Ricardo B. Leite

Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring

BackgroundThe Manila clam, Ruditapes philippinarum, is one of the major aquaculture species in the world and a potential sentinel organism for monitoring the status of marine ecosystems. However, genomic resources for R. philippinarum are still extremely limited. Global analysis of gene expression profiles is increasingly used to evaluate the biological effects of various environmental stressors on aquatic animals under either artificial conditions or in the wild. Here, we report on the development of a transcriptomic platform for global gene expression profiling in the Manila clam.ResultsA normalized cDNA library representing a mixture of adult tissues was sequenced using a ultra high-throughput sequencing technology (Roche 454). A database consisting of 32,606 unique transcripts was constructed, 9,747 (30%) of which could be annotated by similarity. An oligo-DNA microarray platform was designed and applied to profile gene expression of digestive gland and gills. Functional annotation of differentially expressed genes between different tissues was performed by enrichment analysis. Expression of Natural Antisense Transcripts (NAT) analysis was also performed and bi-directional transcription appears a common phenomenon in the R. philippinarum transcriptome. A preliminary study on clam samples collected in a highly polluted area of the Venice Lagoon demonstrated the applicability of genomic tools to environmental monitoring.ConclusionsThe transcriptomic platform developed for the Manila clam confirmed the high level of reproducibility of current microarray technology. Next-generation sequencing provided a good representation of the clam transcriptome. Despite the known limitations in transcript annotation and sequence coverage for non model species, sufficient information was obtained to identify a large set of genes potentially involved in cellular response to environmental stress.

mRNA-Seq and microarray development for the Grooved carpet shell clam, Ruditapes decussatus: a functional approach to unravel host -parasite interaction

BackgroundThe Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis.ResultsA database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed.ConclusionsThis study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported.

Identification of a New pebp2αA2 Isoform From Zebrafish runx2 Capable of Inducing Osteocalcin Gene Expression In Vitro

The zebrafish runx2b transcription factor is an ortholog of RUNX2 and is highly conserved at the structural level. The runx2b pebp2αA2 isoform induces osteocalcin gene expression by binding to a specific region of the promoter and seems to have been selectively conserved in the teleost lineage.

Solea senegalensis vasa transcripts: molecular characterisation, tissue distribution and developmental expression profiles

The Vasa protein is an RNA helicase belonging the DEAD (Asp-Glu-Ala-Asp)-box family. The crucial role played by the vasa gene in the germ-cell lineage of both vertebrates and invertebrates has made this gene a useful molecular marker for germinal cells and a useful tool in surrogate broodstock production using primordial germ cell transplantation. With the aim of establishing a novel approach to improving Solea senegalensis broodstock management, the vasa gene in this species was characterised. Four S. senegalensis vasa transcripts were isolated: Ssvasa1, Ssvasa2, Ssvasa3 and Ssvasa4. Their phylogenetic relationship with other vasa homologues was determined confirming the high degree of conservation of this helicase throughout evolution. Our qPCR results showed that S. senegalensis vasa transcripts are prevalently expressed in gonads, with ovary-specific expression for Ssvasa3 and Ssvasa4. During embryonic and larval development, a switch between the longest and the shortest transcripts was observed. While Ssvasa1 and Ssvasa2 were maternally supplied, Ssvasa3 and Ssvasa4 depended on the de novo expression program of the growing juveniles, suggesting that vasa mRNA could be involved in Senegalese sole gonad differentiation. In situ hybridisation and immunohistochemical analysis performed in 150-days after hatching (DAH) larvae showed vasa product expression in the germinal region of early gonads. In our work we demonstrated the usefulness of Ssvasa mRNAs as molecular markers for primordial germ cells and germinal cells during embryonic development, larval ontogenesis and gonad differentiation. Furthermore, our results confirmed the potential of vasa to help investigate germinal cell biotechnology for Senegalese sole reproduction.

An oxygen molecular sensor, the HIF prolyl 4-hydroxylase, in the marine protist Perkinsus olseni.

Adaptations to changes in oxygen availability are crucial for survival and have been shown to be implicated in several disease states. Such adaptations are known to rely upon gene expression and regulation of hypoxia-inducible factors (HIF) and are mainly promoted by HIF Prolyl Hydroxylases (HPHs). These enzymes are involved in intracellular signaling and function as oxygen sensors. In the presence of molecular oxygen, HIF1alpha becomes hydroxylated at two specific prolyl residues and is targeted for destruction, whereas in the absence of oxygen (hypoxia conditions), HIF is not hydroxylated and thus remains free to activate HIF target genes. We have cloned the cDNAs corresponding to four different HPH transcripts from the bivalve parasite Perkinsus olseni, a unicellular eukaryotic organism, and determined the corresponding gene structure. Furthermore, we have analyzed the relationship between expression of this gene and parasite virulence. HPH-like genes were previously identified in other microorganisms, but only through comparative genomic analysis, and exclusively in pathogenic bacteria, thus suggesting that they may be involved in pathogen activity. To investigate a possible correlation between Perkinsus pathogenicity and HPH gene expression, pure cultures of this parasite were exposed to hemolymph from different bivalve species, either susceptible or resistant to Perkinsus, and HPH gene expression analyzed by real-time PCR. Our data show a massive increase in HPH expression in the presence of the susceptible hosts hemolymph, indicative of the importance of HPH in infection mechanisms. In addition, the dependence of HIF-like enzymes on other factors or substrates known to be associated with their biological function, such as iron, 2-oxoglutarate (2OG), glycolytic enzymes and oxygen, was also investigated through the use of iron chelators, 2OG antagonists, glycolytic inhibitors and hypoxia conditions. A clear correlation was observed between the production of reactive oxygen species and HPH activity, in agreement with studies that indicate a stabilization of HIF by inhibition of HPH. Interestingly, it was also possible to conclude, through phylogenetic analysis, that Perkinsus HPH is closer to the actual HPH2 from mammals, thus providing additional evidence supporting previous data pointing to HPH2 as the most ancestral of the three HPH isoforms known in metazoans.

Genomic Approaches in Aquaculture and Fisheries

Despite the enormous input into the worldwide development of fish and shellfish farming in the recent decades, in part as an attempt to minimize the impact of fishing on already overexploited natural populations, the application of genomics to aquaculture and fisheries remains poorly developed. Improving state-of-the-art genomics research in various aquaculture systems, as well as its industrial applications, remains one of the major challenges in this area and should be the focus of well developed strategies to be implemented in the next generation of projects. This chapter will first provide an overview of the genomic tools and resources available, then discuss the application of genomic approaches to the improvement of fish and shellfish farming (e.g. breeding, reproduction, growth, nutrition and product quality), including the evaluation of stock diversity and the use of selection procedures. The chapter will also discuss the use of genomic approaches to study and monitor natural fish and shellfish populations and to understand interactions within their ecosystems.

Peroxides with antiplasmodial activity inhibit proliferation of Perkinsus olseni, the causative agent of Perkinsosis in bivalves

Perkinsus olseni, the causative agent of Perkinsosis, can drastically affect the survival of target marine mollusks, with dramatic economic consequences for aquaculture. P. olseni is a member of the Alveolata group, which also comprises parasites that are highly relevant for medical and veterinary sciences such as Plasmodium falciparum and Toxoplasma. P. olseni shares several unique metabolic pathways with those pathological parasites but is not toxic to humans. In this work, six antimalarially active peroxides, derived from the natural product artemisinin or synthetic trioxolanes, were synthesized and tested on P. olseni proliferation and survival. All peroxides tested revealed an inhibitory effect on P. olseni proliferation at micromolar concentrations. The relevance of the peroxide functionality on toxicity and the effect of Fe(II)-intracellular concentration on activity were also evaluated. Results demonstrated that the peroxide functionality is the toxofore and intracellular iron concentration also proved to be a crucial co-factor on the activation of peroxides in P. olseni. These data points to a mechanism of bioactivation in P. olseni sharing similarities with the one proposed in P. falciparum parasites. Preliminary studies on bioaccumulation were conducted using fluorescent-labeled peroxides. Results show that synthetic trioxolanes tend to accumulate on a vacuole while the labeled artemisinin accumulates in the cytoplasm. Preliminary experiments on differential genes expression associated to Fe(II) transport protein (Nramp) and calcium transport protein (ATP6/SERCA) were also conducted by qPCR. Results point to a fourfold increase in expression of both genes upon exposure to trioxolanes and approximately twofold upon exposure to artemisinin derivatives. Data obtained in this investigation is relevant for better understanding of the biology of Perkinsus and may also be important in the development of new strategies for Perkinsosis prevention and control.

Four-and-a-half LIM domains protein 2 (FHL2) is associated with the development of craniofacial musculature in the teleost fish Sparus aurata.

Four-and-a-half LIM domains protein 2 (FHL2) is involved in major cellular mechanisms such as regulation of gene transcription and cytoskeleton modulation, participating in physiological control of cardiogenesis and osteogenesis. Knowledge on underlying mechanisms is, however, limited. We present here new data on FHL2 protein and its role during vertebrate development using a marine teleost fish, the gilthead seabream (Sparus aurata L.). In silico comparison of vertebrate protein sequences and prediction of LIM domain three-dimensional structure revealed a high degree of conservation, suggesting a conserved function throughout evolution. Determination of sites and levels of FHL2 gene expression in seabream indicated a central role for FHL2 in the development of heart and craniofacial musculature, and a potential role in tissue calcification. Our data confirmed the key role of FHL2 protein during vertebrate development and gave new insights into its particular involvement in craniofacial muscle development and specificity for slow fibers.

MAQ - A BIOINFORMATICS TOOL FOR AUTOMATIC MACROARRAY ANALYSIS

This article presents a prototype of an application for the analysis of cDNA/gene expression data and contribution for genome annotation from macroarrays. The application described here allows the quantification of a set of experiments with macroarrays of the same size. It generates html reports, easily shared, showing the expression of genes contained in each membrane and a global report comparing films (expositions) and probes. Although initially the objective of this application was macroarray analysis, with minor adjustments, it can be used for other densitometric analysis involving circular dish plates such as petri dish plates, or multiple cell plates. It aims to be a simple, but effective, tool that does not require the user to have previous knowledge in image processing.

Herbicides and Protozoan Parasite Growth Control: Implications for New Drug Development

Modern chromalveolates were originated through a series of events of endosymbiosis of microalgae by an eukaryote, leading to the retention of its plastid by this new host. These complex events, which took place multiple times during the course of evolution, originated new organisms that either lost or retained parts of the metabolisms present in their ancestral microalgae symbionts, achieved by transferring some of their genes into the nucleus of the host cell or even maintaining a relic organelle, a plastid circular DNA. The presence of this relic DNA was discovered in some apicomplexas, and thus named apicomplast. It was later found that a large number of parasites belonging to Alveolata also contained a relic nonphotosynthetic plastid, homologous to the chloroplast of plants and algae, thus expanding the apicomplast concept outside the apicomplexas. During the 90’s, some important metabolic pathways just known to occur in plants, algae, fungus and some bacteria were also identified in a series of important human parasites like Plasmodium falciparum and Toxoplasma gondii, the agents of malaria and toxoplasmosis respectively, opening a new era in drug prevention/control. Some of those protozoan parasites are responsible for millions of disease cases worldwide and hence a major concern for human health. The growing understanding of the biology and biochemistry of protozoan parasites, which has considerably increased over the past two decades, has paved the way to the discovery of many potential targets for new parasite drugs. The decrypted genomes of several species and the new post-genomic tools considerably improved our ability to identify and study the different metabolic pathways at the molecular level, and consequently contribute to validate potential drug targets. In 1998 the journal Nature published the discovery of a group of protozoan parasites that shared a metabolic pathway (shikimate pathway) essential for their survival with many plants, fungi and bacteria, but not found in mammals. Furthermore, researchers demonstrated that the herbicide glyphosate, known to interfere with this pathway in plants, could be used successfully to inhibit the in vitro growth of these parasites, opening the possibility of using available herbicides as a start point to develop new drugs to control parasite growth. Various studies have since then highlighted the importance of plastid or algae-like pathways for other parasite metabolisms such as fatty-acid FAS II, heme and isoprenoid