Speciation, evolution and phylogeny of some shallow-water octocorals (Cnidaria: Anthozoa)

Shallow-water octocorals are among the most abundant macro-benthic organisms inhabiting tropical subtropical and temperate communities. In spite of being worldwide distributed and highly diverse, the systematics of many octocorals remains controversial and the understanding of the processes who led to their diversification is largely unexplored. This study includes five chapters, each dealing with different aspects of the systematics, phylogeny and evolution of six soft coral genera such as Lobophytum, Sarcophyton, Paramuricea, Leptogorgia, Muricea and Pacifigorgia. One of the main goals of the study was to explore, through the use of molecular methods, the genetic variation within species notoriously difficult to identify. Besides the use of standard molecular methods for phylogenetic reconstruction and species delimitation, the effectiveness of Next Generation Sequencing (NGS) technologies was tested for mitogenomic and genotyping analyses. In the first chapter the use of single-locus markers (e.g. COI, mtMutS and 28S rDNA) was investigated and different automated species delimitation methods (e.g. ABGD, bPTP) were employed to assess species richness among soft coral genera from Western Australia. The methods used appeared suitable for preliminary and rapid diversity assessments especially in the presence of species-rich genera such as Lobophytum and Sarcophyton where morphological identification is particularly difficult and time consuming. In the second chapter, along with the sequencing of complete mitogenomes of Mediterranean Paramuricea species (P. clavata and P. macrospina), the biogeography of the genus was investigated. The results revealed nucleotide and genome size polymorphisms, while the biogeographic predictions suggested that the Mediterranean species have resulted from independent speciation events, explaining in part the high phylogenetic divergence detected. In the third chapter, the sequencing of complete mitogenomes of five Leptogorgia species from different geographic areas (eastern Pacific, eastern Atlantic and Mediterranean) was followed by phylogenetic reconstructions based on an extended mtMutS dataset. The phylogenetic tree recovered Leptogorgia polyphyletic with a clear segregation between the eastern Pacific and eastern Atlantic forms. A time calibrated phylogeny provided insights into the evolution of the genus. In chapter four, using NGS approaches, the complete mitochondrial genome of two eastern Pacific Muricea species (M. crassa and M. purpurea) has been sequenced. The recovery of complete mitogenomes allowed to evaluate the presence of variable and informative regions and to infer a more robust phylogeny. Overall, the results showed high nucleotide diversity in the intergenic spacers, making these regions new potential molecular markers for species-level identifications. In the last chapter a genome-wide Single Nucleotide Polymorphisms (SNPs) and a Bayes Factor Delimitation method were used to infer the genetic relationships within species of the genus Pacifigorgia. The data obtained showed incongruence between molecular and morphological investigations suggesting the possibility of alternative taxonomic assignments for these species. This study provides information on the evolution and speciation of ecologically important soft corals, which distribution range from the littoral and sublittoral zones of the Mediterranean to the tropical and subtropical reefs of Western Australia (WA) and eastern Pacific (EP). The use of mitochondrial markers such as MutS allowed to shed some light on the biogeography and evolutionary history of widespread gorgonians with special emphasis on the Mediterranean endemics and the Atlantic species. Concerning the Western Australia, the obtained results will support the management and conservation of under-investigated marine biodiversity hotspots and potentially species-rich localities such as the Kimberley. In terms of species delimitation, the application of genome-wide SNPs and the use of NGS technologies showed a higher resolution when compared with the traditional methods based on DNA barcoding and single-locus phylogenies. The data generated have been used to clarify the systematics of the species investigated and will be considered as a baseline for future studies on population genetics with a closer look on the adaptive processes

Speciation, evolution and phylogeny of some shallow-water octocorals (Cnidaria: Anthozoa)

Shallow-water octocorals are among the most abundant macro-benthic organisms inhabiting tropical subtropical and temperate communities. In spite of being worldwide distributed and highly diverse, the systematics of many octocorals remains controversial and the understanding of the processes who led to their diversification is largely unexplored. This study includes five chapters, each dealing with different aspects of the systematics, phylogeny and evolution of six soft coral genera such as Lobophytum, Sarcophyton, Paramuricea, Leptogorgia, Muricea and Pacifigorgia. One of the main goals of the study was to explore, through the use of molecular methods, the genetic variation within species notoriously difficult to identify. Besides the use of standard molecular methods for phylogenetic reconstruction and species delimitation, the effectiveness of Next Generation Sequencing (NGS) technologies was tested for mitogenomic and genotyping analyses. In the first chapter the use of single-locus markers (e.g. COI, mtMutS and 28S rDNA) was investigated and different automated species delimitation methods (e.g. ABGD, bPTP) were employed to assess species richness among soft coral genera from Western Australia. The methods used appeared suitable for preliminary and rapid diversity assessments especially in the presence of species-rich genera such as Lobophytum and Sarcophyton where morphological identification is particularly difficult and time consuming. In the second chapter, along with the sequencing of complete mitogenomes of Mediterranean Paramuricea species (P. clavata and P. macrospina), the biogeography of the genus was investigated. The results revealed nucleotide and genome size polymorphisms, while the biogeographic predictions suggested that the Mediterranean species have resulted from independent speciation events, explaining in part the high phylogenetic divergence detected. In the third chapter, the sequencing of complete mitogenomes of five Leptogorgia species from different geographic areas (eastern Pacific, eastern Atlantic and Mediterranean) was followed by phylogenetic reconstructions based on an extended mtMutS dataset. The phylogenetic tree recovered Leptogorgia polyphyletic with a clear segregation between the eastern Pacific and eastern Atlantic forms. A time calibrated phylogeny provided insights into the evolution of the genus. In chapter four, using NGS approaches, the complete mitochondrial genome of two eastern Pacific Muricea species (M. crassa and M. purpurea) has been sequenced. The recovery of complete mitogenomes allowed to evaluate the presence of variable and informative regions and to infer a more robust phylogeny. Overall, the results showed high nucleotide diversity in the intergenic spacers, making these regions new potential molecular markers for species-level identifications. In the last chapter a genome-wide Single Nucleotide Polymorphisms (SNPs) and a Bayes Factor Delimitation method were used to infer the genetic relationships within species of the genus Pacifigorgia. The data obtained showed incongruence between molecular and morphological investigations suggesting the possibility of alternative taxonomic assignments for these species. This study provides information on the evolution and speciation of ecologically important soft corals, which distribution range from the littoral and sublittoral zones of the Mediterranean to the tropical and subtropical reefs of Western Australia (WA) and eastern Pacific (EP). The use of mitochondrial markers such as MutS allowed to shed some light on the biogeography and evolutionary history of widespread gorgonians with special emphasis on the Mediterranean endemics and the Atlantic species. Concerning the Western Australia, the obtained results will support the management and conservation of under-investigated marine biodiversity hotspots and potentially species-rich localities such as the Kimberley. In terms of species delimitation, the application of genome-wide SNPs and the use of NGS technologies showed a higher resolution when compared with the traditional methods based on DNA barcoding and single-locus phylogenies. The data generated have been used to clarify the systematics of the species investigated and will be considered as a baseline for future studies on population genetics with a closer look on the adaptive processes

Pathogenicity of Beauveria bassiana (Bals.-Criv.) Vuill. and Metarhizium anisopliae (Metschn.) Sorokin against Galleria mellonella L. and Tenebrio molitor L. in laboratory assays

The pathogenicity of 23 isolates of Beauveria bassiana (Ascomycota, Hypocreales: Cordycipitaceae) and four of Metarhizium anisopliae (Ascomycota, Hypocreales: Clavicipitaceae) was tested against Galleria mellonella (Lepidoptera: Galleriidae) and Tenebrio molitor (Coleoptera: Tenebrionidae) larvae in laboratory assays, using 2•106 conidia mL-1 fungal suspensions. The commercial myco-insecticide Naturalis (Intrachem Bio Italia, Italy), containing the ATCC 74040 B. bassiana strain, was included in the assays for comparison. Mycosed larvae were counted 1, 2, 3, 7, 10, 14 and 17 days after inoculation and the cumulative mortality data were used to calculate mean survival time (MST) and lethal times (LT50 and LT95). No difference between B. bassiana and M. anisopliae were detected in the pathogenicity against the two insect species. However, a wide variability occurred among fungal isolates within species. The two B. bassiana isolates AL1 and ALB55 killed G. mellonella larvae within the shortest time (MST of 2.2 and 2.3 days, respectively), as well as the ALB55 did against T. molitor larvae (MST of 2.8 days). Naturalis was superior to these two B. bassiana isolates, causing a MST of 1.1 day or shorter on the insect larvae. Overall, G. mellonella resulted more sensitive than T. molitor, as showed also by the non-inoculated controls, for which MSTs were 7.7 and 8.4 days, respectively. Due to the rapid and effective insecticide action, the ALB55 B. bassiana isolate can be considered as a new promising candidate for the microbial pest control

Occurrence of Acute Pulmonary Embolism in COVID-19—A case series

[No abstract available

Mitochondrial RNA processing in absence of tRNA punctuations in octocorals

Background: Mitogenome diversity is staggering among early branching animals with respect to size, gene density, content and order, and number of tRNA genes, especially in cnidarians. This last point is of special interest as tRNA cleavage drives the maturation of mitochondrial mRNAs and is a primary mechanism for mt-RNA processing in animals. Mitochondrial RNA processing in non-bilaterian metazoans, some of which possess a single tRNA gene in their mitogenomes, is essentially unstudied despite its importance in understanding the evolution of mitochondrial transcription in animals. Results: We characterized the mature mitochondrial mRNA transcripts in a species of the octocoral genus Sinularia (Alcyoniidae: Octocorallia), and defined precise boundaries of transcription units using different molecular methods. Most mt-mRNAs were polycistronic units containing two or three genes and 5' and/or 3' untranslated regions of varied length. The octocoral specific, mtDNA-encoded mismatch repair gene, the mtMutS, was found to undergo alternative polyadenylation, and exhibited differential expression of alternate transcripts suggesting a unique regulatory mechanism for this gene. In addition, a long noncoding RNA complementary to the ATP6 gene (lncATP6) potentially involved in antisense regulation was detected. Conclusions: Mt-mRNA processing in octocorals possessing a single mt-tRNA is complex. Considering the variety of mitogenome arrangements known in cnidarians, and in general among non-bilaterian metazoans, our findings provide a first glimpse into the complex mtDNA transcription, mt-mRNA processing, and regulation among early branching animals and represent a first step towards understanding its functional and evolutionary implications

The Proto-Oncogene LRF Is under Post-Transcriptional Control of MiR-20a: Implications for Senescence

MicroRNAs (miRNAs) are short 20–22 nucleotide RNA molecules that act as negative regulators of gene expression via translational repression: they have been shown to play a role in development, proliferation, stress response, and apoptosis. The transcriptional regulator LRF (Leukemia/lymphoma Related Factor) has been shown to prevent p19ARF transcription and consequently to inhibit senescence in mouse embryonic fibroblasts (MEF). Here we report, for the first time, that LRF is post-transcriptionally regulated by miR-20a. Using a gene reporter assay, direct interaction of miR-20a with the LRF 3′UTR is demonstrated. To validate the interaction miR-20a/3′UTR LRF miR-20a was over-expressed, either by transient transfection or retroviral infection, in wild type mouse embryo fibroblasts and in LRF-null MEF derived from LRF knock-out mice. We observed LRF decrease, p19ARF increase, inhibition of cell proliferation and induction of senescence. The comparison of miR-20a activity in wt and LRF-null MEF indicates that LRF is the main mediator of the miR-20a-induced senescence and that other targets are cooperating. As LRF down-regulation/p19ARF induction is always accompanied by E2F1 down-regulation and increase of p16, we propose that all these events act in synergy to accomplish miR-20a-induced senescence in MEF. Senescence has been recently revaluated as a tumor suppressor mechanism, alternative to apoptosis; from this point of view the discovery of new physiological “senescence inducer” appears to be promising as this molecule could be used as anticancer drug

The Pseudogenes of Barley

Pseudogenes have a reputation of being ‘evolutionary relics’ or ‘junk DNA’. While they are well characterized in mammals, studies in more complex plant genomes were so far hampered by the absence of reference genome sequences. Barley is one of the economically most important cereals and has a genome size of 5.1 Gb. With the first high-quality genome reference assembly available for a Triticeae crop, we conducted a whole genome assessment of pseudogenes on the barley genome. We identified, characterized, and classified 89,440 gene fragments and pseudogenes, scattered along the chromosomes with occasional hotspots and higher densities at the chromosome ends. Full-length pseudogenes (11,015) have preferentially retained their exon-intron structure. Retrotransposition of processed mRNAs only plays a marginal role in their creation. However, the distribution of retroposed pseudogenes reflects the Rabl configuration of barley chromosomes and thus hints towards founding mechanisms. While defense-response related parent genes were found under-represented in cultivated barley, we detected several defense related pseudogenes in wild barley accessions. 7.2% of the pseudogenes are transcriptionally active and may potentially adopt new regulatory roles.The barley genome is rich in pseudogenes and small gene fragments mainly located towards chromosome tips or as tandemly repeated units. Our results indicate non-random duplication and pseudogenization preferences and improve our understanding of gene birth and death dynamics in large plant genomes and the mechanisms that lead to evolutionary innovations201

New deep-water cnidarian sites in the southern Adriatic Sea

Recent ROV (Remotely Operated Vehicle) exploration and bottom sampling in the southern Adriatic Sea (Apulian and Montenegrin margins) resulted in the discovery of cnidarian-rich deep-sea habitats in the depth range of ca. 400-700 m. In particular, ROV inspection of Montenegrin canyons reveals the existence of megabenthic communities dominated by a variety of cnidarians, including scleractinians (Madrepora oculata, Lophelia pertusa, Dendrophyllia cornigera),antipatharians (Leiopathes glaberrima) and gorgonians (Callogorgia verticillata) as major habitat forming taxa, often in association with sponges and, subordinately, serpulids. All such cnidarians are new records for the south-eastern side of the Adriatic Sea. Our investigation indicates that an almost continuous belt of patchy cold water coral sites occurs along the entire south-western margin (Apulian),basically connecting the Adriatic populations with those inhabiting the Ionian margin (Santa Maria di Leuca coral province)

A Network of MicroRNAs and mRNAs Involved in Melanosome Maturation and Trafficking Defines the Lower Response of Pigmentable Melanoma Cells to Targeted Therapy

Simple Summary Selective inhibitors of mutant BRAFV600E (BRAFi) have revolutionized the treatment of metastatic melanoma patients and represent a powerful example of the efficacy of targeted therapy. However, one of the main limitations of BRAFi is that treated cells put in place several adaptive response mechanisms, which initially confer drug tolerance and later provide a gateway for the insurgence of genetically acquired resistance mechanisms. We previously discovered that pigmentation is one of these adaptive response mechanisms. Upon BRAFi treatment, those cells that increase their pigmentation level are more resistant to BRAFi than those that do not. Here, we demonstrate that pigmentation limits BRAFi activity through an increase in the number of intracellular mature melanosomes. We also show that this increase derives from increased maturation and/or trafficking. In addition, we identify the miRNAs and mRNAs that are involved in these biological processes. Finally, we provide the rationale for testing a new combinatorial therapeutic strategy that aims at increasing BRAFi efficacy by blocking the adaptive responses that they elicit. This strategy is based on the combined use of BRAFi with inhibitors of pigmentation, specifically inhibitors of melanosome maturation and/or trafficking. Background: The ability to increase their degree of pigmentation is an adaptive response that confers pigmentable melanoma cells higher resistance to BRAF inhibitors (BRAFi) compared to non-pigmentable melanoma cells. Methods: Here, we compared the miRNome and the transcriptome profile of pigmentable 501Mel and SK-Mel-5 melanoma cells vs. non-pigmentable A375 melanoma cells, following treatment with the BRAFi vemurafenib (vem). In depth bioinformatic analyses (clusterProfiler, WGCNA and SWIMmeR) allowed us to identify the miRNAs, mRNAs and biological processes (BPs) that specifically characterize the response of pigmentable melanoma cells to the drug. Such BPs were studied using appropriate assays in vitro and in vivo (xenograft in zebrafish embryos). Results: Upon vem treatment, miR-192-5p, miR-211-5p, miR-374a-5p, miR-486-5p, miR-582-5p, miR-1260a and miR-7977, as well as GPR143, OCA2, RAB27A, RAB32 and TYRP1 mRNAs, are differentially expressed only in pigmentable cells. These miRNAs and mRNAs belong to BPs related to pigmentation, specifically melanosome maturation and trafficking. In fact, an increase in the number of intracellular melanosomes-due to increased maturation and/or trafficking-confers resistance to vem. Conclusion: We demonstrated that the ability of pigmentable cells to increase the number of intracellular melanosomes fully accounts for their higher resistance to vem compared to non-pigmentable cells. In addition, we identified a network of miRNAs and mRNAs that are involved in melanosome maturation and/or trafficking. Finally, we provide the rationale for testing BRAFi in combination with inhibitors of these biological processes, so that pigmentable melanoma cells can be turned into more sensitive non-pigmentable cells