Calcareous sponge genomes reveal complex evolution of alpha-carbonic anhydrases and two key biomineralization enzymes

Background: Calcium carbonate biominerals form often complex and beautiful skeletal elements, including coral exoskeletons and mollusc shells. Although the ability to generate these carbonate structures was apparently gained independently during animal evolution, it sometimes involves the same gene families. One of the best-studied of these gene families comprises the a-carbonic anhydrases (CAs), which catalyse the reversible transformation of CO2 to HCO3- and fulfill many physiological functions. Among Porifera -the oldest animal phylum with the ability to produce skeletal elements- only the class of calcareous sponges can build calcitic spicules, which are the extracellular products of specialized cells, the sclerocytes. Little is known about the molecular mechanisms of their synthesis, but inhibition studies suggest an essential role of CAs. In order to gain insight into the evolution and function of CAs in biomineralization of a basal metazoan species, we determined the diversity and expression of CAs in the calcareous sponges Sycon ciliatum and Leucosolenia complicata by means of genomic screening, RNA-Seq and RNA in situ hybridization expression analysis. Active biomineralization was located with calcein-staining. Results: We found that the CA repertoires of two calcareous sponge species are strikingly more complex than those of other sponges. By characterizing their expression patterns, we could link two CAs (one intracellular and one extracellular) to the process of calcite spicule formation in both studied species. The extracellular biomineralizing CAs seem to be of paralogous origin, a finding that advises caution against assuming functional conservation of biomineralizing genes based upon orthology assessment alone. Additionally, calcareous sponges possess acatalytic CAs related to human CAs X and XI, suggesting an ancient origin of these proteins. Phylogenetic analyses including CAs from genomes of all non-bilaterian phyla suggest multiple gene losses and duplications and presence of several CAs in the last common ancestor of metazoans. Conclusions: We identified two key biomineralization enzymes from the CA-family in calcareous sponges and propose their possible interaction in spicule formation. The complex evolutionary history of the CA family is driven by frequent gene diversification and losses. These evolutionary patterns likely facilitated the numerous events of independent recruitment of CAs into biomineralization within Metazoa

Diversifying selection and host adaptation in two endosymbiont genomes

Background: The endosymbiont Wolbachia pipientis infects a broad range of arthropod and filarial nematode hosts. These diverse associations form an attractive model for understanding host: symbiont coevolution. Wolbachia's ubiquity and ability to dramatically alter host reproductive biology also form the foundation of research strategies aimed at controlling insect pests and vector-borne disease. The Wolbachia strains that infect nematodes are phylogenetically distinct, strictly vertically transmitted, and required by their hosts for growth and reproduction. Insects in contrast form more fluid associations with Wolbachia. In these taxa, host populations are most often polymorphic for infection, horizontal transmission occurs between distantly related hosts, and direct fitness effects on hosts are mild. Despite extensive interest in the Wolbachia system for many years, relatively little is known about the molecular mechanisms that mediate its varied interactions with different hosts. We have compared the genomes of the Wolbachia that infect Drosophila melanogaster, wMel and the nematode Brugia malayi, wBm to that of an outgroup Anaplasma marginale to identify genes that have experienced diversifying selection in the Wolbachia lineages. The goal of the study was to identify likely molecular mechanisms of the symbiosis and to understand the nature of the diverse association across different hosts. Results: The prevalence of selection was far greater in wMel than wBm. Genes contributing to DNA metabolism, cofactor biosynthesis, and secretion were positively selected in both lineages. In wMel there was a greater emphasis on DNA repair, cell division, protein stability, and cell envelope synthesis. Conclusion: Secretion pathways and outer surface protein encoding genes are highly affected by selection in keeping with host: parasite theory. If evidence of selection on various cofactor molecules reflects possible provisioning, then both insect as well as nematode Wolbachia may be providing substances to hosts. Selection on cell envelope synthesis, DNA replication and repair machinery, heat shock, and two component switching suggest strategies insect Wolbachia may employ to cope with diverse host and intra-host environments

Spicule formation in calcareous sponges: Coordinated expression of biomineralization genes and spicule-type specific genes

The ability to form mineral structures under biological control is widespread among animals. In several species, specific proteins have been shown to be involved in biomineralization, but it is uncertain how they influence the shape of the growing biomineral and the resulting skeleton. Calcareous sponges are the only sponges that form calcitic spicules, which, based on the number of rays (actines) are distinguished in diactines, triactines and tetractines. Each actine is formed by only two cells, called sclerocytes. Little is known about biomineralization proteins in calcareous sponges, other than that specific carbonic anhydrases (CAs) have been identified, and that uncharacterized Asx-rich proteins have been isolated from calcitic spicules. By RNA-Seq and RNA in situ hybridization (ISH), we identified five additional biomineralization genes in Sycon ciliatum: two bicarbonate transporters (BCTs) and three Asx-rich extracellular matrix proteins (ARPs). We show that these biomineralization genes are expressed in a coordinated pattern during spicule formation. Furthermore, two of the ARPs are spicule-type specific for triactines and tetractines (ARP1 or SciTriactinin) or diactines (ARP2 or SciDiactinin). Our results suggest that spicule formation is controlled by defined temporal and spatial expression of spicule-type specific sets of biomineralization genes

The protective role of carbon monoxide (CO) produced by heme oxygenases and derived from the CO-releasing molecule CORM-2 in the pathogenesis of stress-induced gastric lesions : evidence for non-involvement of nitric oxide (NO)

Carbon monoxide (CO) produced by heme oxygenase (HO)-1 and HO-2 or released from the CO-donor, tricarbonyldichlororuthenium (II) dimer (CORM-2) causes vasodilation, with unknown efficacy against stress-induced gastric lesions. We studied whether pretreatment with CORM-2 (0.1–10 mg/kg oral gavage (i.g.)), RuCl3 (1 mg/kg i.g.), zinc protoporphyrin IX (ZnPP) (10 mg/kg intraperitoneally (i.p.)), hemin (1–10 mg/kg i.g.) and CORM-2 (1 mg/kg i.g.) combined with NG-nitro-l-arginine (l-NNA, 20 mg/kg i.p.), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 mg/kg i.p.), indomethacin (5 mg/kg i.p.), SC-560 (5 mg/kg i.g.), and celecoxib (10 mg/kg i.g.) affects gastric lesions following 3.5 h of water immersion and restraint stress (WRS). Gastric blood flow (GBF), the number of gastric lesions and gastric CO and nitric oxide (NO) contents, blood carboxyhemoglobin (COHb) level and the gastric expression of HO-1, HO-2, hypoxia inducible factor 1α (HIF-1α), tumor necrosis factor α (TNF-α), cyclooxygenase (COX)-2 and inducible NO synthase (iNOS) were determined. CORM-2 (1 mg/kg i.g.) and hemin (10 mg/kg i.g.) significantly decreased WRS lesions while increasing GBF, however, RuCl3 was ineffective. The impact of CORM-2 was reversed by ZnPP, ODQ, indomethacin, SC-560 and celecoxib, but not by l-NNA. CORM-2 decreased NO and increased HO-1 expression and CO and COHb content, downregulated HIF-1α, as well as WRS-elevated COX-2 and iNOS mRNAs. Gastroprotection by CORM-2 and HO depends upon CO’s hyperemic and anti-inflammatory properties, but is independent of NO

An expressed sequence tag (EST) library for Drosophila serrata, a model system for sexual selection and climatic adaptation studies

The native Australian fly Drosophila serrata belongs to the highly speciose montium subgroup of the melanogaster species group. It has recently emerged as an excellent model system with which to address a number of important questions, including the evolution of traits under sexual selection and traits involved in climatic adaptation along latitudinal gradients. Understanding the molecular genetic basis of such traits has been limited by a lack of genomic resources for this species. Here, we present the first expressed sequence tag (EST) collection for D. serrata that will enable the identification of genes underlying sexually-selected phenotypes and physiological responses to environmental change and may help resolve controversial phylogenetic relationships within the montium subgroup

The Role of Homology and Orthology in the Phylogenomic Analysis of Metazoan Gene Content

Resolving the relationships of animals (Metazoa) is crucial to our understanding of the origin of key traits such as muscles, guts, and nerves. However, a broadly accepted metazoan consensus phylogeny has yet to emerge. In part, this is because the genomes of deeply diverging and fast-evolving lineages may undergo significant gene turnover, reducing the number of orthologs shared with related phyla. This can limit the usefulness of traditional phylogenetic methods that rely on alignments of orthologous sequences. Phylogenetic analysis of gene content has the potential to circumvent this orthology requirement, with binary presence/absence of homologous gene families representing a source of phylogenetically informative characters. Applying binary substitution models to the gene content of 26 complete animal genomes, we demonstrate that patterns of gene conservation differ markedly depending on whether gene families are defined by orthology or homology, that is, whether paralogs are excluded or included. We conclude that the placement of some deeply diverging lineages may exceed the limit of resolution afforded by the current methods based on comparisons of orthologous protein sequences, and novel approaches are required to fully capture the evolutionary signal from genes within genomes.W.P. was supported by the French National Research Agency (ANR) grant Ancestrome (ANR-10-BINF-01-01), and by grants from the National Science Foundation (DEB1556615, DEB-1256993). D.P. was supported by a NERC BETR (NE/P013643/1) grant. G.W. was supported by LMU Munich’s Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative, and the German Research Foundation (DFG) grant Wo896/19-1, and from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 764840 (ITN IGNITE

Wound healing and regeneration in the reef building coral Acropora millepora

Branching scleractinian corals are niche-constructing organisms, providing continuously-growing, structural foundation for spectacularly biodiverse coral reef ecosystems. A large part of their success lies in the ability to quickly regenerate following mechanical damage. Even now, when the corals undergo great decline due to anthropogenic weather and storm extremes, it is surprising how little is known about molecular mechanisms governing regeneration in these iconic organisms. In this study, we used RNA-seq to identify genes involved in the regeneration of Acropora millepora, starting with the initial wound closure up to complete rebuilding of lost structures. Many of the differentially expressed genes we found in the wound healing steps are homologues of genes known to be involved in wound healing and regeneration of bilaterian and other cnidarian species, prominently including multiple components of FGF and Wnt signalling pathways. Comparison between genes involved in wound healing and continuous growth of the colony demonstrates both similarity and distinctiveness of the genetic programmes controlling these processes. A striking example is specific expression of c-Fos, a transcription factor with conserved role in early injury response, during the earliest stages of wound healing of A. millepora. By comparing results obtained in diverse experimental conditions including a closed-loop, recirculating aquarium and a flow-through system of marine station, we have demonstrated feasibility of using zooxanthellate scleractinian corals as experimental models in fundamental biology research, including studies of regeneration

Prevalence of electrocardiographic left ventricular hypertrophy among patients with coronary artery disease and diabetes mellitus

Introduction. Electrocardiography (ECG) is a widely used non-invasive diagnostic method for assessment of patients with cardiovascular diseases. Numerous different electrocardiographic criteria exist for detection of left ventricular hypertrophy (LVH). LVH is an important risk factor in patients with coronary artery disease (CAD) or diabetes mellitus and its presence is associated with increased cardiovascular morbidity and mortality. The aim of this study was to evaluate the prevalence of the most frequently used electrocardiographic left ventricular hypertrophy (ECG-LVH) criteria among patients with CAD and diabetes. Methods. A cross-sectional, multicenter study was conducted in outpatient clinics across Poland. Family physicians performed physical examinations and collected relevant information about: onset of CAD and diabetes, presence and onset of hypertension, dyslipidemia, heart failure, diabetic complications, history of acute coronary syndrome and pharmacotherapy. In order to detect LVH, we used seven ECG criteria: 1) the Sokolow-Lyon voltage, 2) the Gubner voltage, 3) the criterion of the R wave amplitude on the leads V5–V6 and 4) aVL, 5) the gender specific Cornell voltage and 6) product, and 7) the Romhilt-Estes point score. Centralized manual assessment of the obtained ECG tracings were performed. Results. We enrolled 1001 patients (48.5% women, 51.5% men, mean age 65 ± 11 years) into the study. At least one ECG-LVH criterion was met in 20.0% (n = 200) of the study participants. The ECG-LVH diagnosis was the most common when using the Romhilt-Estes point score (n = 138; 13.8%). The corresponding prevalence rates for the Cornell voltage, the Cornell product, the R wave amplitude on the lead aVL, the Sokolow-Lyon voltage, the Gubner voltage and the R wave amplitude on the leads V5-V6 criteria were 5.5% (n = 55), 5.2% (n = 52), 3.2% (n = 32), 2.2% (n = 22), 1.9% (n = 19) and 1.3% (n = 13) respectively. Subsequently, the prevalence of the three most frequently used in clinical practice electrocardiographic criteria for LVH (the Sokolow-Lyon voltage, the Cornell voltage and the Romhilt-Estes point score) was analyzed. At least one of them was fulfilled in 185 ECGs. All three criteria at the same time were met only in 5 ECGs (2.7% of 185). Two and only one out of three criteria were fulfilled in 20 (10.8%) and 160 (86.5%) ECGs respectively. Conclusions. The co-occurrence of all assessed ECG-LVH criteria, including the three most frequently applied in clinical practice, is very low in diabetic CAD patients. The Romhilt-Estes point score identifies the highest number of ECG-LVH cases in this setting. However, it seems reasonable to use routinely several ECG criteria for detection of LVH. Further studies are needed to compare diagnostic values of ECG-LVH criteria with imaging methods and to assess prognostic values of various ECG-LVH criteria

Early divergence, broad distribution, and high diversity of animal chitin synthases

Even though chitin is one of themost abundant biopolymers in nature, current knowledge on chitin formation is largely based only on data from fungi and insects. This study reveals unanticipated broad taxonomic distribution and extensive diversification of chitin synthases (CSs) in Metazoa, shedding newlight on the relevance of chitin in animals and suggesting unforeseen complexity of chitin synthesis in many groups. We uncovered robust orthologs to insect type CSs in several representatives of deuterostomes, which generally are not thought to possess chitin. This suggests a broader distribution and function of chitin in this branch of the animal kingdom. We characterize a new CS type present not only in basal metazoans such as sponges and cnidarians but also in several bilaterian representatives. Themost extensive diversification of CSs took place during emergence of lophotrochozoans, the third large group of protostomes next to arthropods and nematodes, resulting in coexistence of up to ten CS paralogs inmolluscs. Independent fusion to different kinds of myosinmotor domains in fungi and lophotrochozoans points toward high relevance of CS interaction with the cytoskeleton for fine-tuned chitin secretion. Given the fundamental role that chitin plays in themorphology of many animals, the here presented CS diversification revealsmany evolutionary complexities. Our findings strongly suggest a very broad andmultifarious occurrence of chitin and question an ancestral role as cuticular component. The molecular mechanisms underlying regulation of animal chitin synthesis are most likely far more complex and diverse than existing data from insects suggest