Monodelphis Research Articles

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.

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.

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.

Spatial and temporal patterns of the emerging tick-borne pathogen Borrelia miyamotoi in blacklegged ticks (Ixodes scapularis) in New York

Borrelia miyamotoi, a bacterium that causes relapsing fever, is found in ixodid ticks throughout the northern hemisphere. The first cases of human infection with B. miyamotoi were identified in 2011. In the eastern USA, blacklegged ticks (Ixodes scapularis) become infected by feeding on an infected vertebrate host, or through transovarial transmission. We surveyed B. miyamotoi prevalence in ticks within forested habitats in Dutchess County, New York, and identified possible reservoir hosts. To assess spatial variation in infection, we collected questing nymphal ticks at > 150 sites. To assess temporal variation in infection, we collected questing nymphs for 8 years at a single study site. We collected questing larval ticks from nine plots to estimate the amount of transovarial transmission. To evaluate potential reservoir hosts, we captured 14 species of mammal and bird hosts naturally infested with larval blacklegged ticks and held these hosts in the laboratory until ticks fed to repletion and molted to nymphs. We determined infection for all ticks using quantitative polymerase chain reaction. The overall infection prevalence of questing nymphal ticks across all sites was ~ 1%, but prevalence at individual sites was as high as 9.1%. We detected no significant increase in infection through time. Only 0.4% of questing larval ticks were infected. Ticks having fed as larvae from short-tailed shrews, red squirrels, and opossums tended to have higher infection prevalence than did ticks having fed on other hosts. Further studies of the role of hosts in transmission are warranted. The locally high prevalence of B. miyamotoi in the New York/New England landscape suggests the importance of vigilance by health practitioners and the public.Graphical abstract

The hard task of a short-tailed mouse opossum (Monodelphis) to prey a harvestman (Arachnida: Opiliones)

ABSTRACT Despite the great diversity of small insectivorous mammals and the use of scent gland secretions as a defense mechanism by harvestmen, there is no observation about the effectiveness of scent glands against predators such as small mammals. We report a remarkable harvestman defense mechanism against a small-mammal attack. When a harvestman and a mouse opossum confronted each other inside a cage, the harvestman knocked out the mouse opossum two sequential times before it could attack the harvestman. Although it is a unique observation and there is no information about its frequency in nature, this report stimulates the study of agonistic behavior between small, cryptic species, which are difficult to observe in the field.

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).

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.

Available Sensory Input Determines Motor Performance and Strategy in Early Blind and Sighted Short-Tailed Opossums.

SummaryThe early loss of vision results in a reorganized neocortex, affecting areas of the brain that process both the spared and lost senses, and leads to heightened abilities on discrimination tasks involving the spared senses. Here, we used performance measures and machine learning algorithms that quantify behavioral strategy to determine if and how early vision loss alters adaptive sensorimotor behavior. We tested opossums on a motor task involving somatosensation and found that early blind animals had increased limb placement accuracy compared with sighted controls, while showing similarities in crossing strategy. However, increased reliance on tactile inputs in early blind animals resulted in greater deficits in limb placement and behavioral flexibility when the whiskers were trimmed. These data show that compensatory cross-modal plasticity extends beyond sensory discrimination tasks to motor tasks involving the spared senses and highlights the importance of whiskers in guiding forelimb control.

A single-cell transcriptome atlas of marsupial embryogenesis and Xinactivation.

Single-cell RNA sequencing of embryos can resolve the transcriptional landscape of development at unprecedented resolution. To date, single-cell RNA-sequencing studies of mammalian embryos have focused exclusively on eutherian species. Analysis of mammalian outgroups has the potential to identify deeply conserved lineage specification and pluripotency factors, and can extend our understanding of Xdosage compensation. Metatherian (marsupial) mammals diverged from eutherians around 160million years ago. They exhibit distinctive developmental features, including late implantation1 and imprinted Xchromosome inactivation2, which is associated with expression of the XIST-like noncoding RNA RSX3. Here we perform a single-cell RNA-sequencing analysis of embryogenesis and Xchromosome inactivation in a marsupial, the grey short-tailed opossum (Monodelphis domestica). We resolve the developmental trajectory and transcriptional signatures of the epiblast, primitive endoderm and trophectoderm, and identify deeply conserved lineage-specific markers that pre-date the eutherian-marsupial divergence. RSX coating and inactivation of the Xchromosome occurs early and rapidly. This observation supports the hypothesis that-in organisms with early Xchromosome inactivation-imprinted Xchromosome inactivation prevents biallelic Xsilencing. We identify XSR, an RSXantisense transcript expressed from the active Xchromosome, as a candidate for the regulator of imprinted Xchromosome inactivation. Our datasets provide insights into the evolution of mammalian embryogenesis and Xdosage compensation.

Performance and Behavioral Flexibility on a Complex Motor Task Depend on Available Sensory Inputs in Early Blind and Sighted Short-Tailed Opossums

The early loss of vision results in a reorganization of visual cortex, which becomes commandeered by the somatosensory and auditory systems. Recent studies in the short-tailed opossum found that the connections and response properties of neurons in somatosensory cortex of blind animals are also altered. While studies in humans and other mammals show that early vision loss leads to heightened abilities on discrimination tasks involving the spared senses, if and how this superior discrimination leads to adaptive sensorimotor behavior has yet to be determined. Moreover, little is known about the extent to which blind animals rely on the spared senses. Here, we tested opossums on a sensorimotor task involving somatosensation. We found that early blind animals had increased limb placement accuracy compared to sighted controls, while showing similarities in crossing strategy. Whisker trimming nullified this advantage, resulting in decreases in limb placement accuracy and more variable forelimb movements in both groups. However, the increased reliance on tactile inputs in early blind animals led to greater deficits in limb placement and behavioral flexibility when the whiskers were trimmed. This data demonstrates that incoming sensory input from the whiskers is processed differently in the brains of early blind compared to sighted opossums, likely due, in part, to the dramatic changes to visual and somatosensory cortex. These results also provide direct evidence for the prevailing theory that the whiskers are critical for skilled forelimb control in whisking mammals, and highlight the importance of studying both performance and strategy when assessing differences in behavior.

Opossum Mammary Maturation as It Relates to Immune Cell Infiltration and Nutritional Gene Transcription.

SynopsisThe mammary gland has evolved to accommodate the developmental needs of offspring in species-specific ways. This is particularly true for marsupials. Marsupial milk content changes dramatically throughout lactation in ways appearing timed with neonatal ontogeny and behavior. Here we investigate morphological restructuring within the mammaries throughout lactation in the gray short-tailed opossum, Monodelphis domestica. Substantial remodeling of the mammaries occurs throughout the first half of active lactation. It is not until the latter half of lactation that opossum mammaries appear histologically similar to active eutherian mammaries. Noteworthy was the presence of eosinophils in early developing mammary tissue, which correlated with elevated abundance of transcripts encoding the chemokine IL-16. The presence and abundance of whey protein transcripts within the opossum mammaries were also quantified. Whey acidic protein (WAP) transcript abundance peaked in the latter half of lactation and remained elevated through weaning. Minimal transcripts for the marsupial-specific Early and Late Lactation Proteins (ELP/LLP) were detected during active lactation. Elevated abundance of LLP transcripts was only detected prior to parturition. Overall, the results support the role of eosinophils in mammary restructuring appearing early in mammalian evolution, and describe key similarities and differences in nutritional protein transcript abundance among marsupial species.

Experimental infection of Monodelphis domestica with Rickettsia parkeri

This study aimed to evaluate the infection of the marsupial Monodelphis domestica (the gray short-tailed opossum) by Rickettsia parkeri and its role as an amplifier of the bacterium for Amblyomma ticks. Ten M. domestica males were inoculated with phosphate-buffered saline (PBS) and 106 Vero cells infected with R. parkeri. In seven animals, inoculation was intramuscular, and in three intraperitoneal. One male (control) received 1 ml of the same vehicle used for inoculation intraperitoneally. The three animals inoculated intraperitoneally were infested with uninfected A. sculptum larvae and nymphs between the 2nd and 9th day post-infection (DPI). Parasitemia was monitored from the 3rd to 9th DPI by polymerase chain reaction, using primers for 17 kDa and ompA. The animals were also clinically evaluated. Of the animals infected intramuscularly, only one was blood-positive by the 5th DPI. The three animals infected intraperitoneally were blood-positive on the 2nd, 5th, 7th, and 9th DPI. Of the ten pools of recovered engorged ticks, six had positive bands. The kidney, liver, heart, and spleen of an intramuscularly infected animal were also positive. The rectal temperature of the animals tested increased only in the first three DPI. The animals inoculated intraperitoneally showed prostration, bristled hair, and weight loss. The study found that R. parkeri was capable of infecting M. domestica, which developed rickettsemia and caused infection in xenodiagnostic ticks.

Influence of biological sex on social behavior, individual recogntion, and non-associative learning in the adult gray short-tailed opossum (Monodelphis domestica)

Social behavior is critical for relationship formation and is influenced by myriad environmental and individual factors. Basic and preclinical research typically relies on rodent models to identify the mechanisms that underlie behavior; however, it is important to use non-rodent models as well. A major objective of the present study was to test the hypothesis that biological sex and social experience modulate the expression of social behavior in the adult gray short-tailed opossum (Monodelphis domestica), a non-traditional model. We also investigated the non-associative learning abilities of these animals. Following a period of social isolation, animals of both sexes were paired with a non-familiar, same-sex partner for 10 min on three different occasions, with 24-hour inter-trial intervals. We are the first research group to find significant sex differences in submissive and nonsocial behaviors in Monodelphis. Females displayed significantly higher durations of nonsocial behavior that increased over trials. Males were more aggressive; their latencies to the first attack and submissive behavior decreased over trials whereas these latencies increased for females; males' duration of submissive behavior increased over trials whereas it decreased for females. A different group of subjects habituated in response to repeated presentations to neutral odors and dishabituated in response to novel odors. In addition, both males and females demonstrated the ability to form social memories in a standard individual (social) recognition test. Our results contribute to the characterization of this marsupial species, an important first step in developing it as a model of complex social behaviors.

Endometrial recognition of pregnancy occurs in the grey short-tailed opossum ( Monodelphis domestica).

In human pregnancy, recognition of an embryo within the uterus is essential to support the fetus through gestation. In most marsupials, such as the opossums, pregnancy is shorter than the oestrous cycle and the steroid hormone profile during pregnancy and oestrous cycle are indistinguishable. For these reasons, it was assumed that recognition of pregnancy, as a trait, evolved in the eutherian (placental) stem lineage and independently in wallabies and kangaroos. To investigate whether uterine recognition of pregnancy occurs in species with pregnancy shorter than the oestrous cycle, we examined reproduction in the short-tailed opossum ( Monodelphis domestica), a marsupial with a plesiomorphic mode of pregnancy. We examined the morphological and gene expression changes in the uterus of females in the non-pregnant oestrous cycle and compared these to pregnancy. We found that the presence of an embryo did not alter some aspects of uterine development but increased glandular activity. Transcriptionally, we saw big differences between the uterus of pregnant and cycling animals. These differences included an upregulation of genes involved in transport, inflammation and metabolic-activity in response to the presence of a fetus. Furthermore, transcriptional differences reflected protein level differences in transporter abundance. Our results suggest that while the uterus exhibits programmed changes after ovulation, its transcriptional landscape during pregnancy responds to the presence of a fetus and upregulates genes that may be essential for fetal support. These results are consistent with endometrial recognition of pregnancy occurring in the opossum. While the effects on maternal physiology appear to differ, recognition of pregnancy has now been observed in eutherian mammals, as well as, Australian and American marsupials.

Unraveling the LRC Evolution in Mammals: IGSF1 and A1BG Provide the Keys.

Receptors of the leukocyte receptor cluster (LRC) play a range of important functions in the human immune system. However, the evolution of the LRC remains poorly understood, even in m\\ammals not to mention nonmammalian vertebrates. We conducted a comprehensive bioinformatics analysis of the LRC-related genes in the publicly available genomes of six species that represent eutherian, marsupial, and monotreme lineages of mammals. As a result, the LRCs of African elephant and armadillo were characterized, two new genes, IGSF1 and A1BG, were attributed to the LRC of eutherian mammals, the LRC gene content was substantially extended in the short-tailed opossum and Tasmanian devil and, finally, four LRC genes were identified in the platypus genome. These findings have for the first time provided a solid basis for inference of the LRC phylogeny across mammals. Our analysis suggests that the mammalian LRC family likely derived from two ancestral genes, which evolved in a lineage-specific manner by expansion/contraction, extensive exon shuffling, and sequence divergence. The striking structural and functional diversity of eutherian LRC molecules appears largely lineage specific. The only family member retained in all the three mammalian lineages is a collagen-binding receptor OSCAR. Strong sequence conservation of a transmembrane domain known to associate with FcRγ suggests an adaptive role of this domain subtype in the LRC evolution.

Discovery of the rare Handley’s short-tailed opossum, Monodelphis handleyi, in the threatened southern Amazonian savanna of Brazil

We report a new locality for Monodelphis handleyi, a rare short-tailed opossum species, previously known only from its type locality, in Loreto, northeastern Peru. One adult male was collected using pitfall trap disposed in Humaitá Amazonian savanna of southern Amazonas state, Brazil. Voucher specimen had their identification confirmed by molecular data (mitochondrial gene Cytochrome b) and morphological comparison. We provide external and cranial measurements of this specimen and comment on its morphology. The specimen reported here represents the first record of M. handleyi in Brazil and is the second known locality for the species, and there is high level of divergence found between the two distant localities (5.1%). This record extends the species range at least 1200 km eastern beyond the type locality and denotes the importance of use of complementary methods for sampling small nonvolant mammals.