Browsing by Author "Fonseca, V. G."
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- Feeding and the rhodopsin family g-protein coupled receptors in nematodes and arthropodsPublication . Cardoso, João CR; Félix, Rute C.; Fonseca, V. G.; Power, DeborahIn vertebrates, receptors of the rhodopsin G-protein coupled superfamily (GPCRs) play an important role in the regulation of feeding and energy homeostasis and are activated by peptide hormones produced in the brain-gut axis.These peptides regulate appetite and energy expenditure by promoting or inhibiting food intake. Sequence and function homologs of human GPCRs involved in feeding exist in the nematode roundworm, Caenorhabditis elegans (C. elegans), and the arthropod fruit fly, Drosophila melanogaster (D. melanogaster), suggesting that the mechanisms that regulate food intake emerged early and have been conserved during metazoan radiation. Nematodes and arthropods are the most diverse and successful animal phyla on Earth. They can survive in a vast diversity of environments and have acquired distinct life styles and feeding strategies.The aim of the present review is to investigate if this diversity has affected the evolution of invertebrate GPCRs. Homologs of the C. elegans and D. melanogaster rhodopsin receptorswere characterized in the genome of other nematodes and arthropods and receptor evolution compared.With the exception of bombesin receptors (BBR) that are absent from nematodes, a similar gene complement was found. In arthropods, rhodopsin GPCR evolution is characterized by species-specific gene duplications and deletions and in nematodes by gene expansions in species with a free-living stage and gene deletions in representatives of obligate parasitic taxa. Based upon variation in GPCR gene number and potentially divergent functions within phyla we hypothesize that life style and feeding diversity practiced by nematodes and arthropods was one factor that contributed to rhodopsin GPCR gene evolution. Understanding how the regulation of food intake has evolved in invertebrates will contribute to the development of novel drugs to control nematodes and arthropods and the pests and diseases that use them as vectors.
- Identification of a new cartilage-specific S100-like protein up-regulated during endo/perichondral mineralization in gilthead seabreamPublication . Fonseca, V. G.; Rosa, Joana; Laizé, Vincent; Gavaia, Paulo J.; Cancela, LeonorCalcium ions and calcium-binding proteins play a major role in many cellular processes, in particular skeletogenesis and bone formation. We report here the discovery of a novel S100 protein in fish and the analysis of its gene expression patterns. A 648-bp full-length cDNA encoding an 86-amino acid S100-like calcium-binding protein was identified through the subtractive hybridization of a gilthead seabream (Sparus aurata) cDNA library constructed to identify genes associated with in vitro mineralization. Deduced protein lacks an identifiable signal peptide and exhibits two EF-hand motifs characteristic of S100 proteins. Phylogenetic and bioinformatic analyses of S100 sequences suggested that gilthead seabream protein represents a novel and fish-specific member of the S100 protein family. Expression of S100-like gene was up-regulated during the in vitro mineralization of bone-derived cell lines and during seabream development, from larvae throughout adulthood, reflecting skeletogenesis. Restriction of S100-like gene expression to chondrocytes of cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish further confirmed the mineralogenic role of the protein in fish and emphasized the potential of S100-like as a marker of mineralizing cartilage in developing fish.
- Identification of an osteopontin-like protein in fish associated with mineral formationPublication . Fonseca, V. G.; Laizé, Vincent; Valente, M. S.; Cancela, LeonorFish has been recently recognized as a suitable vertebrate model and represents a promising alternative to mammals for studying mechanisms of tissue mineralization and unravelling specific questions related to vertebrate bone formation. The recently developed Sparus aurata (gilthead seabream) osteoblast-like cell line VSa16 was used to construct a cDNA subtractive library aimed at the identification of genes associated with fish tissue mineralization. Suppression subtractive hybridization, combined with mirror orientation selection, identified 194 cDNA clones representing 20 different genes up-regulated during the mineralization of the VSa16 extracellular matrix. One of these genes accounted for 69% of the total number of clones obtained and was later identified as theS. aurata osteopontin-like gene. The 2138-bp full-length S. aurata osteopontin-like cDNA was shown to encode a 374 amino-acid protein containing domains and motifs characteristic of osteopontins, such as an integrin receptor-binding RGD motif, a negatively charged domain and numerous post-translational modifications (e.g. phosphorylations and glycosylations). The common origin of mammalian osteopontin and fish osteopontin-like proteins was indicated through an in silico analysis of available sequences showing similar gene and protein structures and was further demonstrated by their specific expression in mineralized tissues and cell cultures. Accordingly, and given its proven association with mineral formation and its characteristic protein domains, we propose that the fish osteopontin-like protein may play a role in hard tissue mineralization, in a manner similar to osteopontin in higher vertebrates.
- Isolation of marine meiofauna from sandy sediments From decanting to DNA extractionPublication . Fonseca, V. G.; Packer, Margaret; Carvalho, Gary; Power, Deborah; Lambshead, John; Creer, S.This protocol describes the separation of marine meiofauna from sediment and subsequent environmental DNA extraction. In this study meiofauna samples were taken with a 45 mm core from the upper 5 to 10 cm of sediment layer. Separation from sediment was achieved using a decantation process followed by isolation from fine silt using repetitive centrifugation steps with a 1.16 specific gravity (sg) LUDOX-TM solution. Meiofauna were deliberately separated from macrofauna by using a 1 mm sieve on top of a bottle-top sterile 45 !M sieve. High quality DNA was subsequently obtained using the QIAamp DNA Blood Maxi Kit (Qiagen) with minor adjustments to the manufacturer’s protocol. This procedure allowed efficient isolation of meiofaunal representatives from marine sediments and also extraction of high quality environmental DNA that can be used for downstream metagenetic analysis.
- Predicting the future of our oceans-evaluating genomic forecasting approaches in marine speciesPublication . Layton, K. K. S.; Brieuc, M. S. O.; Castilho, Rita; Diaz‐Arce, N.; Estévez‐Barcia, D.; Fonseca, V. G.; Fuentes‐Pardo, A. P.; Jeffery, N. W.; Jiménez-Mena, B.; Junge, C.; Leinonen, T.; Maes, S. M.; McGinnity, P.; Reed, T. E.; Reisser, C. M. O.; Silva, G.; Vasemägi, A.; Bradbury, I. R.; Kaufmann, J.Climate change is restructuring biodiversity on multiple scales and there is a pressing need to understand the downstream ecological and genomic consequences of this change. Recent advancements in the field of eco-evolutionary genomics have sought to include evolutionary processes in forecasting species' responses to climate change (e.g., genomic offset), but to date, much of this work has focused on terrestrial species. Coastal and offshore species, and the fisheries they support, may be even more vulnerable to climate change than their terrestrial counterparts, warranting a critical appraisal of these approaches in marine systems. First, we synthesize knowledge about the genomic basis of adaptation in marine species, and then we discuss the few examples where genomic forecasting has been applied in marine systems. Next, we identify the key challenges in validating genomic offset estimates in marine species, and we advocate for the inclusion of historical sampling data and hindcasting in the validation phase. Lastly, we describe a workflow to guide marine managers in incorporating these predictions into the decision-making process. Predicting climate change impacts is of central importance in marine ecosystems that provide a major source of nutrition to global communities and this work must be based on a sound understanding of both ecological and genomic impacts. This opinion synthesizes knowledge about the genomic basis of adaptation in marine species, highlights the few examples where genomic forecasting has been applied in marine systems, identifies the key challenges in validating genomic offset estimates in marine species, and provides a workflow to guide marine managers in incorporating these predictions into the decision-making process.image
- Rapid identification of differentially expressed genes by in situ screening of bacteriaPublication . Fonseca, V. G.; Lago-Lestón, Asunción; Laizé, Vincent; Cancela, LeonorThe identification of differentially expressed genes is a key step in the understanding of specific molecular mechanisms. Various methods have been developed to search for differences in expression but most of them are time or money consuming. We present here an alternative technique that connects standard suppression subtractive hybridization with in situ screening of bacteria to isolate and identify differentially expressed transcripts. The in situ differential screening is based on the transfer of bacteria directly from cultures onto nylon membranes with no need of phenol/chloroform extraction, colony lifting, or polymerase chain reaction amplification. This improved method was successfully applied and must be seen as a simple, low-cost, time-saving, and reproducible approach to identify differentially expressed genes.
- Revealing higher than expected meiofaunal diversity in Antarctic sediments: a metabarcoding approachPublication . Fonseca, V. G.; Sinniger, F.; Gaspar, J. M.; Quince, C.; Creer, S.; Power, Deborah M.; Peck, Lloyd S.; Clark, Melody S.An increasing number of studies are showing that Antarctic mega- and macrofauna are highly diverse, however, little is known about meiofaunal biodiversity in sediment communities, which are a vital part of a healthy and functional ecosystem. This is the first study to analyse community DNA (targeting meiofauna) using metabarcoding to investigate biodiversity levels in sediment communities of the Antarctic Peninsula. The results show that almost all of the meiofaunal biodiversity in the benthic habitat has yet to be characterised, levels of biodiversity were higher than expected and similar to temperate regions, albeit with the existence of potentially new and locally adapted species never described before at the molecular level. The Rothera meiofaunal sample sites showed four dominant eukaryotic groups, the nematodes, arthropods, platyhelminthes, and the annelids; some of which could comprise species complexes. Comparisons with deep-sea data from the same region suggest little exchange of Operational Taxonomic Units (OTUs) between depths with the nematodes prevalent at all depths, but sharing the shallow water benthos with the copepods. This study provides a preliminary analysis of benthic Antarctic Peninsula meiofauna using high throughput sequencing which substantiates how little is known on the biodiversity of one of the most diverse, yet underexplored communities of the Antarctic: the benthos.
- Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analysesPublication . Fonseca, V. G.; Nichols, B.; Lallias, D.; Quince, C.; Carvalho, Gary; Power, Deborah; Creer, S.Eukaryotic diversity in environmental samples is often assessed via PCR-based amplification of nSSU genes. However, estimates of diversity derived from pyrosequencing environmental data sets are often inflated, mainly because of the formation of chimeric sequences during PCR amplification. Chimeras are hybrid products composed of distinct parental sequences that can lead to the misinterpretation of diversity estimates. We have analyzed the effect of sample richness, evenness and phylogenetic diversity on the formation of chimeras using a nSSU data set derived from 454 Roche pyrosequencing of replicated, large control pools of closely and distantly related nematode mock communities, of known intragenomic identity and richness. To further investigate how chimeric molecules are formed, the nSSU gene secondary structure was analyzed in several individuals. For the first time in eukaryotes, chimera formation proved to be higher in both richer and more genetically diverse samples, thus providing a novel perspective of chimera formation in pyrosequenced environmental data sets. Findings contribute to a better understanding of the nature and mechanisms involved in chimera formation during PCR amplification of environmentally derived DNA. Moreover, given the similarities between biodiversity analyses using amplicon sequencing and those used to assess genomic variation, our findings have potential broad application for identifying genetic variation in homologous loci or multigene families in general.
- Second-generation environmental sequencing unmasks marine metazoan biodiversityPublication . Fonseca, V. G.; Carvalho, Gary; Sung, W.; Johnson, Harriet F.; Power, Deborah; Neill, Simon P.; Packer, Margaret; Blaxter, M. L.; Lambshead, P. J. D.; Thomas, W. K.; Creer, S.Biodiversity is of crucial importance for ecosystem functioning, sustainability and resilience, but the magnitude and organization of marine diversity at a range of spatial and taxonomic scales are undefined. In this paper, we use second-generation sequencing to unmask putatively diverse marine metazoan biodiversity in a Scottish temperate benthic ecosystem. We show that remarkable differences in diversity occurred at microgeographical scales and refute currently accepted ecological and taxonomic paradigms of meiofaunal identity, rank abundance and concomitant understanding of trophic dynamics. Richness estimates from the current benchmarked Operational Clustering of Taxonomic Units from Parallel UltraSequencing analyses are broadly aligned with those derived from morphological assessments. However, the slope of taxon rarefaction curves for many phyla remains incomplete, suggesting that the true alpha diversity is likely to exceed current perceptions. The approaches provide a rapid, objective and cost-effective taxonomic framework for exploring links between ecosystem structure and function of all hitherto intractable, but ecologically important, communities.
- Ultrasequencing of the meiofaunal biosphere practice, pitfalls and promisesPublication . Creer, S.; Fonseca, V. G.; Porazinska, D. L.; Giblin-Davis, R. M.; Sung, W.; Power, Deborah; Packer, Margaret; Carvalho, Gary; Blaxter, M. L.; Lambshead, P. J. D.; Thomas, W. K.Biodiversity assessment is the key to understanding the relationship between biodiversity and ecosystem functioning, but there is a well-acknowledged biodiversity identification gap related to eukaryotic meiofaunal organisms. Meiofaunal identification is confounded by the small size of taxa, morphological convergence and intraspecific variation. However, the most important restricting factor in meiofaunal ecological research is the mismatch between diversity and the number of taxonomists that are able to simultaneously identify and catalogue meiofaunal diversity. Accordingly, a molecular operational taxonomic unit (MOTU)-based approach has been advocated for en mass meiofaunal biodiversity assessment, but it has been restricted by the lack of throughput afforded by chain termination sequencing. Contemporary pyrosequencing offers a solution to this problem in the form of environmental metagenetic analyses, but this represents a novel field of biodiversity assessment. Here, we provide an overview of meiofaunal metagenetic analyses, ranging from sample preservation and DNA extraction to PCR, sequencing and the bioinformatic interrogation of multiple, independent samples using 454 Roche sequencing platforms. We report two examples of environmental metagenetic nuclear small subunit 18S (nSSU) analyses of marine and tropical rainforest habitats and provide critical appraisals of the level of putative recombinant DNA molecules (chimeras) in metagenetic data sets. Following stringent quality control measures, environmental metagenetic analyses achieve MOTU formation across the eukaryote domain of life at a fraction of the time and cost of traditional approaches. The effectiveness of Roche 454 sequencing brings substantial advantages to studies aiming to elucidate the molecular genetic richness of not only meiofaunal, but also all complex eukaryotic communities.