Monodelphis Domestica Research Articles

A Burst of Numt Insertion in the Dasyuridae Family During Marsupial Evolution

Nuclear pseudogenes of mitochondrial origin (numts) are common in all eukaryotes. Our previous scan of numts in sequenced nuclear genomes suggested that the highest numt content currently known in animals is that in the gray short-tailed opossum. The present work sought to determine numt content in marsupials and to compare it to those in placental and monothematic mammals as well as in non-mammalian vertebrates. To achieve this, 70 vertebrate species with available nuclear and mitochondrial genomes were scanned for numt content. An extreme numt content was found in the Dasyuridae, with 3,450 in Sarcophilus harrisii (1,955 kb) and 2,813 in Antechinus flavipes (847 kb). The evolutionarily closest species analyzed, the extinct Thylacinus cynocephalus belonging to the Thylacindae family, had only 435 numts (238 kb). These two Dasyuridae genomes featured the highest numt content identified in animals to date. A phylogenetic analysis of numts longer than 300 bp, using a Diprotodonita mitochondrial tree, indicated a burst of numt insertion that began before the divergence of the Dasyurini and Phascogalini, reaching a peak in the early evolution of the two tribes. No comparable increase was found in the early divergent species T. cynocephalus. Divergence of the Dasyuridae tribes has been previously dated to shortly after the Miocene climate transition, characterized by a rapid temperature decline. Interestingly, deviation from optimal growth temperature is one of the environmental factors reported to increase numt insertions in a laboratory setting.

Proteomic analysis of opossum Monodelphis domestica spinal cord reveals the changes of proteins related to neurodegenerative diseases during developmental period when neuroregeneration stops being possible

One of the major challenges of modern neurobiology concerns the inability of the adult mammalian central nervous system (CNS) to regenerate and repair itself after injury. It is still unclear why the ability to regenerate CNS is lost during evolution and development and why it becomes very limited in adult mammals. A convenient model to study cellular and molecular basis of this loss is neonatal opossum (Monodelphis domestica). Opossums are marsupials that are born very immature with the unique possibility to successfully regenerate postnatal spinal cord after injury in the first two weeks of their life, after which this ability abbruptly stops. Using comparative proteomic approach we identified the proteins that are differentially distributed in opossum spinal tissue that can and cannot regenerate after injury, among which stand out the proteins related to neurodegenerative diseases (NDD), such as Huntington, Parkinson and Alzheimer's disease, previously detected by comparative transcriptomics on the analog tissue. The different distribution of the selected proteins detected by comparative proteomics was further confirmed by Western blot (WB), and the changes in the expression of related genes were analysed by quantitative reverse transcription PCR (qRT-PCR). Furthermore, we explored the cellular localization of the selected proteins using immunofluorescent microscopy.To our knowledge, this is the first report on proteins differentially present in developing, non-injured mammalian spinal cord tissue with different regenerative capacities. The results of this study indicate that the proteins known to have an important role in the pathophysiology of neurodegeneration in aged CNS, could also have an important phyisological role during CNS postnatal development and in neuroregeneration process.

Genetic and genomic architecture in eight strains of the laboratory opossum Monodelphis domestica.

The gray short-tailed opossum (Monodelphis domestica) is an established laboratory-bred marsupial model for biomedical research. It is a critical species for comparative genomics research, providing the pivotal phylogenetic outgroup for studies of derived vs ancestral states of genomic/epigenomic characteristics for eutherian mammal lineages. To characterize the current genetic profile of this laboratory marsupial, we examined 79 individuals from eight established laboratory strains. Double digest restriction site-associated DNA sequencing and whole-genome resequencing experiments were performed to investigate the genetic architecture in these strains. A total of 66,640 high-quality single nucleotide polymorphisms (SNPs) were identified. We analyzed SNP density, average heterozygosity, nucleotide diversity, and population differentiation parameter Fst within and between the eight strains. Principal component and population structure analysis clearly resolve the strains at the level of their ancestral founder populations, and the genetic architecture of these strains correctly reflects their breeding history. We confirmed the successful establishment of the first inbred laboratory opossum strain LSD (inbreeding coefficient F > 0.99) and a nearly inbred strain FD2M1 (0.98 < F < 0.99), each derived from a different ancestral background. These strains are suitable for various experimental protocols requiring controlled genetic backgrounds and for intercrosses and backcrosses that can generate offspring with informative SNPs for studying a variety of genetic and epigenetic processes. Together with recent advances in reproductive manipulation and CRISPR/Cas9 techniques for Monodelphis domestica, the existence of distinctive inbred strains will enable genome editing on different genetic backgrounds, greatly expanding the utility of this marsupial model for biomedical research.

Targeted gene disruption in a marsupial, Monodelphis domestica, by CRISPR/Cas9 genome editing

Marsupials represent one of three extant mammalian subclasses with very unique characteristics not shared by other mammals. Most notably, much of the development of neonates immaturely born after a relatively short gestation takes place in the external environment. Among marsupials, the gray short-tailed opossum (Monodelphis domestica; hereafter "the opossum") is one of very few established laboratory models. Due to many biologically unique characteristics and experimentally advantageous features, the opossum is used as a prototype species for basic research on marsupial biology.1,2 However, invivo studies of gene function in the opossum, and thus marsupials in general, lag far behind those of eutherian mammals due to the lack of reliable means to manipulate their genomes. In this study, we describe the successful generation of genome edited opossums by a combination of refined methodologies in reproductive biology and embryo manipulation. We took advantage of the opossum's resemblance to popular rodent models, such as the mouse and rat, in body size and breeding characteristics. First, we established a tractable pipeline of reproductive technologies, from induction of ovulation, timed copulation, and zygote collection to embryo transfer to pseudopregnant females, that warrant an essential platform to manipulate opossum zygotes. Further, we successfully demonstrated the generation of gene knockout opossums at the Tyr locus by microinjection of pronuclear stage zygotes using CRISPR/Cas9 genome editing, along with germline transmission of the edited alleles to the F1 generation. This study provides a critical foundation for venues to expand mammalian reverse genetics into the metatherian subclass.

Hello Possums!

Hello Possums!

Single-cell transcriptome analysis of the B-cell repertoire reveals the usage of immunoglobulins in the gray short-tailed opossum (Monodelphis domestica)

B-cells are key to humoral immunity, are found in multiple lymphoid organs, and have the unique ability to mediate the production of antigen-specific antibodies in the presence of pathogens. The marsupial immunoglobulin (Ig) heavy (H) chain locus encodes four constant region isotypes, IgA, IgG, IgM and IgE, but no IgD, and there are two light (L) chain isotypes, lambda (Igλ) and kappa (Igκ). To gain an understanding of the marsupial humoral immune system, B-cell transcriptomes generated by single-cell RNA sequencing from gray short-tailed opossum (Monodelphis domestica) splenocytes, and peripheral blood mononuclear cells were analysed. The cells used were from a single unimmunized animal and the majority of B-cells were transcribing IgM heavy chains. The ratio of Ig light chain use was roughly 2:1, Igλ:Igκ in this individual. This was not predicted due to Igκ being the more complex of the two L chain loci. The variable (V) gene segment pairs used in individual B-cells confirm greater diversity provided by the L chain V. This study is the first to report on using single cell analysis to investigate Ig repertoires in a marsupial and confirms a number of prior hypothesis, as well as revealing some surprises.

The Element of Paaw in Marsupials and the Ear Region of Philander opossum (Linnaeus, 1758) (Didelphimorphia, Didelphidae)

A small piece of cartilage or bone, the element of Paaw, occurs in the tendon of the stapedius muscle in some extant marsupial and placental mammals. It has been nearly a century since the last comprehensive treatment of the distribution of the element of Paaw in mammals. The current report updates knowledge on this structure by synthesizing the subsequent literature and providing new observations of extant marsupials from the collections of the Section of Mammals, Carnegie Museum of Natural History, and two online resources for CT scanned data: DigiMorph.org and MorphoSource.org. We found an element of Paaw in some representatives of all seven extant marsupial orders: Didelphimorphia, Microbiotheria, Notoryctemorphia, Peramelemorphia, Paucituberculata, Dasyuromorphia, and Diprotodontia. In the first four orders, the element is substantial, longer than the long axis of the fenestra vestiuli (oval window), which holds the stapedial footplate; it is smaller than the long axis of the fenestra vestibuli in Paucituberculata and we do not have measures to report for the last two orders. In most marsupials examined, the element of Paaw contacts the petrosal behind the oval window, suggesting it functions as a sesamoid bone, increasing the lever arm of the stapedius muscle. Although there is some variability in the presence of the bone both between and within individual museum specimens, we interpret this as the result of preparation techniques rather than true variation.To place the element of Paaw in its anatomical context, we describe in detail the ear region and middle-ear auditory apparatus of the gray four-eyed opossum, Philander opossum (Linnaeus, 1758), a didelphid from Central and South America, based on a CT scanned specimen from Carnegie Museum of Natural History. It has an ossified element of Paaw with a volume greater than the stapes. Comparisons are made with petrosals of Didelphis marsupialisLinnaeus, 1758, and Monodelphis domestica (Wagner, 1842), also based on CT scanned specimens.

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.

Developmental plasticity of texture discrimination following early vision loss in the marsupial Monodelphis domestica

ABSTRACTBehavioral strategies that depend on sensory information are not immutable; rather they can be shaped by the specific sensory context in which animals develop. This behavioral plasticity depends on the remarkable capacity of the brain to reorganize in response to alterations in the sensory environment, particularly when changes in sensory input occur at an early age. To study this phenomenon, we utilize the short-tailed opossum, a marsupial that has been a valuable animal model to study developmental plasticity due to the extremely immature state of its nervous system at birth. Previous studies in opossums have demonstrated that removal of retinal inputs early in development results in profound alterations to cortical connectivity and functional organization of visual and somatosensory cortex; however, behavioral consequences of this plasticity are not well understood. We trained early blind and sighted control opossums to perform a two-alternative forced choice texture discrimination task. Whisker trimming caused an acute deficit in discrimination accuracy for both groups, indicating the use of a primarily whisker-based strategy to guide choices based on tactile cues. Mystacial whiskers were important for performance in both groups; however, genal whiskers only contributed to behavioral performance in early blind animals. Early blind opossums significantly outperformed their sighted counterparts in discrimination accuracy, with discrimination thresholds that were lower by ∼75 μm. Our results support behavioral compensation following early blindness using tactile inputs, especially the whisker system.

Molecular Evolution of CatSper in Mammals and Function of Sperm Hyperactivation in Gray Short-Tailed Opossum.

Males have evolved species-specifical sperm morphology and swimming patterns to adapt to different fertilization environments. In eutherians, only a small fraction of the sperm overcome the diverse obstacles in the female reproductive tract and successfully migrate to the fertilizing site. Sperm arriving at the fertilizing site show hyperactivated motility, a unique motility pattern displaying asymmetric beating of sperm flagella with increased amplitude. This motility change is triggered by Ca2+ influx through the sperm-specific ion channel, CatSper. However, the current understanding of the CatSper function and its molecular regulation is limited in eutherians. Here, we report molecular evolution and conservation of the CatSper channel in the genome throughout eutherians and marsupials. Sequence analyses reveal that CatSper proteins are slowly evolved in marsupials. Using an American marsupial, gray short-tailed opossum (Monodelphis domestica), we demonstrate the expression of CatSper in testes and its function in hyperactivation and unpairing of sperm. We demonstrate that a conserved IQ-like motif in CatSperζ is required for CatSperζ interaction with the pH-tuned Ca2+ sensor, EFCAB9, for regulating CatSper activity. Recombinant opossum EFCAB9 can interact with mouse CatSperζ despite high sequence divergence of CatSperζ among CatSper subunits in therians. Our finding suggests that molecular characteristics and functions of CatSper are evolutionarily conserved in gray short-tailed opossum, unraveling the significance of sperm hyperactivation and fertilization in marsupials for the first time.

Two New Species of Chiggers (Trombidiformes: Trombiculidae) From Brazil.

Chigger mites are parasites of terrestrial vertebrates, including humans. Here, we describe two new species belonging to the American genera Colicus Brennan and Parasecia Loomis. Both species were described on the base of museum specimens originated from Minas Gerais State, Brazil, Colicus barrosbattestiae n. sp. parasitizing the rodent, Oligoryzomys fornesi and Parasecia jacinaviciusi n. sp. parasitizing the marsupial, Monodelphis domestica.

In non-eutherians, a third type of T cell

Immunology The two established T cell lineages found in jawed vertebrates use either an αβ or a γδ T cell receptor (TCR) to detect antigens. Recently, another type of TCR chain (TCRµ) was found in marsupials and monotremes. Morrissey et al. analyzed T cells from the gray short-tailed opossum and uncovered a third lineage resident in the spleen that uses a γµ TCR (see the Perspective by Criscitiello). The authors then characterized the crystal structures of two different γµ TCRs, which exhibited an architecture distinct from αβ or γδ TCRs in which a highly diverse, unpaired immunoglobulin-like variable domain was predicted to be the major antigen recognition determinant. Like camelid VHH and shark IgNAR antibodies, γµ TCRs could potentially inform future nanobody development. Science , this issue p. [1383][1]; see also p. [1308][2] [1]: /lookup/doi/10.1126/science.abe7070 [2]: /lookup/doi/10.1126/science.abg7639

The molecular assembly of the marsupial γμ T cell receptor defines a third T cell lineage.

αβ and γδ T cell receptors (TCRs) are highly diverse antigen receptors that define two evolutionarily conserved T cell lineages. We describe a population of γμTCRs found exclusively in non-eutherian mammals that consist of a two-domain (Vγ-Cγ) γ-chain paired to a three-domain (Vμ-Vμj-Cμ) μ-chain. γμTCRs were characterized by restricted diversity in the Vγ and Vμj domains and a highly diverse unpaired Vμ domain. Crystal structures of two distinct γμTCRs revealed the structural basis of the association of the γμTCR heterodimer. The Vμ domain shared the characteristics of a single-domain antibody within which the hypervariable CDR3μ loop suggests a major antigen recognition determinant. We define here the molecular basis underpinning the assembly of a third TCR lineage, the γμTCR.

Establishment of Long-Term Primary Cortical Neuronal Cultures From Neonatal Opossum Monodelphis domestica.

Primary dissociated neuronal cultures have become a standard model for studying central nervous system (CNS) development. Such cultures are predominantly prepared from the hippocampus or cortex of rodents (mice and rats), while other mammals are less used. Here, we describe the establishment and extensive characterization of the primary dissociated neuronal cultures derived from the cortex of the gray South American short-tailed opossums, Monodelphis domestica. Opossums are unique in their ability to fully regenerate their CNS after an injury during their early postnatal development. Thus, we used cortex of postnatal day (P) 3–5 opossum to establish long-surviving and nearly pure neuronal cultures, as well as mixed cultures composed of radial glia cells (RGCs) in which their neurogenic and gliogenic potential was confirmed. Both types of cultures can survive for more than 1 month in vitro. We also prepared neuronal cultures from the P16–18 opossum cortex, which were composed of astrocytes and microglia, in addition to neurons. The long-surviving opossum primary dissociated neuronal cultures represent a novel mammalian in vitro platform particularly useful to study CNS development and regeneration.

A brief report on the bioaccumulation of small terrestrial mammals: a suggestion for a new line of research in Brazil

Small mammals are potential elements to evaluate mining impacts because they occupy key positions in food webs and can be sampled using relatively inexpensive, easy and quick methods. They have been used as environmental indicators for various purposes, such as ecological succession gradients and pollution, among others. However, little is mentioned about bioaccumulation in South America. Our objective is to present data from a heavy metal accumulation test using the Brazilian gracile opossum Gracilinanus microtarsus (J. A. Wagner, 1842) as an experimental model. We concluded that the contents of heavy metals found in the animals’ tissues showed differences when compared to other individuals of the same species from an area without the influence of heavy metals. As a result, we encourage the scientific community to carry out more studies in this little mentioned line in the literature in South America and incipient in Brazil.

Helicobacter monodelphidis sp. nov. and Helicobacter didelphidarum sp. nov., isolated from grey short-tailed opossums (Monodelphis domestica) with endemic cloacal prolapses.

In a search for potential causes of increased prolapse incidence in grey short-tailed opossum colonies, samples from the gastrointestinal tracts of 94 clinically normal opossums with rectal prolapses were screened for Helicobacter species by culture and PCR. Forty strains of two novel Helicobacter species which differed from the established Helicobacter taxa were isolated from opossums with and without prolapses. One of the Helicobacter species was spiral-shaped and urease-negative whereas the other Helicobacter strain had fusiform morphology with periplasmic fibres and was urease-positive. 16S rRNA gene sequence analysis revealed that all the isolates had over 99 % sequence identity with each other, and were most closely related to Helicobacter canadensis. Strains from the two novel Helicobacter species were subjected to gyrB and hsp60 gene and whole genome sequence analyses. These two novel Helicobacter species formed separate phylogenetic clades, divergent from other known Helicobacter species. The bacteria were confirmed as novel Helicobacter species based on digital DNA-DNA hybridization and average nucleotide identity analysis of their genomes, for which we propose the names Helicobacter monodelphidis sp. nov. with the type strain MIT 15-1451T (=LMG 29780T=NCTC 14189T) and Helicobacter didelphidarum sp. nov with type strain MIT 17-337T (=LMG 31024T=NCTC 14188T).

J. P. Hill and Katherine Watson's studies of the neural crest in marsupials.

In the early part of the 20th century, J. P. Hill and K. P. Watson embarked on a comprehensive study of the development of the brain in Australian marsupials. Their work included series from three major groups: dasyurids, peramelids, and diprotodonts, covering early primitive streak through brain closure and folding stages. While the major part of the work was on the development of the brain, in the course of this work they documented that cellular proliferations from the neural plate provided much of the mesenchyme of the branchial arches. These proliferations are now known to be the neural crest. However, except for a very brief note, published shortly after Hill's death, this work was never published. In this study, I present Hill and Watson's work on the development of the early neural plate and the neural crest in marsupials. I compare their findings with published work on the South American marsupial, Monodelphis domestica and demonstrate that patterns reported in Monodelphis are general for marsupials. Further, using their data I demonstrate that in dasyurids, which are ultra-altricial at birth, the neural crest migrates early and in massive quantities, even relative to other marsupials. Finally, I discuss the historical context and speculate on reasons for why this work was unpublished. I find little support for ideas that Hill blocked publication because of loyalty to the germ layer theory. Instead, it appears primarily to have been a very large project that was simply orphaned as Watson and Hill pursued other activities.

Bone biomarkers in koalas: validation of assays and preliminary analyses.

Traumatic injury, including bone fracture, is, to date, one of the leading causes of koala mortality in the South East Queensland region of Australia. Further, the specialist diet of koalas, which is restricted to certain Eucalyptus spp., may impact their normal bone physiology. Considering the dramatic koala population decline and high incidence of trauma, a greater understanding of koala bone physiology may support conservation. We retrieved from GenBank the protein sequences of parathyroid hormone (PTH), osteocalcin (OCN), and tissue-nonspecific alkaline phosphatase (TNALP) in human, dog, cattle, horse, koala, and gray short-tailed opossum. After homology was determined, plasma samples from 13 koalas were analyzed with human PTH, OCN, and bone-specific ALP (BALP) assay kits. Although koala PTH exhibited relatively low sequence homology with placental mammals, high sequence homology between humans and koalas was observed for both OCN and TNALP, and successful cross-reactivity was achieved using human enzyme immunoassay kits for detection of OCN and BALP biomarkers in koala plasma. However, we identified no correlation between OCN and BALP concentrations of healthy and trauma-affected koalas (p = 0.66 and p = 0.79, respectively). Further analysis of OCN and BALP in healthy and diseased koalas will allow a better understanding of bone physiology in this unique marsupial.

Downregulation of TrkC Receptors Increases Dendritic Arborization of Purkinje Cells in the Developing Cerebellum of the Opossum, Monodelphis domestica.

In therian mammals, the cerebellum is one of the late developing structures in the brain. Specifically, the proliferation of cerebellar granule cells occurs after birth, and even in humans, the generation of these cells continues during the first year of life. The main difference between marsupials and eutherians is that the majority of the brain structures in marsupials develop after birth. Herein, we report that in the newborn laboratory opossum (Monodelphis domestica), the cerebellar primordium is distinguishable in Nissl-stained sections. Additionally, bromodeoxyuridine birthdating experiments revealed that the first neurons form the deep cerebellar nuclei (DCN) and Purkinje cells, and are generated within postnatal days (P) 1 and 5. Three weeks after birth, progenitors of granule cells in the external germinal layer (EGL) proliferate, producing granule cells. These progenitor cells persist for a long time, approximately 5 months. Furthermore, to study the effects of neurotrophic tropomyosin receptor kinase C (TrkC) during cerebellar development, cells were obtained from P3 opossums and cultured for 8 days. We found that TrkC downregulation stimulates dendritic branching of Purkinje neurons, which was surprising. The number of dendritic branches was higher in Purkinje cells transfected with the shRNA TrkC plasmid. However, there was no morphological change in the number of dendritic branches of granule cells transfected with either control or shRNA TrkC plasmids. We suggest that inhibition of TrkC activity enables NT3 binding to the neurotrophic receptor p75NTR that promotes dendritic arborization of Purkinje cells. This effect of TrkC receptors on dendritic branching is cell type specific, which could be explained by the strong expression of TrkC in Purkinje cells but not in granule cells. The data indicate a new role for TrkC receptors in Monodelphis opossum.

Understory use by terrestrial small mammals in an unflooded forest patch in the Pantanal floodplain

Abstract To date, there have been no studies that have evaluated small mammal utilization of the understory of forests. In this study, we described the use of vertical strata by small mammals in patches of unflooded forests, known as “cordilheiras”, in the Nhecolândia sub-region of the Pantanal, Brazil. We collected all species using the ground and understory, including the terrestrial didelphid Monodelphis domestica. We suppose that local habitat features (e.g., Acuri palms), rather than intrinsic species characteristics, may be more conducive to the use of understory vegetation by small mammals in the Nhecolândia region.