X Chromosome Inactivation during Grasshopper Spermatogenesis

Regulation of transcriptional activity during meiosis depends on the interrelated processes of recombination and synapsis. In eutherian mammal spermatocytes, transcription levels change during prophase-I, being low at the onset of meiosis but highly increased from pachytene up to the end of diplotene. However, X and Y chromosomes, which usually present unsynapsed regions throughout prophase-I in male meiosis, undergo a specific pattern of transcriptional inactivation. The interdependence of synapsis and transcription has mainly been studied in mammals, basically in mouse, but our knowledge in other unrelated phylogenetically species is more limited. To gain new insights on this issue, here we analyzed the relationship between synapsis and transcription in spermatocytes of the grasshopper Eyprepocnemis plorans. Autosomal chromosomes of this species achieve complete synapsis; however, the single X sex chromosome remains always unsynapsed and behaves as a univalent. We studied transcription in meiosis by immunolabeling with RNA polymerase II phosphorylated at serine 2 and found that whereas autosomes are active from leptotene up to diakinesis, the X chromosome is inactive throughout meiosis. This inactivation is accompanied by the accumulation of, at least, two repressive epigenetic modifications: H3 methylated at lysine 9 and H2AX phosphorylated at serine 139. Furthermore, we identified that X chromosome inactivation occurs in premeiotic spermatogonia. Overall, our results indicate: (i) transcription regulation in E. plorans spermatogenesis differs from the canonical pattern found in mammals and (ii) X chromosome inactivation is likely preceded by a process of heterochromatinization before the initiation of meiosis.

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 (ddRAD-seq) 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 M. 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.

Sensitivity of The Stripe-Faced Dunnart, Sminthopsis Macroura (Gould 1845), To The Phenyl Pyrazole Insecticide, Fipronil, Toxicological Signs And Implications For Pesticide Risk Assessments In Australia

A lack of toxicity data quantifying responses of Australian native mammals to agricultural pesticides prompted an investigation into the sensitivity of the stripe-faced dunnart, Sminthopsis macroura (Gould 1845) to the insecticide, fipronil (5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylsulfinyl pyrazole, CAS No. 120068-37-3). Using the Up-And-Down method for determining acute oral toxicity in mammals, derived by the Organisation for Economic Cooperation and Development (OECD), median lethal dose estimates of 990 mg kg− 1 (95% confidence interval (CI) = 580.7–4770.0 mg kg− 1) and 270.4 mg kg− 1 (95% CI = 0.0 - >20000.0 mg kg− 1) were resolved for male and female S. macroura respectively. The difference between median lethal dose estimates for males and females may have been influenced by the increased age of two female dunnarts. Further modelling of female responses to fipronil doses used the following assumptions: (a) death at 2000 mg kg− 1, (b) survival at 500 mg kg− 1 and (c) a differential response (both survival and death) at 990 mg kg− 1. This modelling revealed median lethal dose estimates for female S. macroura of 669.1 mg kg− 1 (95% CI = 550–990 mg kg− 1; assuming death at 990 mg kg− 1) and 990 mg kg− 1 (95% CI = 544.7–1470 mg kg− 1; assuming survival at 990 mg kg− 1). These median lethal dose estimates are 3–10-fold higher than the only available LD50 value for a similarly sized eutherian mammal, Mus musculus (L. 1758; 94 mg kg− 1) and that available for Rattus norvegicus (Birkenhout 1769; 97 mg kg− 1). Implications for pesticide risk assessments in Australia are discussed.

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

The gray short-tailed opossum is an established laboratory-bred marsupial model for biomedical research. It serves as a critical species for comparative genomics research, providing the pivotal phylogenetic outgroup for studies of derived vs. ancestral states of genomic/epigenomic characteristics for all 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 (ddRAD-seq) 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 M. 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.

Ancient Viral Integrations in Marsupials: A Potential Antiviral Defence

Marsupial viruses are understudied compared to their eutherian mammal counterparts, although they may pose severe threats to vulnerable marsupial populations. Genomic viral integrations, termed endogenous viral elements (EVEs) could protect the host from infection. It is widely known past viral infections and EVEs play an active role in antiviral defence in invertebrates and plants. This study aimed to characterise actively transcribed EVEs in Australian marsupial species, because they may play an integral role in cellular defence against viruses. This study screened publicly available RNA sequencing datasets (n=35) and characterised 200 viral transcripts from thirteen Australian marsupial species. Of the 200 transcripts, 188 originated from either Bornaviridae, Filoviridae or Parvoviridae EVEs. The other 12 transcripts were from putative active infections from members of the Herpesviridae and Anelloviridae, and Hepadnaviridae. EVE transcripts (n=188) were mapped to marsupial genomes (where available, n=5/13) to identify the genomic insertion sites. Of the 188 transcripts, 117 mapped to 39 EVEs within the koala, bare-nosed wombat, tammar wallaby, brushtail possum and Tasmanian devil genomes. The remaining eight animals had no available genome (transcripts n=71). Every marsupial have Bornaviridae, Filoviridae and Parvoviridae EVEs, a trend widely observed in eutherian mammals. Whilst eutherian bornavirus EVEs are predominantly nucleoprotein-derived, marsupial bornavirus EVEs demonstrate a surprising replicase gene bias. We predicted these widely distributed EVEs were conserved within marsupials from ancient germline integrations, as many were over 65 million years old. One bornavirus replicase EVE, present in six marsupial genomes, was estimated to be 160 million years old, predating the American-Australian marsupial split. We considered transcription of these EVEs through small non-coding RNA as an ancient viral defence. Consistent with this, in koala small RNA sequence datasets we detected Bornaviridae replicase and Filoviridae nucleoprotein produced piRNA. These were enriched in testis tissue, suggesting they could protect marsupials from vertically transmitted viral integrations.

Apical Sperm Hook Morphology Is Linked to Sperm Swimming Performance and Sperm Aggregation in Peromyscus Mice

Mammals exhibit a tremendous amount of variation in sperm morphology and despite the acknowledgement of sperm structural diversity across taxa, its functional significance remains poorly understood. Of particular interest is the sperm of rodents. While most Eutherian mammal spermatozoa are relatively simple cells with round or paddle-shaped heads, rodent sperm are often more complex and, in many species, display a striking apical hook. The function of the sperm hook remains largely unknown, but it has been hypothesized to have evolved as an adaptation to inter-male sperm competition and thus has been implicated in increased swimming efficiency or in the formation of collective sperm movements. Here we empirically test these hypotheses within a single lineage of Peromyscus rodents, in which closely related species naturally vary in their mating systems, sperm head shapes, and propensity to form sperm aggregates of varying sizes. We performed sperm morphological analyses as well as in vitro analyses of sperm aggregation and motility to examine whether the sperm hook (i) morphologically varies across these species and (ii) associates with sperm competition, aggregation, or motility. We demonstrate inter-specific variation in the sperm hook and then show that hook width negatively associates with sperm aggregation and sperm swimming speed, signifying that larger hooks may be a hindrance to sperm movement within this group of mice. Finally, we confirmed that the sperm hook hinders motility within a subset of Peromyscus leucopus mice that spontaneously produced sperm with no or highly abnormal hooks. Taken together, our findings suggest that any adaptive value of the sperm hook is likely associated with a function other than inter-male sperm competition, such as interaction with ova or cumulous cells during fertilization, or migration through the complex female reproductive tract.

Petrosal Anatomy of the Paleocene Eutherian Mammal Deltatherium fundaminis (Cope, 1881)

We describe the tympanic anatomy of the petrosal of Deltatherium fundaminis, an enigmatic Paleocene mammal based on cranial specimens recovered from New Mexico, U.S.A. Although the ear region of Deltatherium has previously been described, there has not been a comprehensive, well-illustrated contribution using current anatomical terminology. The dental and cranial anatomy of Deltatherium is a chimera, with morphological similarities to both ‘condylarth’ and ‘cimolestan’ taxa. As such, the phylogenetic relationships of this taxon have remained elusive since its discovery, and it has variably been associated with Arctocyonidae, Pantodonta and Tillodontia. The petrosal of Deltatherium is anteriorly bordered by an open space comprising a contiguous carotid opening and pyriform fenestra. The promontorium features both a small rostral tympanic process and small epitympanic wing but lacks well-marked sulci. A large ventral facing external aperture of the canaliculus cochleae is present and bordered posteriorly by a well-developed caudal tympanic process. The hiatus Fallopii opens on the ventral surface of the petrosal. The tegmen tympani is mediolaterally broad and anteriorly expanded, and its anterior margin is perforated by a foramen for the ramus superior of the stapedial artery. The tympanohyal is small but approximates the caudal tympanic process to nearly enclose the stylomastoid notch. The mastoid is widely exposed on the basicranium and bears an enlarged mastoid process, separate from the paraoccipital process. These new observations provide novel anatomical data corroborating previous hypotheses regarding the plesiomorphic eutherian condition but also reveal subtle differences among Paleocene eutherians that have the potential to help inform the phylogeny of Deltatherium.

An enigmatic specialized new eutherian mammal from the Late Cretaceous of Western Europe (Northern Pyrenees)

We report the discovery of a new Late Cretaceous eutherian mammal, Azilestes ragei n. gen., n. sp. from the Mas-d’Azil northern Pyrenean site (France), which is among the largest known. It is only known from a broken lower jaw found in uppermost levels of the Grès de Labarre Formation (early Maastrichtian). Despite its poor preservation, it displays distinctive specialized features with respect to known Cretaceous eutherians. This includes a reduced premolar formula and shortened and robust jaw, an incipient hypolophid, and a cingular-like postcristid and hypoconulid. The phylogenetic analysis suggests indeed a possible stem relationship between Azilestes n. gen. and some clades of herbivorous Cenozoic placentals, but with weak support. Several molar features reminiscent of the Zhelestidae, especially Valentinella Tabuce, Vianey-Liaud & Garcia, 2004, support instead that Azilestes n. gen. is a basal eutherian showing early specialization in a herbivorous diet convergent with some crown placentals. Whatever the suprageneric position of Azilestes n. gen., which remains to be clarified with additional material, its discovery highlights a significant diversity of European Cretaceous eutherians in contrast to their very poor fossil record.

Evolution of Fc Receptor-Like Scavenger in Mammals

Fc receptor-like (FCRL) molecules comprise a large family of receptors, homologous to the receptors for the Fc portion of immunoglobulins (FCR). Within this family, an unusual gene known to exist in mice, rats and dogs, termed FCRLS, encodes a chimeric protein with both Ig-like FCRL and type B scavenger-receptor cysteine-rich (SRCR)-like domains. In mice, FCRLS is located next to the CD5L and KIRREL1 genes. Here, we show that the curious FCRLS gene is actually present across major mammalian groups, but its annotation is generally incorrect or absent. Anchored on mouse FCRLS and FCRL2 genomic sequence alignments, phylogenetic analyses demonstrated that many mammalian sequences currently annotated as FCRL2 cluster with FCRLS, supported by a conserved genetic synteny among organisms. This analysis shows that FCRLS is present in Rodentia, some Carnivora (Canidae and Ursidae), Chiroptera, Arctiodactyla, Proboscidae, and some Primata. Thus, the FCRLS most likely originated in a eutherian mammal ancestor since it is not present in Monotremata or Marsupialia. FCRLS has a peculiar distribution pattern across mammalian lineages, being present in some species, but absent in others from the same family, as in carnivores for example. The most parsimonious hypothesis to explain this FCRLS evolution is that it was convergently lost in several independent mammalian lineages. Analyses of branch-specific nucleotide evolutionary rates, show that FCRL2 and FCRLS have similar ranges of rates across mammals, suggesting that both genes have crucial, but separate functions in the immune system. Bayesian estimates of evolutionary rates for FCRLS in mammalian lineages revealed that carnivores display the highest mutation rate after rodents. Additionally, positive diversifying selection was detected for both FCRL2 and FCRLS. Our results show that the presence of the FCRLS gene is older and more widespread across mammals than previously thought and appears to be functional, being under positive selection. Its precise physiologic role should thus be investigated.

Evolution of the Human Chromosome 13 Synteny: Evolutionary Rearrangements, Plasticity, Human Disease Genes and Cancer Breakpoints

The history of each human chromosome can be studied through comparative cytogenetic approaches in mammals which permit the identification of human chromosomal homologies and rearrangements between species. Comparative banding, chromosome painting, Bacterial Artificial Chromosome (BAC) mapping and genome data permit researchers to formulate hypotheses about ancestral chromosome forms. Human chromosome 13 has been previously shown to be conserved as a single syntenic element in the Ancestral Primate Karyotype; in this context, in order to study and verify the conservation of primate chromosomes homologous to human chromosome 13, we mapped a selected set of BAC probes in three platyrrhine species, characterised by a high level of rearrangements, using fluorescence in situ hybridisation (FISH). Our mapping data on Saguinus oedipus, Callithrix argentata and Alouatta belzebul provide insight into synteny of human chromosome 13 evolution in a comparative perspective among primate species, showing rearrangements across taxa. Furthermore, in a wider perspective, we have revised previous cytogenomic literature data on chromosome 13 evolution in eutherian mammals, showing a complex origin of the eutherian mammal ancestral karyotype which has still not been completely clarified. Moreover, we analysed biomedical aspects (the OMIM and Mitelman databases) regarding human chromosome 13, showing that this autosome is characterised by a certain level of plasticity that has been implicated in many human cancers and diseases.