Monodelphis Research Articles

Characterization of cochlear nucleus principal cells of Meriones unguiculatus and Monodelphis domestica by use of calcium-binding protein immunolabeling

Antibodies directed against calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k and calretinin were used as neuronal markers to identify and characterize different principal cell types in the mammalian cochlear nucleus. For this purpose, double immunofluorescence labeling and the combination of CaBP-labeling with pan-neuronal markers were applied to analyze the CaBPs distribution in neurons of the cochlear nucleus (CN) of the Mongolian gerbil ( Meriones unguiculatus) and the gray short-tailed opossum ( Monodelphis domestica). Despite of the fact, that these two mammalian species are not closely related, principal cell types in the CN of the two species showed many corresponding morphological features and similarities in immunolabeling of the CaBPs. Parvalbumin seems not to be suited as a differential neuronal marker in the CN since it is expressed by almost all neurons. In contrast, calbindin and calretinin were more restricted to specific cell types and showed a mostly complementary labeling pattern. As one of the most interesting findings, calbindin and calretinin were predominantly found in subpopulations of globular bushy cells and octopus cells in the ventral CN. Such a neuron-specific CaBP-expression in subpopulations of morphologically defined cell types argues for a more refined classification of CN cell types in Meriones and Monodelphis. Additionally, other cell types (cartwheel cells, unipolar brush cells, fusiform cells) were marked with calbindin or calretinin as well. Calretinin staining was predominantly observed in auditory nerve fibers and their endings including endbulbs of Held in Meriones. Spherical bushy cells showed a different calretinin-immunolabeling in Meriones and Monodelphis. This species-specific difference may be related to adaptive differences in auditory function.

The genome of a Gondwanan mammal

Australia is thought of as the home of marsupials, but South America has 60 or so species of these interesting mammals. The genome of one of these, the South American grey short-tailed opossum, Monodelphis domestica, has just been sequenced and published in June.1 The high quality 6x coverage is the first marsupial genome completed, pipping the 2x coverage of the Australian tammar wallaby at the post by half a year. The opossum genome has an unusual structure with fewer chromosomes than the human genome (9 pairs versus 23 pairs) but a longer total length (3.4 billion versus 3 billion bases). The opossum autosomes, like those of all marsupials, are extremely large but, in contrast, the X chromosome is only 76 Mb long. The opossum genome has turned up several surprises and provided critical new information on the evolution of mammalian genomes.

THE SUGAR GLIDER (PETAURUS BREVICEPS): A LABORATORY HOST FOR THE NEMATODE PARASTRONGYLOIDES TRICHOSURI

Parastrongyloides trichosuri is a nematode parasite of the Australian brush-tailed possums that can be propagated through many generations in vitro. This makes P. trichosuri uniquely suited for genetic investigations, including those involving transgenesis. However, an obstacle to its use as an experimental model has been the fact that its host is limited to Australia and New Zealand and that it cannot be exported because of its status as a protected species or agricultural pest, respectively. In previous studies, conventional laboratory animals such as rats, mice, rabbits, ferrets, and chickens have failed to support infections. In the present study, gerbils and short-tailed opossums proved similarly refractory to infection. In contrast, the sugar glider (Petaurus breviceps, family Petauridae) proved to be a good host for P. trichosuri. Patent infections resulted using as few as 6 infective larvae (L3i) and as many as 2,000 L3i. Large numbers of L3i (1,000-2,000) produced patent infections of much shorter duration than those seen when 100 L3i were initially given to the sugar glider. In one case, an infection initiated with 100 L3i was patent for over 1 yr. Parastrongyloides trichosuri is easily cryopreserved using a method developed for Strongyloides stercoralis. Thus, we have identified an experimental host for P. trichosuri that will make it possible to conduct research on this parasite in laboratories outside the endemic sites.

Isolation and characterization of melanopsin (Opn4) from the Australian marsupial Sminthopsis crassicaudata (fat-tailed dunnart).

Melanopsin confers photosensitivity to a subset of retinal ganglion cells and is responsible for many non-image-forming tasks, like the detection of light for circadian entrainment. Recently, two melanopsin genes, Opn4m and Opn4x, were described in non-mammalian vertebrates. However, only one form, Opn4m, has been described in the mammals, although studies to date have been limited to the placentals and have not included the marsupials. We report here the isolation and characterization of an Opn4 gene from an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), and present evidence which suggests that the Opn4x gene was lost before the placental/marsupial split. In situ hybridization shows that the expression of Opn4 in the dunnart eye is restricted to a subset of ganglion cells, a pattern previously reported for rodents and primates. These Opn4-positive cells are randomly distributed across the dunnart retina. We also undertook a comparative analysis with the South American marsupial, the grey short-tailed opossum (Monodelphis domestica), and two placental mammals, mouse and human. This approach reveals that the two marsupials show a higher sequence identity than that seen between rodents and primates, despite separating at approximately the same point in time, some 65-85 Myr ago.

Cranial endocasts from a growth series of Monodelphis domestica (Didelphidae, Marsupialia): A study of individual and ontogenetic variation

Intraspecific variation (e.g., ontogenetic, individual, sexual dimorphic) is rarely examined among cranial endocasts (infillings of the braincase cavity) because of the difficulty in obtaining multiple specimens of a species, particularly fossil taxa. We extracted digital cranial endocasts from CT scans of a growth series of skulls of Monodelphis domestica, the gray short-tailed opossum, as a preliminary assessment of the amount of intraspecific variation in mammalian endocranial morphology. The goals of this study were 1) to provide an anatomical description to document developmental changes in endocranial morphology of M. domestica and 2) to examine ontogenetic and individual variation with respect to phylogenetic characters of endocranial cavities that are known to be variable between different mammalian taxa. In this study, "ontogenetic variation" refers to variation between specimens of different ages whereas "individual variation" (i.e., polymorphism) is restricted to variation between specimens of comparable age. Aside from size, changes in shape account for the greatest amount of morphological variation between the endocasts of different ages. Endocast length, width, and volume increase with age for the growth series. Relative olfactory bulb cast size increases with age in the growth series, but the relative size of the parafloccular casts shows a slight negative allometric trend through ontogeny. More than one-third of the phylogenetic characters of the endocranial cavity we examined showed some sort of variation (ontogenetic, individual, or both). This suggests that although endocasts are potentially informative for systematics, both ontogenetic and individual variation affect how endocranial characters are scored for phylogenetic analysis. Further studies such as this are necessary to determine the taxonomic extent of significant intraspecific variation of these endocranial characters.

Towards a ZP‐based contraceptive for marsupials: Comparative analysis and developmental expression of marsupial ZP genes

Fertility control in the form of a zona pellucida (ZP)-based immunocontraceptive has shown potential as a humane form of control for overabundant marsupials including the brushtail possum and macropods. Further refinement and development of a ZP-based vaccine requires detailed knowledge of the protein structure and expression in order to ensure maximum efficacy and specificity. Sequencing and comparative analysis of the ZP3 protein from three marsupial orders in this study found a high overall level of conservation; within order Diprotodontia, the ZP3 protein is 86.9-98.9% identical. ZP3 identity falls to 56.6-57.2%, when the grey, short-tailed opossum (a Didelphimorphian) is compared to dasyurid and diprotodontan marsupials. This is similar to its amino acid identity with ZP3 from eutherian species (50.7-52.8%). Comparison of a 21 amino acid epitope in marsupial ZP3 that has shown contraceptive effects, reveals 95-100% identity between the four macropodid species, 81-86% amino acid identity between brushtail possum and the macropods and 67-71% identity between the diprotodontans and the fat-tailed dunnart (a dasyurid). This is comparable to the level of identity between related eutherian mammals. The expression pattern of three ZP genes during brushtail possum and tammar wallaby pouch young development was examined by RT-PCR. This analysis of ZP gene expression has confirmed that ZP mRNA transcription begins in the ovary during pouch young development by about 51 days of age. The presence of ZP transcripts at this stage in pouch young development suggests that marsupial ZP gene transcription begins before the onset of follicular development.

Molecular characterisation and expression of CD4 in two distantly related marsupials: The gray short-tailed opossum ( Monodelphis domestica) and tammar wallaby ( Macropus eugenii)

The gene and corresponding cDNA for CD4 in the gray short-tailed opossum, Monodelphis domestica, and the cDNA sequence for CD4 in the tammar wallaby, Macropus eugenii, have been characterised. The opossum CD4 homolog reveals conserved synteny, preserved genomic organisation and analogous structural arrangement to human and mouse CD4. Opossum and tammar CD4 exhibit typical eutherian CD4 features including the highly conserved p56 lck binding motif in the cytoplasmic region and the invariant cysteine residues in extracellular domains 1 and 4. Interestingly, the marsupial CD4 sequences substitute a tryptophan for the first cysteine in domain 2 negating the formation of a disulphide bond as seen in other eutherian CD4 sequences except human and mouse. Overall the marsupial CD4 sequences share amino acid identity of 59% to each other and 37–41% with eutherian mammals. However, in contrast to eutherian homologs, the marsupial CD4 sequences were found to be truncated at the terminal end of the cytoplasmic tail. This is the first report confirming the presence of CD4 in a marsupial and describing its key features.

Characterization of the opossum immune genome provides insights into the evolution of the mammalian immune system

The availability of the first marsupial genome sequence has allowed us to characterize the immunome of the gray short-tailed opossum (Monodelphis domestica). Here we report the identification of key immune genes, including the highly divergent chemokines, defensins, cathelicidins, and Natural Killer cell receptors. It appears that the increase in complexity of the mammalian immune system occurred prior to the divergence of the marsupial and eutherian lineages approximately 180 million years ago. Genomes of ancestral mammals most likely contained all of the key mammalian immune gene families, with evolution on different continents, in the presence of different pathogens leading to lineage specific expansions and contractions, resulting in some minor differences in gene number and composition between different mammalian lineages. Gene expansion and extensive heterogeneity in opossum antimicrobial peptide genes may have evolved as a consequence of the newborn young needing to survive without an adaptive immune system in a pathogen laden environment. Given the similarities in the genomic architecture of the marsupial and eutherian immune systems, we propose that marsupials are ideal model organisms for the study of developmental immunology.

Evolutionary dynamics of transposable elements in the short-tailed opossum Monodelphis domestica

The genome of the gray short-tailed opossum Monodelphis domestica is notable for its large size ( approximately 3.6 Gb). We characterized nearly 500 families of interspersed repeats from the Monodelphis. They cover approximately 52% of the genome, higher than in any other amniotic lineage studied to date, and may account for the unusually large genome size. In comparison to other mammals, Monodelphis is significantly rich in non-LTR retrotransposons from the LINE-1, CR1, and RTE families, with >29% of the genome sequence comprised of copies of these elements. Monodelphis has at least four families of RTE, and we report support for horizontal transfer of this non-LTR retrotransposon. In addition to short interspersed elements (SINEs) mobilized by L1, we found several families of SINEs that appear to use RTE elements for mobilization. In contrast to L1-mobilized SINEs, the RTE-mobilized SINEs in Monodelphis appear to shift from G+C-rich to G+C-low regions with time. Endogenous retroviruses have colonized approximately 10% of the opossum genome. We found that their density is enhanced in centromeric and/or telomeric regions of most Monodelphis chromosomes. We identified 83 new families of ancient repeats that are highly conserved across amniotic lineages, including 14 LINE-derived repeats; and a novel SINE element, MER131, that may have been exapted as a highly conserved functional noncoding RNA, and whose emergence dates back to approximately 300 million years ago. Many of these conserved repeats are also present in human, and are highly over-represented in predicted cis-regulatory modules. Seventy-six of the 83 families are present in chicken in addition to mammals.

Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences

We report a high-quality draft of the genome sequence of the grey, short-tailed opossum (Monodelphis domestica). As the first metatherian ('marsupial') species to be sequenced, the opossum provides a unique perspective on the organization and evolution of mammalian genomes. Distinctive features of the opossum chromosomes provide support for recent theories about genome evolution and function, including a strong influence of biased gene conversion on nucleotide sequence composition, and a relationship between chromosomal characteristics and X chromosome inactivation. Comparison of opossum and eutherian genomes also reveals a sharp difference in evolutionary innovation between protein-coding and non-coding functional elements. True innovation in protein-coding genes seems to be relatively rare, with lineage-specific differences being largely due to diversification and rapid turnover in gene families involved in environmental interactions. In contrast, about 20% of eutherian conserved non-coding elements (CNEs) are recent inventions that postdate the divergence of Eutheria and Metatheria. A substantial proportion of these eutherian-specific CNEs arose from sequence inserted by transposable elements, pointing to transposons as a major creative force in the evolution of mammalian gene regulation.

New Species ofMonodelphis(Didelphimorphia: Didelphidae) from Peru, with Notes onM. adusta()

Monodelphis (short-tailed opossums) is the most diverse genus in the family Didelphidae, including at least 20 recognized species. This paper describes a new species of short-tailed opossum from the lowland forests of Loreto, northeastern Peru. The new species is intermediate in size and coloration between M. ronaldi, a species recently described on the basis of 1 specimen from southeastern Peru, and M. adusta, a more common species distributed across western Amazonia. However, the new species is sympatric only with M. emiliae. Diagnostic characters include overall large size, conspicuously wide buffy stripe on the venter, wide rostrum, narrow postorbital constriction, rounded posterior border of the infraorbital foramen, small and well-separated tympanic processes of alisphenoid, conspicuous posttympanic processes, enlarged canines, small 1st upper premolars, and enlarged cingula on premolars and molars. Whereas morphologically the new species closely resembles M. ronaldi and M. adusta, a phylogenetic analysis of mitochondrial DNA sequences (which did not include M. ronaldi) indicates M. osgoodi as its sister taxon. Examination of molecular data also indicates that M. adusta is paraphyletic relative to M. osgoodi and the new species, and that populations currently referred to M. a. adusta and to M. a. peruviana each represent 2 distantly related and clearly distinct species.

Integrated cytogenetic BAC map of the genome of the gray, short-tailed opossum, Monodelphis domestica

The generation of high-quality genome assemblies for numerous species is advancing at a rapid pace. As the number of genome assemblies increases, so does our ability to investigate genome relationships and their contributions to unraveling complex biological, evolutionary, and biomedical processes. A key process in the generation of a genome assembly is to determine and verify the precise physical location and order of the large sequence blocks (scaffolds) that result from the assembly. For organisms of relatively recent common ancestry this process may be achieved largely through comparative sequence alignment. However, as the evolutionary distance between species lengthens, the use of comparative sequence alignment becomes increasingly less reliable. Simultaneous cytogenetic mapping, using multicolor fluorescence in-situ hybridization (FISH) analysis, offers an alternative means to define the cytogenetic location and relative order of DNA sequences, thereby anchoring the genome sequence to the karyotype. In this article we report the molecular cytogenetic locations of 415 bacterial artificial chromosome (BAC) clones that served to anchor sequence scaffolds of the gray, short-tailed opossum (Monodelphis domestica) to its karyotype, which enabled accurate integration of these regions into the genome assembly.

Skin, gland, and urine odors elicit intense investigation by male gray short-tailed opossums, Monodelphis domestica

Chemical signaling between animals of the same species is common among vertebrates. We have reported this phenomenon in the gray short-tailed opossum, Monodelphis domestica (Wagner, 1842). We now find that male opossums investigate odors derived from the mandibular and flank regions and from urine of male and female opossums and from the sternal gland of male opossums significantly more than distilled water. Males investigate female odors significantly more than male odors. They also investigate strange male odors significantly more than their own odors when similar body parts were compared. The males investigate female flank odors significantly longer than female urine odors when these were paired, but the time spent investigating other pairings of female odors was not significantly different. Furthermore, experimental males, unlike females tested in former studies, discriminate between urine of male and of female conspecifics when compared with water control. We hypothesize that male opossums require information about the presence of male conspecifics that recently traversed their home range to avoid potential conflicts. Urine from diestrous females signals the proximity of a female that has not already mated.

Locomotor kinetics on sloped arboreal and terrestrial substrates in a small quadrupedal mammal

Small animals must be capable of moving on a wide variety of surfaces; thus, examining the mechanics of locomotion on a wide variety of substrates is necessary to understand how the animal can utilize its habitat. Therefore, locomotor kinetics are examined on arboreal and terrestrial sloped substrates in the marsupial Monodelphis domestica (gray short-tailed opossum). Substrate reaction forces were measured as opossums moved across four trackways: 30° upslope and 30° downslope trackways, which were flat (“terrestrial”) or cylindrical (“arboreal”). Regardless of substrate slope, medial limb forces were measured on arboreal trackways and usually lateral limb forces on terrestrial trackways. Otherwise the general patterns of vertical and craniocaudal forces and impulses were similar between same-sloped terrestrial and arboreal trackways. Some significant modifications to these gross patterns occurred: on the arboreal upslope trackway, hindlimbs supported more body weight than on the terrestrial uphill, possibly because hindlimbs were more stably positioned on the upslope arboreal trackway than forelimbs. Furthermore, the difference between fore- and hindlimbs with respect to craniocaudal impulses was less on the arboreal sloped trackways. In conclusion, kinetic patterns can usually be explained by body weight support roles and by the placement of the limbs on the arboreal trackway.

Species-specific evolution of the FcR family in endothermic vertebrates

In primates and rodents, the extended FcR family is comprised of three subsets: classical FcRs, structurally diverse cell surface receptors currently designated FCRL1-FCRL6, and intracellular proteins FCRLA and FCRLB. Using bioinformatic analysis, we revealed the FcR-like genes of the same three subsets in the genome of dog, another representative of placental mammals, and in the genome of short-tailed opossum, a representative of marsupials. In contrast, a single FcR-like gene was found in the current version of the chicken genome. This in silico finding was confirmed by the gene cloning and subsequent Southern blot hybridization. The chicken FCRL gene encodes a cell surface receptor with the extracellular region composed of four Ig-like domains of the D1-, D2-, D3-, and D4-subtypes. The gene is expressed in lymphoid and non-lymphoid tissues. Phylogenetic analysis of the mammalian and chicken genes suggested that classical FcRs, FCRLA, and FCRLB emerged after the mammalian-avian split but before the eutherian-marsupial radiation. The data obtained show that the repertoire of the classical FcRs and surface FcR-like proteins in mammalian species was shaped by an extensive recombination process, which resulted in domain shuffling and species-specific gain and loss of distinct exons or entire genes.

Firing Properties of Accessory Olfactory Bulb Mitral/Tufted Cells in Response to Urine Delivered to the Vomeronasal Organ of Gray Short-Tailed Opossums

In comparison with many mammals, there is limited knowledge of the role of pheromones in conspecific communication in the gray short-tailed opossum. Here we report that mitral/tufted (M/T) cells of the accessory olfactory bulb (AOB) of male opossums responded to female urine but not to male urine with two distinct patterns: excitation followed by inhibition or inhibition. Either pattern could be mimicked by application of guanosine 5'-O-3-thiotriphosphate and blocked by guanosine 5'-O-2-thiodiphosphate, indicating that the response of neurons in this pathway is through a G-protein-coupled receptor mechanism. In addition, the inhibitor of phospholipase C (PLC), U73122, significantly blocked urine-induced responses. Male and female urine were ineffective as stimuli for M/T cells in the AOB of female opossums. These results indicate that urine of diestrous females contains a pheromone that directly stimulates vomeronasal neurons through activation of PLC by G-protein-coupled receptor mechanisms and that the response to urine is sexually dimorphic.

A microsatellite-based, physically anchored linkage map for the gray, short-tailed Opossum (Monodelphis domestica)

The genome of the gray, short-tailed opossum, Monodelphis domestica, will be the first of any marsupial to be fully sequenced. The utility of this sequence will be greatly enhanced by construction and integration of detailed genetic and physical maps. Therefore, it is important to verify the unusual recombinational characteristics that were suggested by the 'first-generation' M. domestica linkage map; specifically, very low levels of recombination and severely reduced female recombination, both of which are contrary to patterns in other vertebrates. We constructed a new linkage map based on a different genetic cross, using a new and much larger set of map markers, and physically anchored and oriented the linkage groups onto chromosomes via fluorescence in-situ hybridization mapping. This map includes 150 loci in eight autosomal linkage groups corresponding to the eight autosome pairs, and spans 86-89% of the autosomal genome. The sex-averaged autosomal map covers 715 cM, with a full-length estimate of 866 cM; the shortest full-length linkage map reported for any vertebrate. The sex-specific maps confirmed severely reduced female recombination in all linkage groups, and an overall F/M map ratio = 0.54. These results greatly extend earlier findings, and provide an improved microsatellite-based linkage map for this species.

Suprasternal gland secretion of male short-tailed opossum induces IP 3 generation in the vomeronasal organ

Chemical communication is an important component of mammalian social behaviors. Gray short-tailed opossums ( Monodelphis domestica) communicate by scent marking. The male opossum possesses a prominent suprasternal scent gland, extracts of which strongly attract female opossums. This attractivity remains unaltered following repeated lyophilization. The suprasternal gland secretion functions in a sexually dimorphic manner, i.e., it elicits elevated levels of IP 3 in the vomeronasal (VN) sensory epithelium of female opossums, but suppressed the levels of IP 3 in the VN sensory epithelium of male opossums. The elevated levels of IP 3 induced by suprasternal gland secretion in female vomeronasal sensory epithelium is inhibited by the G i/o specific inhibitor, NF023, but not its inactive analogue, NF007. It is also suppressed by specific antibodies to the alpha subunits of G i and G o proteins, by the phospholipase C inhibitor, U73122, as well as by GDPβS. Surprisingly, GDPβS itself enhances basal levels of IP 3 in female VN sensory epithelium. This GDPβS-induced increase in levels of IP 3 is reduced by the PLC inhibitor, U73122, but not by the G i/o inhibitor, NF023. In addition, GDP also enhances basal levels of IP 3. GDPβS, a known inhibitor of G-protein activation, thus appears to have dual functions: as both stimulator and inhibitor of IP 3 production in the VN sensory epithelium of opossums. In contrast, this nucleotide analogue functions as an inhibitor in the VN sensory epithelium of mice. The mechanism of signal transduction underlying the suprasternal gland secretion-elicited signals in the VN sensory epithelium of opossums appears to involve signals that are generated through activation of G-protein-coupled receptors and transduced via activation of G i/o-proteins and the effector, phospholipase C, resulting in an increased production of the second messenger, IP 3. The extracellular signals are thus amplified.

Discovery of α-defensins in basal mammals

α-Defensins are essential molecules of the innate immune system that have broad spectrum antimicrobial activity against a range of bacteria and viruses. To date, α-defensins have only been identified in the Euarchontoglires branch of the mammals. This has led to speculation that α-defensins may be specific to this group, a somewhat surprising finding, given their importance in the immune system. The mammalian genome project provided us with the opportunity to search for α-defensins in previously unexamined mammalian superorders. Using hidden Markov model (HMM) profile searching, we report the discovery of α-defensins in the African savanna elephant, the lesser hedgehog tenrec, and the nine-banded armadillo genomes representing two of the most basal mammalian superorders, Afrotheria and Xenarthra. Furthermore, we identify an α-defensin-like gene in the gray short-tailed opossum, suggesting that α-defensins may have evolved much earlier than previously thought, before the divergence of placental mammals and marsupials approximately 130 mya.

Phenotypic diversity is the cornerstone of evolution: Variation in cortical field size within short‐tailed opossums

Natural selection operates on phenotypic variation that exists within a population. Variable aspects of cortical organization, such as the size and connections of a cortical field, can generate differences in behavior, which is a target of natural selection. Yet studies pertaining to within-species variability in cortical organization are limited. In the present investigation, we examined variation in brain size, cortical sheet size, and primary sensory cortical field sizes in the adult short-tailed opossum (Monodelphis domestica). Within individuals, we found no significant difference between the right and left hemispheres in the overall size of the dorsolateral cortex or in primary cortical field sizes. Between individuals, we found relatively little intraspecies variation in brain weight, brain volume, and cortical sheet area for the dorsolateral neocortex and pyriform cortex; however, we observed a large degree of variability in body weight and primary sensory cortical field size, as defined by myeloarchitecture. Further, we found that the size of each cortical field correlated with the size of the other cortical fields as well as with the total size of the dorsolateral cortex. Here we discuss the possible sources of variation and examine the relationship between cortical field size and sensory processing abilities and behaviors across species. Since behavior is the target of natural selection, variation in cortical field size across individuals may supply the raw material necessary for cortical field evolution.