Expression and cellular distribution of ubiquitin in response to injury in the developing spinal cord of Monodelphis domestica.

Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellular distribution in spinal cord up to postnatal day P35 in control opossums (Monodelphis domestica) and in response to complete spinal transection (T10) at P7, when axonal growth through site of injury occurs, and P28 when this is no longer possible. Cords were collected 1 or 7 days after injury, with age-matched controls and segments rostral to lesion were studied. Following spinal injury ubiquitin levels (western blotting) appeared reduced compared to controls especially one day after injury at P28. In contrast, after injury mRNA expression (qRT-PCR) was slightly increased at P7 but decreased at P28. Changes in isoelectric point of separated ubiquitin indicated possible post-translational modifications. Cellular distribution demonstrated a developmental shift between earliest (P8) and latest (P35) ages examined, from a predominantly cytoplasmic immunoreactivity to a nuclear expression; staining level and shift to nuclear staining was more pronounced following injury, except 7 days after transection at P28. After injury at P7 immunostaining increased in neurons and additionally in oligodendrocytes at P28. Mass spectrometry showed two ubiquitin bands; the heavier was identified as a fusion product, likely to be an ubiquitin precursor. Apparent changes in ubiquitin expression and cellular distribution in development and response to spinal injury suggest an intricate regulatory system that modulates these responses which, when better understood, may lead to potential therapeutic targets.

The discovery of Foxl2 paralogs in chondrichthyan, coelacanth and tetrapod genomes reveals an ancient duplication in vertebrates

The Foxl2 (forkhead box L2) gene is an important member of the forkhead domain family, primarily responsible for the development of ovaries during female sex differentiation. The evolutionary studies conducted previously considered the presence of paralog Foxl2 copies only in teleosts. However, to search for possible paralog copies in other groups of vertebrates and ensure that all predicted copies were homolog to the Foxl2 gene, a broad evolutionary analysis was performed, based on the forkhead domain family. A total of 2464 sequences for the forkhead domain were recovered, and subsequently, 64 representative sequences for Foxl2 were used in the evolutionary analysis of this gene. The most important contribution of this study was the discovery of a new subgroup of Foxl2 copies (ortholog to Foxl2B) present in the chondrichthyan Callorhinchus milii, in the coelacanth Latimeria chalumnae, in the avian Taeniopygia guttata and in the marsupial Monodelphis domestica. This new scenario indicates a gene duplication event in an ancestor of gnathostomes. Furthermore, based on the analysis of the syntenic regions of both Foxl2 copies, the duplication event was not exclusive to Foxl2. Moreover, the duplicated copy distribution was shown to be complex across vertebrates, especially in tetrapods, and the results strongly support a loss of this copy in eutherian species. Finally, the scenario observed in this study suggests an update for Foxl2 gene nomenclature, extending the actual suggested teleost naming of Foxl2A and Foxl2B to all vertebrate sequences and contributing to the establishment of a new evolutionary context for the Foxl2 gene.

Molecular identification and expression of Lyn tyrosine kinase isoforms in marsupials

Lyn is a tyrosine kinase molecule required for modulation of signalling cascades in cell populations including B lymphocytes of the mammalian immune system. We have characterised the coding domain of the marsupial lyn gene of two macropod marsupials; the Tammar Wallaby (Macropus eugenii) and the Bridled Nailtail Wallaby (Onychogalea fraenata) and show the co-expression of two Lyn isoforms in cells and tissues of these and three other marsupials (Brush-tail Possum, Trichosurus vulpecula; American Grey Short-tailed Opossum, Monodelphis domestica and Red-tailed Phascogale, Phascogale calura). The predicted Lyn proteins (LynA and LynB) were highly conserved across vertebrate species, with amino acid identities of 94% with their human orthologues and conservation of key tyrosine kinase motifs that suggests that marsupial Lyn most likely functions in cell signalling. Comparison of our cDNA data to annotations for Lyn transcripts (available through the Ensembl Genome Browser) for the Tammar Wallaby confirm splice sites for a number of exons in the wallaby transcript that are missing from the current annotation. This is the first report of the expression of kinase signalling molecules that influence immunity in metatherian mammals and provides key information to support ongoing studies of immune regulation in marsupials.

Revision of the family Listropsoralgidae Fain, 1965 (Acariformes: Sarcoptoidea)—skin parasites of marsupials and rodents

The family Listropsoralgidae Fain, 1965 (Acariformes: Sarcoptoidea) is represented by the permanent skin ectoparasites associated with the South American and Australian marsupials (12 species) and the South American rodents of the family Echimyidae (1 species). The phylogenetic relationships of these mites (12 ingroup and 2 outgroup species) are reconstructed on the basis of the maximum parsimony (MP) and Bayesian analyses (BA) of 76 morphological characters. MP analysis confirmed monophyly of the listropsoralgid genera, the strict consensus of 18 trees generated by MP has the following pattern: Petauralges (Listropsoralgoides, Didelphialges, Listropsoralges) with poor resolution among species of the genus Listropsoralges. The same tree was generated by BA. Both successive and implied weighting strategies resulted in 7 MP trees: Petauralges (Listropsoralgoides (Didelphialges (Listropsoralges))). The relationships between species of the genus Listropsoralges received the poorest resolution: L. caenolestes (L. monodelphis, L. vossi, L. faini, L. brevisetosa (L. thylamys (L. marmosa-L. caluromys))). The host-parasite relationships of listropsoralgids are briefly discussed. The family Listropsoralgidae is taxonomically revised and to date includes 13 species in 4 genera. Six species and one genus are described as new: Listropsoralges brevisetosus sp. n. from Marmosa murina (Didelphimorphia: Didelphidae) from Peru, Listropsoralges similis sp. n. from Caluromys derbianus (Didelphidae) from Panama, Listropsoralges thylamys sp. n. from Thylamys venustus (Didelphidae) from Bolivia, Listropsoralges vossi sp. n. from Monodelphis domestica (Didelphidae) from Brazil, Listropsoralges caenolestes sp. n. from Caenolestes fuliginosus (Paucituberculata: Caenolestidae) from Ecuador, and Didelphialges metachirus gen. n., sp. n. from Metachirus nudicaudatus (Didelphidae) from Peru. The female of Listropsoralges faini Bochkov and Wauthy, 2009 is described for the first time.

The evolution of active vibrissal sensing in mammals: evidence from vibrissal musculature and function in the marsupial opossumMonodelphis domestica

Facial vibrissae, or whiskers, are found in nearly all extant mammal species and are likely to have been present in early mammalian ancestors. A sub-set of modern mammals, including many rodents, move their long mystacial whiskers back and forth at high speed whilst exploring in a behaviour known as 'whisking'. It is not known whether the vibrissae of early mammals moved in this way. The grey short-tailed opossum, Monodelphis domestica, is considered a useful species from the perspective of tracing the evolution of modern mammals. Interestingly, these marsupials engage in whisking bouts similar to those seen in rodents. To better assess the likelihood that active vibrissal sensing was present in ancestral mammals, we examined the vibrissal musculature of the opossum using digital miscroscopy to see whether this resembles that of rodents. Although opossums have fewer whiskers than rats, our investigation found that they have a similar vibrissal musculature. In particular, in both rats and opossums, the musculature includes both intrinsic and extrinsic muscles with the intrinsic muscles positioned as slings linking pairs of large vibrissae within rows. We identified some differences in the extrinsic musculature which, interestingly, matched with behavioural data obtained through high-speed video recording, and indicated additional degrees of freedom for positioning the vibrissae in rats. These data show that the whisker movements of opossum and rat exploit similar underlying mechanisms. Paired with earlier results suggesting similar patterns of vibrissal movement, this strongly implies that early therian (marsupial and placental) mammals were whisking animals that actively controlled their vibrissae.

Immunolocalization of cation-chloride cotransporters in the developing and mature spinal cord of opossums, Monodelphis domestica.

Spinal inhibition is required to generate coordinated outputs between antagonistic muscles during locomotion. It relies on low neuronal chloride concentration set by two cation-chloride cotransporters, NKCC1 and KCC2 which, respectively, pumps Cl− in or out of neurons. It is generally accepted that NKCC1 is gradually inactivated during development, while KCC2 is upregulated and activated, resulting in low intracellular [Cl−]. Newborn opossums are very immature but perform rhythmic and alternate movements of the forelimbs to crawl on the mother's belly and attach to a teat. Their hindlimbs are immobile. The alternation of the forelimbs suggests that mechanisms allowing spinal inhibition are present at birth. We studied the anatomical basis of inhibition in the spinal enlargements of postnatal opossums by immunolocalizing NKCC1 and KCC2. In some specimens, motoneurons and sensory afferents were labeled with TRDA prior to immunolabeling. At birth, both NKCC1 and KCC2 are detected in the presumptive gray and white matter of the ventral and the intermediolateral cord of both enlargements, but are sparse in the dorsal horn, where KCC2 is mostly seen on a small bundle of dendrites along primary afferents. KCC2 labeling is bright and has a mesh-like appearance in the gray matter and a radial appearance in the white matter, whereas NKCC1 is pale and diffuse. The subsequent expression of the cotransporters follows general ventrodorsal and mediolateral gradients, with the lumbar segments slightly lagging the cervical segments, until the mature pattern is observed around the 5th week. At all ages studied, KCC2 labeling is strong in the periphery of neurons. NKCC1 labeling decreases and becomes more uniformly distributed in the cells with age. Despite the significant anatomical and motor differences between the forelimbs and the hindlimbs of neonatal opossums, the maturation of KCC2 and NKCC1 is quite similar in both enlargements.

Deformable coregistration of multimodal imaging data of gray short-tailed opossum brain

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Multimodal stereotactic template of the gray short-tailed opossum's brain

Multimodal stereotactic template of the gray short-tailed opossum's brain

Mapping the Prion Protein Distribution in Marsupials: Insights from Comparing Opossum with Mouse CNS

The cellular form of the prion protein (PrPC) is a sialoglycoprotein widely expressed in the central nervous system (CNS) of mammalian species during neurodevelopment and in adulthood. The location of the protein in the CNS may play a role in the susceptibility of a species to fatal prion diseases, which are also known as the transmissible spongiform encephalopathies (TSEs). To date, little is known about PrPC distribution in marsupial mammals, for which no naturally occurring prion diseases have been reported. To extend our understanding of varying PrPC expression profiles in different mammals we carried out a detailed expression analysis of PrPC distribution along the neurodevelopment of the metatherian South American short-tailed opossum (Monodelphis domestica). We detected lower levels of PrPC in white matter fiber bundles of opossum CNS compared to mouse CNS. This result is consistent with a possible role for PrPC in the distinct neurodevelopment and neurocircuitry found in marsupials compared to other mammalian species.

Cephalic sensory influence on forelimb movement in newborn opossums, Monodelphis domestica

Like other marsupials, the opossum Monodelphis domestica is born very immature and crawls, unaided by the mother, from the urogenital opening to a nipple where it attaches and pursues its development. If the alternate, rhythmic movements of the forelimbs which allow this locomotion are generated by the developing spinal motor networks, sensory information is nonetheless needed to guide the newborn to a nipple. Behavioral, anatomical and physiological studies suggest that the auditory and the visual systems are insufficiently developed in newborn opossums to influence spinal motor centers, while the vestibular, trigeminal, and olfactory systems are likelier candidates. The trigeminal, vestibular and olfactory regions of the brain were electrically stimulated to test their relative effectiveness at eliciting forelimb movement in newborn opossums, using in vitro preparations of brain-spinal cord with the limbs attached. The minimal stimulation of the cervical spinal cord needed to induce forelimb movement was considered as threshold (T). Stimulations of the trigeminal ganglion (5G) at ∼2T and of the vestibular complex at ∼20T could induce the same movement, and were not statistically different, in contrast to the ∼600T necessary for the olfactory bulb (OB). Neurofilament-200 immunohistochemistry and retrograde tracing with Texas-Red conjugated Dextran Amines were used to study trigeminal innervation of the facial skin and pathways by which trigeminal inputs may be relayed to the spinal cord. Numerous nerve fibers were observed in the snout dermis, especially in the maxillary region, but also elsewhere in the head skin. Some 5G cells project to the upper spinal cord, but more project to the caudal medulla where they could contact secondary trigeminal neurons or reticular cells projecting to the spinal cord. These results support a significant influence of the trigeminal and the vestibular systems, but not of olfaction, on forelimb movement of neonatal opossums.

Early Postnatal B Cell Ontogeny and Antibody Repertoire Maturation in the Opossum, Monodelphis domestica

Marsupials are a lineage of mammals noted for giving birth to highly altricial young, which complete much of their “fetal” development externally attached to a teat. Postnatal B cell ontogeny and diversity was investigated in a model marsupial species, the gray short-tailed opossum, Monodelphis domestica. The results support the initiation of B cell development late in gestation and progressing into the first two weeks of postnatal life. Transcription of CD79a and CD79b was detected in embryonic tissue prior to birth, while immunoglobulin heavy chain locus transcription was not detected until the first postnatal 24 hours. Transcription of the Ig light chains was not detected until postnatal day 7 at the earliest. The predicted timing of the earliest appearance of mature B cells and completion of gene rearrangements is consistent with previous analyses on the timing of endogenous antibody responses in newborn marsupials. The diversity of early B cell IgH chains is limited, as has been seen in fetal humans and mice, but lacks bias in the gene segments used to encode the variable domains. Newborn light chain diversity is, from the start, comparable to that of the adult, consistent with an earlier hypothesis that light chains contribute extensively to antibody diversity in this species.

Convergent and divergent evolution of genomic imprinting in the marsupial Monodelphis domestica

BackgroundGenomic imprinting is an epigenetic phenomenon resulting in parent-of-origin specific monoallelic gene expression. It is postulated to have evolved in placental mammals to modulate intrauterine resource allocation to the offspring. In this study, we determined the imprint status of metatherian orthologues of eutherian imprinted genes.ResultsL3MBTL and HTR2A were shown to be imprinted in Monodelphis domestica (the gray short-tailed opossum). MEST expressed a monoallelic and a biallelic transcript, as in eutherians. In contrast, IMPACT, COPG2, and PLAGL1 were not imprinted in the opossum. Differentially methylated regions (DMRs) involved in regulating imprinting in eutherians were not found at any of the new imprinted loci in the opossum. Interestingly, a novel DMR was identified in intron 11 of the imprinted IGF2R gene, but this was not conserved in eutherians. The promoter regions of the imprinted genes in the opossum were enriched for the activating histone modification H3 Lysine 4 dimethylation.ConclusionsThe phenomenon of genomic imprinting is conserved in Therians, but the marked difference in the number and location of imprinted genes and DMRs between metatherians and eutherians indicates that imprinting is not fully conserved between the two Therian infra-classes. The identification of a novel DMR at a non-conserved location as well as the first demonstration of histone modifications at imprinted loci in the opossum suggest that genomic imprinting may have evolved in a common ancestor of these two Therian infra-classes with subsequent divergence of regulatory mechanisms in the two lineages.

Visual acuity in the short-tailed opossum (Monodelphis domestica)

Monodelphis domestica (short-tailed opossum) is an emerging animal model for studies of neural development due to the extremely immature state of the nervous system at birth and its subsequent rapid growth to adulthood. Yet little is known about its normal sensory discrimination abilities. In the present investigation, visual acuity was determined in this species using the optokinetic test (OPT), which relies on involuntary head tracking of a moving stimulus and can be easily elicited using a rotating visual stimulus of varying spatial frequencies. Using this methodology, we determined that the acuity of Monodelphis is 0.58 cycles per degree (cpd), which is similar to the acuity of rats using the same methodology, and higher than in mice. However, acuity in the short-tailed opossum is lower than in other marsupials. This is in part due to the methodology used to determine acuity, but may also be due to differences in diel patterns, lifestyle and phylogeny. We demonstrate that for the short-tailed opossum, the OPT is a rapid and reliable method of determining a baseline acuity and can be used to study enhanced acuities due to cortical plasticity.

Response to novelty in the laboratory Wistar rat, wild-captive WWCPS rat, and the gray short-tailed opossum (Monodelphis domestica)

Behavior of the laboratory gray short-tailed opossums (Monodelphis domestica), Warsaw Wild Captive Pisula Stryjek rats (WWCPS) and laboratory rats (Wistar) has been registered in the period of familiarization with a new environment and consecutive confrontation with a novel, innocuous object placed in that familiarized environment. In the new environment the sequence of anxiety, investigation, and habituation was shortest in the opossum, longer in the laboratory rat and longest in the WWCPS rat. When placed in it, gray short-tailed opossums investigated the new environment with the shortest delay and most intensity. In reaction to novel objects, opossums and laboratory rats prolonged the time spent in the proximity of the new object, while the WWCPS rat did not show that reaction. Both opossums and laboratory rats increased the number of contacts with the new object, whereas WWCPS rats reduced those contacts. Behavior of all three species and lines grouped in different clusters. Some other quantitative and qualitative differences in behavior of the investigated animals are also described, showing a higher level of anxiety in both lines of rats than in the opossum. Behavioral differences between species and lines of animals used in this study may be attributed to different ecological adaptations of rats and opossums and to the effect of domestication in the laboratory rats. These behavioral differences make comparisons of opossums vs rat, and wild rat vs laboratory rat interesting models for studying the brain mechanisms of anxiety and neotic motivations.

Recombination, transcription, and diversity of a partially germline-joined VH in a mammal

Full or partially germline-joined V genes have been described in a number of different vertebrate lineages where they can contribute to the expressed antibody repertoire through different mechanisms. Here we demonstrate that VH3.1, a partially germline-joined VH gene in the opossum Monodelphis domestica, can undergo V(D)J recombination to generate productive IgH transcripts. VH3.1 is fused to a DH gene segment in the germline DNA and is the only known example of a germline-joined VH in a mammal. B cells that have recombined VH3.1 were not detected until nearly 2 months of age, around the time of weaning, and much later than B cells using the conventional VH. Compared to opossum IgH transcripts using the conventional VH genes, those with VH3.1 have unusually long CDR3 due to the length of the germline-joined DH.

Rsx is a metatherian RNA with Xist-like properties in X-chromosome inactivation

In female (XX) mammals one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY)1. X-inactivation in eutherian mammals is mediated by the non-coding RNA Xist2. Xist is not found in metatherians3 and how X-inactivation is initiated in these mammals has been the subject of speculation for decades4. Using the marsupial Monodelphis domestica we identify Rsx (RNA-on-the-silent X), an RNA that exhibits properties consistent with a role in X-inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, where both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-inactivation in mammals and pose questions about the mechanisms by which X-inactivation is achieved in eutherians.