Liliana Milani

De Novo Assembly of the Manila Clam Ruditapes philippinarum Transcriptome Provides New Insights into Expression Bias, Mitochondrial Doubly Uniparental Inheritance and Sex Determination

Males and females share the same genome, thus, phenotypic divergence requires differential gene expression and sex-specific regulation. Accordingly, the analysis of expression patterns is pivotal to the understanding of sex determination mechanisms. Many bivalves are stable gonochoric species, but the mechanism of gonad sexualization and the genes involved are still unknown. Moreover, during the period of sexual rest, a gonad is not present and sex cannot be determined. A mechanism associated with germ line differentiation in some bivalves, including the Manila clam Ruditapes philippinarum, is the doubly uniparental inheritance (DUI) of mitochondria, a variation of strict maternal inheritance. Two mitochondrial lineages are present, one transmitted through eggs and the other through sperm, as well as a mother-dependent sex bias of the progeny. We produced a de novo annotation of 17,186 transcripts from R. philippinarum and compared the transcriptomes of males and females and identified 1,575 genes with strong sex-specific expression and 166 sex-specific single nucleotide polymorphisms, obtaining preliminary information about genes that could be involved in sex determination. Then we compared the transcriptomes between a family producing predominantly females and a family producing predominantly males to identify candidate genes involved in regulation of sex-specific aspects of DUI system, finding a relationship between sex bias and differential expression of several ubiquitination genes. In mammalian embryos, sperm mitochondria are degraded by ubiquitination. A modification of this mechanism is hypothesized to be responsible for the retention of sperm mitochondria in male embryos of DUI species. Ubiquitination can additionally regulate gene expression, playing a role in sex determination of several animals. These data enable us to develop a model that incorporates both the DUI literature and our new findings.

A Comparative Analysis of Mitochondrial ORFans: New Clues on Their Origin and Role in Species with Doubly Uniparental Inheritance of Mitochondria

Despite numerous comparative mitochondrial genomics studies revealing that animal mitochondrial genomes are highly conserved in terms of gene content, supplementary genes are sometimes found, often arising from gene duplication. Mitochondrial ORFans (ORFs having no detectable homology and unknown function) were found in bivalve molluscs with Doubly Uniparental Inheritance (DUI) of mitochondria. In DUI animals, two mitochondrial lineages are present: one transmitted through females (F-type) and the other through males (M-type), each showing a specific and conserved ORF. The analysis of 34 mitochondrial major Unassigned Regions of Musculista senhousia F- and M-mtDNA allowed us to verify the presence of novel mitochondrial ORFs in this species and to compare them with ORFs from other species with ascertained DUI, with other bivalves and with animals showing new mitochondrial elements. Overall, 17 ORFans from nine species were analyzed for structure and function. Many clues suggest that the analyzed ORFans arose from endogenization of viral genes. The co-option of such novel genes by viral hosts may have determined some evolutionary aspects of host life cycle, possibly involving mitochondria. The structure similarity of DUI ORFans within evolutionary lineages may also indicate that they originated from independent events. If these novel ORFs are in some way linked to DUI establishment, a multiple origin of DUI has to be considered. These putative proteins may have a role in the maintenance of sperm mitochondria during embryo development, possibly masking them from the degradation processes that normally affect sperm mitochondria in species with strictly maternal inheritance.

A resourceful genome: updating the functional repertoire and evolutionary role of animal mitochondrial DNAs

Recent data from mitochondrial genomics and proteomics research demonstrate the existence of several atypical mitochondrial protein-coding genes (other than the standard set of 13) and the involvement of mtDNA-encoded proteins in functions other than energy production in several animal species including humans. These results are of considerable importance for evolutionary and cellular biology because they indicate that animal mtDNAs have a larger functional repertoire than previously believed. This review summarizes recent studies on animal species with a non-standard mitochondrial functional repertoire and discusses how these genetic novelties represent promising candidates for studying the role of the mitochondrial genome in speciation.

Structure, Transcription, and Variability of Metazoan Mitochondrial Genome: Perspectives from an Unusual Mitochondrial Inheritance System

Despite its functional conservation, the mitochondrial genome (mtDNA) presents strikingly different features among eukaryotes, such as size, rearrangements, and amount of intergenic regions. Nonadaptive processes such as random genetic drift and mutation rate play a fundamental role in shaping mtDNA: the mitochondrial bottleneck and the number of germ line replications are critical factors, and different patterns of germ line differentiation could be responsible for the mtDNA diversity observed in eukaryotes. Among metazoan, bivalve mollusc mtDNAs show unusual features, like hypervariable gene arrangements, high mutation rates, large amount of intergenic regions, and, in some species, an unique inheritance system, the doubly uniparental inheritance (DUI). The DUI system offers the possibility to study the evolutionary dynamics of mtDNAs that, despite being in the same organism, experience different genetic drift and selective pressures. We used the DUI species Ruditapes philippinarum to study intergenic mtDNA functions, mitochondrial transcription, and polymorphism in gonads. We observed: 1) the presence of conserved functional elements and novel open reading frames (ORFs) that could explain the evolutionary persistence of intergenic regions and may be involved in DUI-specific features; 2) that mtDNA transcription is lineage-specific and independent from the nuclear background; and 3) that male-transmitted and female-transmitted mtDNAs have a similar amount of polymorphism but of different kinds, due to different population size and selection efficiency. Our results are consistent with the hypotheses that mtDNA evolution is strongly dependent on the dynamics of germ line formation, and that the establishment of a male-transmitted mtDNA lineage can increase male fitness through selection on sperm function.

Doubly Uniparental Inheritance of Mitochondria As a Model System for Studying Germ Line Formation

Background Doubly Uniparental Inheritance (DUI) of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI). DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos. Methodology/Principal Findings We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow. Conclusions/Significance In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown, they could be a variation of the mechanism regulating the mitochondrial bottleneck in all metazoans.

Paternally Transmitted Mitochondria Express a New Gene of Potential Viral Origin

Mitochondrial ORFans (open reading frames having no detectable homology and with unknown function) were discovered in bivalve molluscs with doubly uniparental inheritance (DUI) of mitochondria. In these animals, two mitochondrial lineages are present, one transmitted through eggs (F-type), the other through sperm (M-type), each showing a specific ORFan. In this study, we used in situ hybridization and immunocytochemistry to provide evidence for the expression of Ruditapes philippinarum male-specific ORFan (orf21): both the transcript and the protein (RPHM21) were localized in spermatogenic cells and mature spermatozoa; the protein was localized in sperm mitochondria and nuclei, and in early embryos. Also, in silico analyses of orf21 flanking region and RPHM21 structure supported its derivation from viral sequence endogenization. We propose that RPHM21 prevents the recognition of M-type mitochondria by the degradation machinery, allowing their survival in the zygote. The process might involve a mechanism similar to that of Modulators of Immune Recognition, viral proteins involved in the immune recognition pathway, to which RPHM21 showed structural similarities. A viral origin of RPHM21 may also support a developmental role, because some integrated viral elements are involved in development and sperm differentiation of their host. Mitochondrial ORFans could be responsible for or participate in the DUI mechanism and their viral origin could explain the acquired capability of M-type mitochondria to avoid degradation and invade the germ line, that is what viruses do best: to elude host immune system and proliferate.

Sex-linked mitochondrial behavior during early embryo development in Ruditapes philippinarum (Bivalvia Veneridae) a species with the Doubly Uniparental Inheritance (DUI) of mitochondria.

In most metazoans mitochondria are inherited maternally. However, in some bivalve molluscs, two mitochondrial lineages are present: one transmitted through females (F-type), the other through males (M-type). This unique system is called Doubly Uniparental Inheritance (DUI) of mitochondria. In DUI species, M-type mitochondria have to invade the germ line of male embryos during development, otherwise sperm would transmit F-type mtDNA and DUI would fail. The mechanisms by which sperm mitochondria enter the germ line are still unknown. To address this question, we traced the movement of spermatozoon mitochondria (M-type) in embryos of the DUI species Ruditapes philippinarum by fertilizing eggs with sperm stained with the mitochondrial-specific vital dye MitoTracker Green. As in Mytilus DUI species, in R. philippinarum the distribution of sperm mitochondria follows two different patterns: an aggregated one in which these organelles locate near the first cleavage furrow, and a dispersed one in which sperm mitochondria are scattered. The presence of the two mitochondrial patterns in these taxa, together with their absence in species with Strictly Maternal Inheritance (SMI), confirms that their occurrence is related to DUI. Moreover, a Real-Time qPCR analysis showed that neither M-type nor F-type mitochondria undergo replication boosts in the earliest embryo development. This is the first study on sex-linked mtDNA copy number carried out by qPCR analysis on embryos of a DUI species and the first time the segregation patterns of sperm mitochondria are described in a DUI system other than Mytilus.

Evidence for somatic transcription of male‑transmitted mitochondrial genome in the DUI species Ruditapes philippinarum (Bivalvia: Veneridae)

In species with doubly uniparental inheritance (DUI), males are heteroplasmic for two sex-linked mitochondrial genomes (M- and F-mtDNA). While a role of M-mtDNA in male gametogenesis and sperm function is evident, there is an ongoing debate on whether it is transcribed or not in male soma. In this work we report a qPCR analysis in the DUI species Ruditapes philippinarum, showing that M-mtDNA is transcribed in somatic tissues. We observed a correlation between DNA copy numbers of the two analyzed genes, cytochrome b and a novel male-specific mitochondrial gene thought to be involved in DUI (orf21), and between their transcription levels. No correlation between a transcript and its DNA copy number was found, supporting the existence of complex regulatory mechanisms of mitochondrial transcription. We found the highest amount of mtDNA and mtRNA in gonads, likely due to the intense cell proliferation and high energy request for gametogenesis, while the observed variation among specimens is probably related to their different stages of gonad development. Finally, orf21 showed a highly variable transcription in advanced stages of gametogenesis. We hypothesize a differential storage of orf21 transcripts in spermatozoa, representing different paternal contributions to progeny, possibly leading to different developmental outcomes. A transcriptional activity does not necessarily imply the translation of M-mtDNA genes, and studies on mitochondrial proteins and their localization are needed to definitively assess the functioning of male-transmitted mitochondria in male soma. All that considered, the male soma of DUI species may represent an intriguing experimental model to study cytoplasmic genetic conflicts.

Nuclear genes with sex bias in Ruditapes philippinarum (Bivalvia, veneridae): Mitochondrial inheritance and sex determination in DUI species

Mitochondria are inherited maternally in most metazoans, but in bivalves with Doubly Uniparental Inheritance (DUI) a mitochondrial lineage is transmitted through eggs (F-type), and another through sperm (M-type). In DUI species, a sex-ratio distortion of the progeny was observed: some females produce a female-biased offspring (female-biased family), others a male-biased progeny (male-biased family), and others a 50:50 sex-ratio. A peculiar segregation pattern of M-type mitochondria in DUI organisms appears to be correlated with the sex bias of these families. According to a proposed model for the inheritance of M-type mitochondria in DUI, the transmission of sperm mitochondria is controlled by three nuclear genes, named W, X, and Z. An additional S gene with different dosage effect would be involved in sex determination. In this study, we analyzed structure and localization of three transcripts (psa, birc, and anubl1) with specific sex and family biases in the Manila clam Ruditapes philippinarum. In situ hybridization confirmed the localization of these transcripts in gametogenic cells. In other animals, homologs of these genes are involved in reproduction and ubiquitination. We hypothesized that these genes may have a role in sex determination and could also be responsible for the maintenance/degradation of spermatozoon mitochondria during embryo development of the DUI species R. philippinarum, so that we propose them as candidate factors of the W/X/Z/S system.

Mitochondrial selfish elements and the evolution of biological novelties

We report the present knowledge about RPHM21, a novel male-specific mitochondrial protein with a putative role in the paternal inheritance of sperm mitochondria in the Manila clam Ruditapes philippinarum, a species with doubly uniparental inheritance of mitochondria (DUI). We review all the available data on rphm21 transcription and translation, analyze in detail its female counterpart, RPHF22, discuss the homology with RPHM21, the putative function and origin, and analyze their polymorphism. The available evidence is compatible with a viral origin of RPHM21 and supports its activity during spermatogenesis. RPHM21 is progressively accumulated in mitochondria and nuclei of spermatogenic cells, and we hypothesize it can influence mitochondrial inheritance and sexual differentiation. We propose a testable model that describes how the acquisition of selfish features by a mitochondrial lineage might have been responsible for the emergence of DUI, and for the evolution of separate sexes (gonochorism) from hermaphroditism. The appearance of DUI most likely entailed the invasion of at least 1 selfish element, and the extant DUI systems can be seen as resolved conflicts. It was proposed that hermaphroditism was the ancestral condition of bivalves, and a correlation between DUI and gonochorism was documented. We hypothesize that DUI might have driven the shift from hermaphroditism to gonochorism, with androdioecy as transition state. The invasion of sex-ratio distorters and the evolution of suppressors can prompt rapid changes among sex-determination mechanisms, and DUI might have been responsible for one of such changes in some bivalve species. If true, DUI would represent the first animal sex-determination system involving mtDNA-encoded proteins.