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Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 12-07-2019, Esta tesis tiene embargado el acceso al texto completo hasta el 12-01-2021, Toll-like receptors (TLRs) play a crucial role in the recognition of pathogen-derived components as a first line of defense against infections. TLR4 and TLR2 receptors play a key role due to their cell surface location and their ability to identify a diversified spectrum of pathogen components. The early signaling activation via TLR4 and TLR2 receptors was studied and the results presented in the first chapter of this work show that TLR2 ligands activated NF-κB and MAPKs earlier and exhibited a higher IL-10 /IL-12 ratio at later time points compared to TLR4 ligands. The results further show the involvement of the phosphatase MKP-1 in the control of the MAPK p38 activation and that MKP-1 contributes to pro-inflammatory cytokine’s upregulation. Furthermore, p38 is critical for IL-10 expression in response to TLR2 ligands, which triggers the macrophage change to a M2 and regulatory phenotype in contrast to the M1 phenotype induced by TLR4 activation. Therefore, the early TLR2-mediated p38 induction contributes for the high IL-10 production as a virulence strategy to suppress host Th1 response against certain types of pathogens. TLR2 activation induces type I interferon expression mediated by MyD88 and TRIF signaling pathways. The role of TRIF-IFN-β-signaling in TLR2-mediated inflammatory responses was investigated and the results presented in the second chapter indicate that TLR2 ligands induce IFN-β expression, dependent of receptor endocytosis which consequently activate the interferon transcription factors IRF3 and IRF7. TRIF signaling was found to be required for IFN-β induction and consequent expression of the cytokine IL-12 in response to TLR2 ligands. Moreover, TLR2 is determinant for Listeria monocytogenes recognition, however, modifications of this bacteria cell wall avoid its recognition. The in vivo administration of TLR2 ligands in a murine model of neonatal listeriosis showed lower levels of bacterial load in neonate’s microglia, and Listeria-infected dendritic cells stimulated with TLR2 ligands presented higher levels of protective TNF-α and IL-12 cytokines, mediated by IFN-β. Therefore, TLR2 ligands exert a modulatory effect on cytokines with beneficial effects on the prevention of Listeria dissemination. This data points to TLR2 ligands as potential adjuvants in vaccine models for this bacterial infection. TLR4 is considered the major receptor to recognize all LPSs. However, some atypical LPS’s structures depart from the well-studied E. coli LPS and induce a TLR2-dependent inflammatory response in immune cells. The results in the third chapter demonstrate that the atypical LPS from Ochrobactrum intermedium is a TLR4/TLR2 agonist, inducing a weaker inflammatory response compared to E. coli LPS. Molecular docking analysis of O. intermedium LPS predicts a favorable formation of a TLR2/TLR4/MD-2 heterodimer, further confirmed by FRET. These imply that atypical LPSs may induce TLR4/TLR2 heterodimerization to decrease the bacteria activation of the innate immune system, Los receptores “Toll-like” (TLRs) tienen un papel clave en el reconocimiento de compuestos derivados de patógenos siendo una primera barrera contra las infecciones. TLR4 y TLR2 son importantes debido a su localización en la superficie celular y por su capacidad de reconocer una variad de moléculas derivadas de patógenos. Se estudió la señalización temprana por la activación de TLR4 y TLR2 fue y los resultados presentados en el capítulo 1 de este trabajo demuestran que ligandos de TLR2 activan NF-κB y MAPKs más tempranamente y exhiben un ratio IL-10/IL-12 más alto que los ligandos TLR4. Además, la fosfatasa MKP-1 controla la fosforilación de p38 y contribuye al incremento de citoquinas proinflamatorias. Nuestros resultados indican que p38 es importante para la expresión de IL-10 en respuesta a ligandos de TLR2, lo que induce un cambio del fenotipo de los macrófagos a tipo M2 y regulatorio, en lugar del tipo M1 que es inducido por la activación de TLR4. Por lo tanto, la activación temprana de p38 mediada por TLR2 contribuye para la alta producción de IL-10 como una estrategia de virulencia para suprimir la respuesta Th1 del hospedero contra determinados patógenos. La activación de TLR2 induce la expresión de interferón tipo I mediado por la señalización dependiente de MyD88 y TRIF. El papel de la señalización TRIF-IFN-β en la respuesta inflamatoria inducida por TLR2 fue estudiado y los resultados descritos en el capítulo 2 indican que la activación de TLR2 lleva a una producción de IFN-β, dependiente de la internalización del receptor, lo que activa los factores de trascripción de interferón IRF3 y IRF7. La señalización por TRIF es necesaria para la inducir IFN-β y la citoquina IL-12 por la activación de TLR2. Además, TLR2 es importante para el reconocimiento de Listeria monocytogenes, sin embargo, modificaciones en la pared de esta bacteria evitan su reconocimiento inmunológico. La administración in vivo de ligandos TLR2 en un modelo murino de listeriosis neonatal disminuye la carga bacteriana en la microglía de los neonatos y su tratamiento en células dendríticas infectadas incrementa los niveles de TNF-α y IL-12, mediado por IFN-β. Por lo tanto, los ligandos TLR2 tienen un efecto modulador en los niveles de citoquinas y previenen la diseminación de la bacteria. Esto apoya el uso de ligandos TLR2 como potenciales adyuvantes en modelos de vacunas para esta bacteria. TLR4 es conocido como el receptor de reconocimiento a los lipopolisacáridos (LPS). Sin embargo, algunos LPSs presentan una estructura distinta del bien estudiado LPS de E. coli y inducen respuestas inflamatorias dependientes de TLR2. Los resultados del capítulo 3 demuestran que el LPS de Ochrobactrum intermedium es un agonista TLR4/TLR2 y induce una respuesta inflamatoria más débil que el LPS de E. coli. Estudios de modelado molecular predicen que el O. intermedium LPS favorece la formación del heterodímero TLR2/TLR4/MD-2, que se confirmó por FRET. Esto indica que estos LPSs atípicos inducen la dimerización de TLR4 y TLR2 para evitar la detección de la bacteria por el sistema inmunológico., The thesis was financed/supported by a predoctoral fellowship under the TOLLerant-ETN action, Marie Sklodowska-Curie grant agreement N° 642157 from the European Union Horizon 2020 program

El estrés oxidativo es una de las principales causas que afecta directamente al correcto desarrollo de los cultivos. La planta, que invierte su energía en defenderse de dicho estrés, presenta problemas de absorción y metabolismo de los nutrientes, reduciendo su potencial de crecimiento considerablemente. Numerosos estudios demuestran que una correcta bioestimulación de la planta es muy beneficiosa para prevenir e incluso revertir dichos estreses, logrando así desarrollar todo el potencial vegetativo de los cultivos. En este trabajo se estudió el efecto de un inductor antioxidante en dos especies, lechuga ‘Matinale’ y naranjo ‘Pineapple’, donde se analizó la actividad de diferentes enzimas relacionadas con la síntesis de ROS. Dicho inductor aumentó significativamente la actividad catalasa, peroxidasa y superóxido dismutasa en ambas especies. En los últimos cincuenta años se ha desarrollado un interés creciente por los problemas relacionados con el estrés oxidativo, los radicales libres, las especies reactivas del oxígeno (ROS por sus siglas en inglés) y los antioxidantes. Todo ello es debido a la importancia que poseen en la bioquímica, la biología, la medicina e incluso en la cosmética. Su aplicación en agronomía ofrece también grandes expectativas. El estrés oxidativo es todo estrés en el que se genera un desequilibrio entre la producción de Especies reactivas del oxígeno (ROS) y la capacidad de un sistema biológico de decodificar rápidamente los reactivos que le hagan frente (antioxidantes) o reparar el daño resultante. Estos desequilibrios ‘ROS vs. antioxidantes’ causan el daño oxidativo cuando los ROS reaccionan con el sustrato oxidable (proteínas, lípidos, hidratos de carbono e incluso las propias moléculas de ADN) (Blokhina y col., 2003; Møller y col., 2007; Imlay, 2008). Es un concepto poco empleado en agronomía pero que, en realidad, se produce con mucha frecuencia debido a sus causas tan comunes: los estreses abióticos. En el mundo vegetal, las principales causas de estrés oxidativo son diferentes agresiones externas o estreses abióticos, como pueden ser el estrés hídrico, tanto por exceso (inundación/encharcamiento) como por defecto (sequía), las temperaturas extremas, la salinidad, la descompensación de nutrientes, la toxicidad por metales pesados, la alta insolación, los ataques de patógenos o incluso causadas por herbívoros, etc.

En el desarrollo de una agricultura más moderna y sostenible la productividad de los cultivos es un factor de vital importancia. En ella, el uso de bioestimulantes que activen el metabolismo de la planta y que logren un cultivo más eficiente es una buena estrategia para aumentar la productividad y la calidad del fruto. Con este objetivo se ha desarrollado un nuevo bioestimulante, SUPRACTYL cuyo efecto sobre plantas de cítricos se ha evaluado en naranjos Salustianos de 3 años de edad, durante dos ciclos vegetativos completos. Durante el primer año se realizó el aporte del producto SUPRACTYL por vía foliar, con 1, 2 ó 3 aplicaciones por árbol y el segundo se realizó una única aplicación del producto. El aporte foliar de SUPRACTYL generó un mejor estado hídrico en la planta debido a una mejor regulación de los estomas y, por tanto, del intercambio gaseoso de la planta, lo que podría repercutir en la resistencia de las plantas al estrés abiótico. Además. La presencia de componentes bioactivos activa el desarrollo celular y provoca un mejor aprovechamiento de la fotosíntesis, posiblemente, a través de la estimulación de los flujos energéticos.

The distribution and demographic patterns of marine organisms in the north Atlantic were largely shaped by climatic changes during the Pleistocene, when recurrent glacial maxima forced them to move south or to survive in northern peri-glacial refugia. These patterns were also influenced by biological and ecological factors intrinsic to each species, namely their dispersion ability. The ballan wrasse (Labrus bergylta), the largest labrid fish along Europe's continental margins, is a target for fisheries and aquaculture industry. The phylogeographic pattern, population structure, potential glacial refugia and recolonization routes for this species were assessed across its full distribution range, using mitochondrial and nuclear markers. The existence of a marked population structure can reflect both recolonization from three distinct glacial refugia and current and past oceanographic circulation patterns. Although isolated in present times shared haplotypes between continental and Azores populations and historical exchange of migrants in both directions point to a common origin of L. bergylta. This situation is likely to be maintained and/or accentuated by current circulation patterns in the north Atlantic, and may lead to incipient speciation in the already distinct Azorean population. Future monitoring of this species is crucial to evaluate how this species is coping with current environmental changes.

The distribution and demographic patterns of marine organisms in the north Atlantic were largely shaped by climatic changes during the Pleistocene, when recurrent glacial maxima forced them to move south or to survive in northern peri-glacial refugia. These patterns were also influenced by biological and ecological factors intrinsic to each species, namely their dispersion ability. The ballan wrasse (Labrus bergylta), the largest labrid fish along Europe's continental margins, is a target for fisheries and aquaculture industry. The phylogeographic pattern, population structure, potential glacial refugia and recolonization routes for this species were assessed across its full distribution range, using mitochondrial and nuclear markers. The existence of a marked population structure can reflect both recolonization from three distinct glacial refugia and current and past oceanographic circulation patterns. Although isolated in present times shared haplotypes between continental and Azores populations and historical exchange of migrants in both directions point to a common origin of L. bergylta. This situation is likely to be maintained and/or accentuated by current circulation patterns in the north Atlantic, and may lead to incipient speciation in the already distinct Azorean population. Future monitoring of this species is crucial to evaluate how this species is coping with current environmental changes.

The distribution and demographic patterns of marine organisms in the north Atlantic were largely shaped by climatic changes during the Pleistocene, when recurrent glacial maxima forced them to move south or to survive in northern peri-glacial refugia. These patterns were also influenced by biological and ecological factors intrinsic to each species, namely their dispersion ability. The ballan wrasse (Labrus bergylta), the largest labrid fish along Europe's continental margins, is a target for fisheries and aquaculture industry. The phylogeographic pattern, population structure, potential glacial refugia and recolonization routes for this species were assessed across its full distribution range, using mitochondrial and nuclear markers. The existence of a marked population structure can reflect both recolonization from three distinct glacial refugia and current and past oceanographic circulation patterns. Although isolated in present times shared haplotypes between continental and Azores populations and historical exchange of migrants in both directions point to a common origin of L. bergylta. This situation is likely to be maintained and/or accentuated by current circulation patterns in the north Atlantic, and may lead to incipient speciation in the already distinct Azorean population. Future monitoring of this species is crucial to evaluate how this species is coping with current environmental changes.

Species with dispersal mediated by planktonic larvae are expected to be more likely to show temporal genetic variation, due to differences in larval mortality and dispersal ability. The shanny Lipophrys pholis is a typical benthic rocky intertidal fish and its dispersion is limited to its long larval stage. In contrast, the sand-smelt Atherina presbyter has a very short planktonic life, small size and weak swimming capabilities, which translates into reduced dispersion potential. A total of 226 specimens of L. pholis (collected in 2003, 2013 and 2014) and 281 of A. presbyter (collected in 2005, 2012, 2013 and 2014) were screened for genetic variation using the mitochondrial control region. Only 12 (out of 171) and 25 (out of 155) haplotypes found were shared between sampling periods for the shanny and the sand smelt, respectively. For both species, haplotype networks showed a deep genealogy with multiple levels of diversification and no temporal structure. Interestingly, some of the previously inferred missing haplotypes were sampled in more recent years. The genetic diversity indices showed little variation among sampling periods and were generally high. For L. pholis significant genetic differentiation was detected between 2013 and 2014, while no significant differences were detected between sampling periods in A. presbyter. The shanny showed lower effective population size per generation when compared to the sand-smelt (which yielded lack of evidence for genetic drift for the first and second period of the study). These results highlight the fact that temporal changes in the gene pool composition need to be considered when evaluating population structure, especially for species with long pelagic larval dispersion, more vulnerable to fluctuations in the recruitment.

Assessing genetic diversity levels and population structure is fundamental for conservation and management of highly threatened species. In this study we use mitochondrial and nuclear markers to address phylogeographic and demographic data on the critically endangered Anaecypris hispanica (Cyprinidae). The populations of this species are strongly differentiated (high and significant ФST and FST values, corroborated by the results from AMOVA and SAMOVA) and genetically diversified. We suggest that the restricted gene flow between populations may have been potentiated by ecological, hydrological and anthropogenic causes. Bayesian skyline plots revealed a signal for expansion for all populations (tMRCA between 68kya and 1.33Mya) and a genetic diversity latitudinal gradient was detected between the populations from the Upper (more diversified) and the Lower (less diversified) Guadiana river basin. Distribution models inferred for this species highlight the significant contribution of temperature seasonality and isothermality for the occurrence of A. hispanica in Guadiana environments and emphasize the importance of stable climatic conditions for the preservation of this species. Given the strong population structure found, high percentage of private haplotypes and virtual absence of inter-basin gene flow we suggest that each A. hispanica population should be considered as an independent Operational Conservation Unit.

Póster presentado en: XV European Congress of Ichthyology (Sep 2015 - Porto, Portugal), Assessing genetic diversity levels and population structure is fundamental for conservation and management of highly threatened species. In this study we use mitochondrial and nuclear markers to address phylogeographic and demographic data on the critically endangered Anaecypris hispanica (Cyprinidae). The populations of this species are strongly differentiated (high and significant ФST and FST values, corroborated by the results from AMOVA and SAMOVA) and genetically diversified. We suggest that the restricted gene flow between populations may have been potentiated by ecological, hydrological and anthropogenic causes. Bayesian skyline plots revealed a signal for expansion for all populations (tMRCA between 68kya and 1.33Mya) and a genetic diversity latitudinal gradient was detected between the populations from the Upper (more diversified) and the Lower (less diversified) Guadiana river basin. Distribution models inferred for this species highlight the significant contribution of temperature seasonality and isothermality for the occurrence of A. hispanica in Guadiana environments and emphasize the importance of stable climatic conditions for the preservation of this species. Given the strong population structure found, high percentage of private haplotypes and virtual absence of inter-basin gene flow we suggest that each A. hispanica population should be considered as an independent Operational Conservation Unit.

Higher education institutions, has an active role in the development of a sustainable future and for this reason, it is essential that they became environmentally sustainable institutions, applying methods such as the Ecological Footprint analysis. This study intent is to strengthen the potential of the ecological footprint as an indicator of the sustainability of students of Lisbon School of Health Technology, and identify the relationship between the ecological footprint and the different socio-demographic variables.

Dissertação de mestrado em Bioquímica apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra. Glycogen is a branched and soluble polymer of glucose, present in cells functioning as energy storage. Its accumulation and utilization, synthesis and degradation are controlled primarily by covalent phosphorylation and allosteric ligand binding of the enzymes involved in glycogen metabolism. It is suggested that such enzymes are controlled by two enzymes, laforin and malin, which form a complex that is able to ubiquitinate those targets and target them for degradation, as a way of controlling glycogen synthesis. Mutations in the genes coding for these two proteins are the cause of Lafora disease, a type of myoclonic progressive epilepsy. Laforin is a unique human dual-specificity phosphatase (DSP), as it contains a carbohydrate binding module (CBM) at its N-terminal along with the Dual-Specificity Phosphatase Domain (DSPD) at its C-terminal, whereas malin is an E3 ubiquitin ligase of RING type. Malin was shown to interact with laforin within the region between the CBM and DSPD domains. Based on this, the aim of this work is the optimization of expression and purification of protein to obtain sufficient amounts of protein for further identification of a minimal laforin amino acid sequence required for the interaction with malin. To achieve this, several constructs coding for either the CBM or DSPD domains with increasing length of the inter-domain region were obtained by molecular biology techniques. The protein expression was performed using E. coli as host system, and the results have shown that, apart from the shorter CBM constructs, most protein is expressed in the form of insoluble inclusion bodies. The attempts to refold such proteins using the protocol previously developed for the refolding of laforin and its CBM proved unsuccessful. Laforin CBM domain was successfully expressed in soluble form and has been purified with a yield of 1,73 mg/L of expression media. Malin expression revealed that the protein is very poorly expressed in E. coli, being only detected by western-blot. Most of the protein was expressed as inclusion bodies, and despite some attempts to obtain soluble protein expression, no protein could be purified. This work has evidenced the difficulties of expressing protein domains and the importance of choosing the correct boundaries of such domains. Nevertheless, this work paves the way for the further optimization necessary for the expression and purification of both malin and the laforin constructs developed during this work. O glicogénio consiste num polímero de glucose, solúvel e ramificado que se encontra presente no citoplasma celular e funciona como reserva energética. A acumulação e utilização, bem como a síntese e degradação são processos controlados pela regulação da atividade das enzimas envolvidas no metabolismo do glicogénio, por reações de fosforilação e interações alostéricas. Duas enzimas, a laforina e a malina, estão envolvidas nesta regulação enzimática. Formam um complexo que promove a ubiquitinação dos respectivos substratos e o seu direcionamento para degradação permitindo uma regulação da síntese do glicogénio. Mutações nos genes que codificam ambas as proteínas levam ao aparecimento da doença da Lafora que consiste num tipo de epilepsia mioclónica progressiva. A laforina é a única fosfatase de dupla especificidade (DSP) humana devido à presença de um respectivo domínio DSP no C-terminal apresentando também um módulo de ligação a carbohidratos (CBM) no N-terminal. Por sua vez, a malina é uma ligase de ubiquitina E3 do tipo RING finger e interage com a laforina na região entre os domínios CBM e DSPD. Com base nesta informação, o objectivo do projecto consistiu na optimização da expressão e purificação das proteínas de modo a obter quantidades suficientes para uma posterior identificação da região mínima da laforina necessária para interagir com a malina. Para isto, várias construções contendo quer o domínio CBM ou o domínio DSP com um aumento da sequência da região entre os domínios foram obtidas por técnicas de biologia molecular. A expressão de proteína foi feita utilizando a E. coli como sistema de expressão e de acordo com os resultados obtidos, à parte das construções mais curtas contendo o domínio CBM, a maior parte da proteína das restantes construções é expressa na forma de corpos de inclusão. Tentativas de refolding destas proteínas com base num protocolo previamente desenvolvido para expressão de laforina e do seu domínio CBM falharam. No entanto, o domínio CBM da laforina foi expresso na forma solúvel e purificado com um rendimento de 1,73 mg/L de meio de expressão. Relativamente à malina, os rendimentos de expressão em E. Coli são muito baixos no qual a proteína só é detectada em western-blot. A maior parte da proteína é expressa como corpos de inclusão e apesar de algumas tentativas no sentido de obter proteína solúvel, ainda assim não foi possível purificar a malina. Este trabalho mostra a dificuldade na expressão de domínios proteicos e na importância de uma escolha correcta dos limites destes domínios. No entanto, este trabalho abre caminho para futuras optimizações necessárias para a expressão e purificação da malina assim como das construções derivadas da laforina desenvolvidas ao longo deste trabalho.

The phylogenetic relationships among theNorth Atlantic Gaidropsarus and between the three Gaidropsarinae genera Gaidropsarus, Ciliata and Enchelyopus are reviewed with the hitherto most comprehensive taxonomic sampling of this group. Phylogenetic results (maximum parsimony, maximum likelihood and Bayesian inference) based on nuclear (rhodopsin) and concatenated mitochondrial (12s, 16s and cytb)markers clearly support this subfamily. For the north-eastern Atlantic species of Gaidropsarus, two previously unreported clades were strongly supported, clarifying the relationships within the genus, and revealing fewer distinct taxa in the north Atlantic Gaidropsarus than previously stipulated. The data challenge the specific status of Gaidropsarus mediterraneus and Gaidropsarus guttatus and raise doubts concerning the distinctiveness of other species. A taxonomic revision of the genus is suggested. This study was funded by the Eco-Ethology Research Unit Strategic Plan (PEst-OE/MAR/UI0331/2011) – Fundação para a Ciência e a Tecnologia (FCT; partially FEDER funded). S.M.F.was supported by an FCT grant (SFRH/BPD/84923/2012). IMAR-Univ Azores was funded through the pluriannual and programmatic funding scheme as research unit #531 and associate laboratory LARSyS #9, with financial support from OE and COMPETE, through FCT and DRCTC/GovAzores. S.S. was a researcher contracted by MAR/DOP under the Ciência 2007 recruitment programme funded by FCT and is now a research fellow supported by the Marie Curie grant cofunded by the EU under the FP7-People-2012-COFUND; Co-funding of Regional, National and International Programmes, GA n.600407 and the Bandiera Project RITMARE.

In this paper, we compare the populations of the Lusitanian toadfish Halobatrachus didactylus along the Portuguese shore, using a putative fragment of the mitochondrial control region and the first intron of the S7 ribosomal protein gene. This demersal species ranges from the tropical West African coast to the Iberian Peninsula and the Mediterranean, having its northern limit in the Tagus River. For the putative control region, a single haplotype occurs in all fish from Tagus and Sado (the location immediately to the south) with a clear increase in diversity to the South, peaking at Algarve (south Portugal). The data seem to point to a very recent Holocenic colonization of Tagus and Sado from the South (possibly Algarve). We suggest that even small oscillations in sea surface temperature (SST) may cause local extinctions and subsequent recolonizations in populations of thermophilic fish that are at the cold limit of their distributions.

The study of the phylogeography of inshore fish from West Europe is revealing diverse geographical and demographical patterns. Some species conform to the phylogeographic patterns typical of terrestrial organisms, with marked signatures of the last glaciation and a decline of genetic diversity to the north of the species range. Other species, however, reveal no decline in diversity with latitude and signatures of expansions older than the last glaciation. The shanny Lipophrys pholis is a common intertidal resident fish in west European rocky shores. It is unable to leave the rocky stretch where it settled as a juvenile, so that dispersal depends entirely on the planktonic larval stage. These life-history and behavioural traits make the shanny an interesting species for phylogeographical analysis, as long-range movements by adults, which could blur historical signals, are absent. In this paper the phylogeography of L. pholis was studied using a fragment of the mitochondrial control region and one from the first intron of the S7 ribosomal protein gene. The European samples (ranging from SW Spain to the Netherlands) did not display population differentiation, isolation-by-distance or latitudinal declines in genetic diversity. Iberia was proposed as having operated as the main glacial refugium for the shanny. The genealogy of the European population showed that the largest expansion detected was older than the last glaciation, with lineages persisting from the early Pleistocene, which does not conform to colonisation by a few founders in the current interglacial. It is argued that if fishes have very large population sizes and high dispersal rates, populations can efficiently track climatic shifts so that little or no genetic structure remains after each range expansion and latitudinal gradients of genetic diversity tend to be weak or non-existent.

Artificial benthic collectors have been widely used for the assessment of settlement rates of decapod crustaceans. However, to date no consistent works have addressed spatial patterns of settlement in different estuarine habitats, and no specific studies targeted the interaction of artificial surfaces with the surrounding natural substrate. It may be expected that the artificial surface may produce a different thigmotactic response when compared to the natural substrate, which may limit the use of this technique for assessment of natural settlement rates. In this study the settlement rates of megalopae of the estuarine crab Carcinus maenas were addressed, specifically deploying artificial benthic collectors in different habitats both intertidal and subtidal in the lower Mira estuary. A number of experiments were performed concerning stratification and temporal fluctuations of settlement. Further, the interaction of collector surface with the surrounding substrate was investigated, by comparing settlement rates in natural and artificial substrates in different habitats. Results have shown significant differences in settlement between different estuarine habitats, both in spatially replicated experiments and in a high-resolution temporal experiment. However, comparison between settlement rates in artificial and natural substrates has shown that there is a strong interference between collectors and surrounding substrate, limiting interpretation of results concerning settlement rates in artificial substrate alone.