First report of Ciguatoxins in two starfish species: Ophidiaster ophidianus and Marthasterias glacialis

Ciguatera fish poisoning (CFP) is a syndrome caused by the ingestion of fish contaminated with Ciguatoxins (CTXs). These phycotoxins are produced mainly by dinoflagellates that belong to the genus Gambierdiscus that are transformed in more toxic forms in predatory fish guts, and are more present in the Indo-Pacific and Caribbean areas. It is estimated that CFP causes per year more than 10,000 intoxications worldwide. With the rise of water temperature and anthropogenic intervention, it is important to study the prevalence of CFP in more temperate waters. Through inter- and subtidal sampling, 22 species of organisms were collected, in Madeira and Azores archipelagos and in the northwestern Moroccan coast, during September of 2012 and June and July of 2013. A total of 94 samples of 22 different species of bivalves, gastropods, echinoderms and crustaceans where analyzed by Ultra Performance Liquid Chromatography-Mass Spectometry-Ion Trap-Time of Flight (UPLC-MS-IT-TOF) and Ultra Performance Chromatography- Mass Spectrometry (UPLC-MS). Our main aim was to detect new vectors and ascertain if there were some geographical differences. We detected for the first time putative CTXs in echinoderms, in two starfish species—M. glacialis and O. ophidianus. We detected differences regarding uptake values by organisms and geographical location. Toxin amounts were significant, showing the importance and the need for continuity of these studies to gain more knowledge about the prevalence of these toxins, in order to better access human health risk. In addition, we suggest monitoring of these toxins should be extended to other vectors, starfish being a good alternative for protecting and accessing human health risk.

New invertebrate vectors of Okadaic acid from the North Atlantic Waters - Portugal (Azores and Madeira) and Morocco

Okadaic acid and its analogues are potent phosphatase inhibitors that cause Diarrheic Shellfish Poisoning (DSP) through the ingestion of contaminated shellfish by humans. This group of toxins is transmitted worldwide but the number of poisoning incidents has declined over the last 20 years due to legislation and monitoring programs that were implemented for bivalves. In the summer of 2012 and 2013, we collected a total of 101 samples of 22 different species that were made up of benthic and subtidal organisms such echinoderms, crustaceans, bivalves and gastropods from Madeira, São Miguel Island (Azores archipelago) and the northwestern coast of Morocco. The samples were analyzed by UPLC-MS/MS. Our main objective was to detect new vectors for these biotoxins. We can report nine new vectors for these toxins in the North Atlantic: Astropecten aranciacus, Arbacia lixula, Echinaster sepositus, Holothuria sanctori, Ophidiaster ophidianus, Onchidella celtica, Aplysia depilans, Patella spp., and Stramonita haemostoma. Differences in toxin contents among the species were found. Even though low concentrations were detected, the levels of toxins that were present, especially in edible species, indicate the importance of these types of studies. Routine monitoring should be extended to comprise a wider number of vectors other than for bivalves of okadaic acid and its analogues.

Benthic nematodes biodiversity patterns along the gradient of the Tagus estuary (Western Coast of Portugal): Link with abundances distribution pattern of the nonindigenous bivalve Ruditapes philippinarum

The non-indigenous bivalve Ruditapes philippinarum is native to the western Pacific Ocean and it has been one of the most widely introduced species for aquaculture purposes in Europe. In Portugal its presence is known in several estuaries for more than two decades and its populations have increased greatly along the last years1. Currently it’s one of the dominant benthic species in some areas of the Tagus estuary (264,1±470,8 ind./30s tow). Studies on the impacts of invasive bivalves on meiofaunal communities are scarce and restricted to the harvesting effect. Meiofauna of the Tagus estuary is poorly known and possible impacts of the introduction of R. philippinarum were never assessed.

Contributo para o conhecimento paleontológico do Devónico do Anticlinal de Valongo (Portugal)

O estudo de uma jazida fossilifera descoberta recentemente no Devónico do Anticlinal de Valongo(Portugal) forneceu novos elementos paleofaunisticos, biostratigrilficos e paleoambientais sobre este contexto estratigráfico. Efetuou-se uma extensa amostragem paleontológica, acompanhada por estudos de estratigrafia em que se focaram aspetos detaxonomia,tafonomia,biostratigrafia e paleoecologia de invertebrados marinhos. Durante o trabalho de campo foram descobertos vários grupos taxonómicos de invertebrados tipicos deste Sistema, entre os quais se contam corais rugosos e tabulados, briozodrios, braquiópodes, bivalves, tentaculites, crinóides, trilobites e vários fósseis problemáticos. As litofacies, a presença de ripple marks com concentrações de tentaculites reorientadas e as carateristicas paleoecológicas da associação fóssil amostrada permitem inferir a presença de um ambiente litoral, de pouca profundidade, com sedimentação siliciclástica fina e correntes de deriva de intensidade moderada. A ocorr6ncia do bizarro género Tiaracrinus, um crinóide, sugere idade compreendida entre o Lochkoviano (Devónico Inferior) e o Eifeliano (Devónico Médio). Este género é reconhecido pela primeira vez no Maciço Ibérico.

Ocorrência e controle de lesmas do gênero Omalonyx (Gastropoda, Succineidae), pragas de capim-elefante Pennisetum purpureum (Poaceae) em Rio Preto da Eva, Amazonas.

Lesmas do gênero Omalonyx d'Orbigny, 1837 são hermafroditas, herbívoras, de distribuição neotropical e vivem em plantas aquáticas, nas demais vegetações adjacentes e em solo úmido próximo a ambientes de água doce. No presente trabalho reporta-se a ocorrência atípica de O. pattersonae Tillier, 1981 e de Omalonyx sp. em área de terra firme, distante de ambiente aquático.

Molecular mechanisms controlling physiological plasticity in marine mussels under the influence of natural and anthropogenic stress factors

Marine mussels are exceptionally well-adapted to live in transitional habitats where they are exposed to fluctuating environmental parameters and elevated levels of natural and anthropogenic stressors throughout their lifecycle. However, there is a dearth of information about the molecular mechanisms that assist in dealing with environmental changes. This project aims to investigate the molecular mechanisms governing acclimatory and stress responses of the Mediterranean mussel (Mytilus galloprovincialis) by addressing relevant life stages and environmental stressors of emerging concern. The experimental approach consisted of two phases to explore (i) the physiological processes at early life history and the consequences of plastic pollution and (ii) the adult physiology processes under natural habitats. As the first phase, I employed a plastic leachate (styrene monomer), and polystyrene microplastics to understand the modulation of cytoprotective mechanisms during the early embryo stages. Results revealed the onset of transcriptional impairments of genes involved in MXR-related transporters and other physiological processes induced by styrene and PS-MPs. In the second phase, as a preliminary analysis, microbiota profile of adult mussels at the tissue scale and its surrounding water was explored to understand microbiota structures that may reflect peculiar adaptations to the respective tissue functions. The broader experiment has been implemented to understand the variability of transcriptional profiles in the mussel digestive glands in the natural setting. All the genes employed in this study have shown possibilities to use as molecular biomarker responses throughout the year for monitoring the physiology of mussels living in a particular environment and, in turn, more properly detecting changes in the environment. As a whole, my studies provide insights into the interactions between environmental parameters, and intrinsic characters, and physiology of marine bivalves, and it could help to interpretation of responses correctly under stress conditions and climate change scenarios.

Mitochondrial Inheritance, Mito-nuclear Coevolution, and Sex-associated Genes in Bivalve Molluscs: Transcriptomics and Molecular Evolution

Bivalvia represents an ancient taxon including around 25,000 living species that have adapted to a wide range of environmental conditions, and show a great diversity in body size, shell shapes, and anatomic structure. Bivalves are characterized by highly variable genome sizes and extremely high levels of heterozygosity, which obstacle complete and accurate genome assemblies and hinder further genomic studies. Moreover, some bivalve species presented a stable evolutionary exception to the strictly maternal inheritance of mitochondria, namely doubly uniparental inheritance (DUI), making these species a precious model to study mitochondrial biology. During my PhD, I focused on a DUI species, the Manila clam Ruditapes philippinarum, and my work was two-folded. First, taking advantage of a newly assembled draft genome and a large RNA-seq dataset from different tissues of both sexes, I investigated 1) the role of gene expression and alternative splicing in tissue differentiation; 2) the relationship across tissue specificity, regulatory network connectivity, and sequence evolution; 3) sexual contrasting genetic markers potentially associated with sexual differentiation. The detailed information for this part is in Chapter 2. Second, using the same RNA-seq data, I investigated how nuclear oxidative phosphorylation (OXPHOS) genes coordinate with two divergent mitochondrial genomes in DUI species (mito-nuclear coordination and coevolution). To address this question, I compared transcription, polymorphism, and synonymous codon usage in the mitochondrial and nuclear OXPHOS genes of R. philippinarum in Chapter 3. To my knowledge, this thesis represents the first study exploring the role of alternative splicing in tissue differentiation, and the first study analyzing both transcriptional regulation and sequence evolution to investigate the coordination of OXPHOS genes in bivalves.