Chiara Manfrin

The C1q domain containing proteins of the Mediterranean mussel Mytilus galloprovincialis: A widespread and diverse family of immune-related molecules

The key component of the classical complement pathway C1q is regarded as a major connecting link between innate and acquired immunity due to the highly adaptive binding properties of its trimeric globular domain gC1q. The gC1q domain also characterizes many non-complement proteins involved in a broad range of biological processes including apoptosis, inflammation, cell adhesion and cell differentiation. In molluscs and many other invertebrates lacking of adaptive immunity, C1q domain containing (C1qDC) proteins are abundant, they most probably emerged as lectins and subsequently evolved in a specialized class of pattern recognition molecules through the expanding interaction properties of gC1q. Here we report the identification of 168 C1qDC transcript sequences of Mytilus galloprovincialis. The remarkable abundance of C1qDC transcripts in the Mediterranean mussel suggests an evolutionary strategy of gene duplication, functional diversification and selection of many specific C1qDC variants. A comprehensive transcript sequence survey in Protostomia also revealed that the C1qDC family expansion observed in mussel could have occurred in some specific taxa independently from the events leading to the establishment of a large complement of C1qDC genes in the Chordates lineage.

Big defensins and mytimacins, new AMP families of the Mediterranean mussel Mytilus galloprovincialis.

Antimicrobial peptides (AMPs) play a fundamental role in the innate immunity of invertebrates, preventing the invasion of potential pathogens. Mussels can express a surprising abundance of cysteine-rich AMPs pertaining to the defensin, myticin, mytilin and mytimycin families, particularly in the circulating hemocytes. Based on deep RNA sequencing of Mytilus galloprovincialis, we describe the identification, molecular diversity and constitutive expression in different tissues of five novel transcripts pertaining to the macin family (named mytimacins) and eight novel transcripts pertaining to the big defensins family (named MgBDs). The predicted antimicrobial peptides exhibit a N-terminal signal peptide, a positive net charge and a high content in cysteines, allegedly organized in intra-molecular disulfide bridges. Mytimacins and big defensins therefore represent two novel AMP families of M. galloprovincialis which extend the repertoire of cysteine-rich AMPs in this bivalve mollusk.

Bivalve omics: state of the art and potential applications for the biomonitoring of harmful marine compounds.

The extraordinary progress experienced by sequencing technologies and bioinformatics has made the development of omic studies virtually ubiquitous in all fields of life sciences nowadays. However, scientific attention has been quite unevenly distributed throughout the different branches of the tree of life, leaving molluscs, one of the most diverse animal groups, relatively unexplored and without representation within the narrow collection of well established model organisms. Within this Phylum, bivalve molluscs play a fundamental role in the functioning of the marine ecosystem, constitute very valuable commercial resources in aquaculture, and have been widely used as sentinel organisms in the biomonitoring of marine pollution. Yet, it has only been very recently that this complex group of organisms became a preferential subject for omic studies, posing new challenges for their integrative characterization. The present contribution aims to give a detailed insight into the state of the art of the omic studies and functional information analysis of bivalve molluscs, providing a timely perspective on the available data resources and on the current and prospective applications for the biomonitoring of harmful marine compounds.

DNA Damage and Transcriptional Changes in the Gills of Mytilus galloprovincialis Exposed to Nanomolar Doses of Combined Metal Salts (Cd, Cu, Hg)

Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism Mytilus galloprovincialis, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes). Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programs.

The eyestalk transcriptome of red swamp crayfish Procambarus clarkii

The red swamp crayfish (Procambarus clarkii, Girard 1852) is among the most economically important freshwater crustacean species, and it is also considered one of the most aggressive invasive species worldwide. Despite its commercial importance and being one of the most studied crayfish species, its genomic and transcriptomic layout has only been partially studied. Illumina RNA-sequencing was applied to characterize the eyestalk transcriptome and identify its most characterizing genes. A collection of 83,170,732 reads from eyestalks was obtained using Illumina paired-end sequencing technology. A de novo assembly was performed with the Trinity assembly software generating 119,255 contigs (average length of 1,007 bp) and identifying the first sequenced transcriptome in this species. The eyestalk is a major site for the production of neurohormones and controls a variety of physiological functions such as osmotic regulation, molting, epidermal color patterns and reproduction. Hence, its transcriptomic characterization is interesting and potentially instrumental to the elucidation of genes which have not been comprehensively described yet. Moreover, the availability of such a large amount of information supported the characterization of molecular families which have never been described before. The P. clarkii eyestalk transcriptome reported here provides a resource for improving the knowledge of the still incompletely defined neuroendocrinology of this species and represents an important source of data for all the interested carcinologists.

Mediterranean Mussel Gene Expression Profile Induced by Okadaic Acid Exposure

Seasonal seawater temperature increases define optimal growth conditions for Dinoflagellate species which can reach high concentrations in water column and also in filter-feeding organisms like Mytilus galloprovincialis. Commonly produced by Dinophysis and Prorocentrum spp., okadaic acid (OA) and its analogues are responsible for the Diarrheic Shellfish Poisoning (DSP) syndrome in humans. Closure of shellfishing grounds is therefore recommended by the EU when DSP toxin levels in shellfish exceed 16 μg OA 100 g(-1) flesh. Despite not being responsible for casualties either in humans or mussels, DSP outbreaks are considered natural events causing health and economic issues due to the frequency of their occurrence. Since gene expression studies offer a wide range of different solutions, we used a mussel cDNA microarray to evaluate gene expression changes in the digestive gland of mussels fed for five weeks with OA-contaminated nutrient. Among the differentially expressed genes we observed a general up-regulation of transcripts coding for stress proteins, proteins involved in cellular synthesis, and a few not annotated proteins. Overall, at the first time point analyzed we identified 58 candidate transcripts for OA-induced stress in mussels, half of which have unknown function. In this paper we present the first gene expression analysis performed on Mediterranean mussels exposed to okadaic acid. The characterization of these transcripts could be useful for the identification of an early physiological response to OA exposure.

Expression of cytoskeletal and molt-related genes is temporally scheduled in the hypodermis of the crayfish Procambarus clarkii during premolt

The rigid crustacean exoskeleton, the cuticle, is composed of the polysaccharide chitin, structural proteins and mineral deposits. It is periodically replaced to enable growth and its construction is an energy-demanding process. Ecdysis, the shedding event of the old cuticle, is preceded by a preparatory phase, termed premolt, in which the present cuticle is partially degraded and a new one is formed underneath it. Procambarus clarkii (Girard 1852), an astacid crustacean, was used here to comprehensively examine the changing patterns of gene expression in the hypodermis underlying the cuticle of the carapace at seven time points along ~14 premolt days. Next generation sequencing was used to construct a multi-tissue P. clarkii transcript sequence assembly for general use in a variety of transcriptomic studies. A reference transcriptome was created here in order to perform digital transcript expression analysis, determining the gene expression profiles in each of the examined premolt stages. The analysis revealed a cascade of sequential expression events of molt-related genes involved in chitin degradation, synthesis and modification, as well as synthesis of collagen and four groups of cuticular structural genes. The new description of major transcriptional events during premolt and the determination of their timing provide temporal markers for future studies of molt progress and regulation. The peaks of the expression of the molt-related genes were preceded by expression peaks of cytoskeletal genes that are hypothesized to be essential for premolt progress through regulating protein synthesis and/or transport, probably by remodeling the cytoskeletal structure.

Application of D-Crustacean Hyperglycemic Hormone Induces Peptidases Transcription and Suppresses Glycolysis-Related Transcripts in the Hepatopancreas of the Crayfish Pontastacus leptodactylus — Results of a Transcriptomic Study

The crustacean Hyperglycemic Hormone (cHH) is a neuropeptide present in many decapods. Two different chiral isomers are simultaneously present in Astacid crayfish and their specific biological functions are still poorly understood. The present study is aimed at better understanding the potentially different effect of each of the isomers on the hepatopancreatic gene expression profile in the crayfish Pontastacus leptodactylus, in the context of short term hyperglycemia. Hence, two different chemically synthesized cHH enantiomers, containing either L- or D-Phe3, were injected to the circulation of intermolt females following removal of their X organ-Sinus gland complex. The effects triggered by the injection of the two alternate isomers were detected after one hour through measurement of circulating glucose levels. Triggered changes of the transcriptome expression profile in the hepatopancreas were analyzed by RNA-seq. A whole transcriptome shotgun sequence assembly provided the assumedly complete transcriptome of P. leptodactylus hepatopancreas, followed by RNA-seq analysis of changes in the expression level of many genes caused by the application of each of the hormone isomers. Circulating glucose levels were much higher in response to the D-isoform than to the L-isoform injection, one hour from injection. Similarly, the RNA-seq analysis confirmed a stronger effect on gene expression following the administration of D-cHH, while just limited alterations were caused by the L-isomer. These findings demonstrated a more prominent short term effect of the D-cHH on the transcription profile and shed light on the effect of the D-isomer on specific functional gene groups. Another contribution of the study is the construction of a de novo assembly of the hepatopancreas transcriptome, consisting of 39,935 contigs, that dramatically increases the molecular information available for this species and for crustaceans in general, providing an efficient tool for studying gene expression patterns in this organ.

Crustacean oxi-reductases protein sequences derived from a functional genomic project potentially involved in ecdysteroid hormones metabolism – A starting point for function examination

A transcriptomic assembly originated from hypodermis and Y organ of the crustacean Pontastacus leptodactylus is used here for in silico characterization of oxi-reductase enzymes potentially involved in the metabolism of ecdysteroid molting hormones. RNA samples were extracted from male Y organ and its neighboring hypodermis in all stages of the molt cycle. An equimolar RNA mix from all stages was sequenced using next generation sequencing technologies and de novo assembled, resulting with 74,877 unique contigs. These transcript sequences were annotated by examining their resemblance to all GenBank translated transcripts, determining their Gene Ontology terms and their characterizing domains. Based on the present knowledge of arthropod ecdysteroid metabolism and more generally on steroid metabolism in other taxa, transcripts potentially related to ecdysteroid metabolism were identified and their longest possible conceptual protein sequences were constructed in two stages, correct reading frame was deduced from BLASTX resemblances, followed by elongation of the protein sequence by identifying the correct translation frame of the original transcript. The analyzed genes belonged to several oxi-reductase superfamilies including the Rieske non heme iron oxygenases, cytochrome P450s, short-chained hydroxysteroid oxi-reductases, aldo/keto oxireductases, lamin B receptor/sterol reductases and glucose-methanol-cholin oxi-reductatses. A total of 68 proteins were characterized and the most probable participants in the ecdysteroid metabolism where indicated. The study provides transcript and protein structural information, a starting point for further functional studies, using a variety of gene-specific methods to demonstrate or disprove the roles of these proteins in relation to ecdysteroid metabolism in P. leptodactylus.

Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis

We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling.