Ecological drivers of a vector borne pathogen: distribution and abundance of Borrelia burgdorferi sensu lato and its vector Ixodes ricinus in Scotland

Vector-borne disease emergence in recent decades has been associated with different environmental drivers including changes in habitat, hosts and climate. Lyme borreliosis is among the most important vector-borne diseases in the Northern hemisphere and is an emerging disease in Scotland. Transmitted by Ixodid tick vectors between large numbers of wild vertebrate host species, Lyme borreliosis is caused by bacteria from the Borrelia burgdorferi sensu lato species group. Ecological studies can inform how environmental factors such as host abundance and community composition, habitat and landscape heterogeneity contribute to spatial and temporal variation in risk from B. burgdorferi s.l. In this thesis a range of approaches were used to investigate the effects of vertebrate host communities and individual host species as drivers of B. burgdorferi s.l. dynamics and its tick vector Ixodes ricinus. Host species differ in reservoir competence for B. burgdorferi s.l. and as hosts for ticks. Deer are incompetent transmission hosts for B. burgdorferi s.l. but are significant hosts of all life-stages of I. ricinus. Rodents and birds are important transmission hosts of B. burgdorferi s.l. and common hosts of immature life-stages of I. ricinus. In this thesis, surveys of woodland sites revealed variable effects of deer density on B. burgdorferi prevalence, from no effect (Chapter 2) to a possible ‘dilution’ effect resulting in lower prevalence at higher deer densities (Chapter 3). An invasive species in Scotland, the grey squirrel (Sciurus carolinensis), was found to host diverse genotypes of B. burgdorferi s.l. and may act as a spill-over host for strains maintained by native host species (Chapter 4). Habitat fragmentation may alter the dynamics of B. burgdorferi s.l. via effects on the host community and host movements. In this thesis, there was lack of persistence of the rodent associated genospecies of B. burgdorferi s.l. within a naturally fragmented landscape (Chapter 3). Rodent host biology, particularly population cycles and dispersal ability are likely to affect pathogen persistence and recolonization in fragmented habitats. Heterogeneity in disease dynamics can occur spatially and temporally due to differences in the host community, habitat and climatic factors. Higher numbers of I. ricinus nymphs, and a higher probability of detecting a nymph infected with B. burgdorferi s.l., were found in areas with warmer climates estimated by growing degree days (Chapter 2). The ground vegetation type associated with the highest number of I. ricinus nymphs varied between studies in this thesis (Chapter 2 & 3) and does not appear to be a reliable predictor across large areas. B. burgdorferi s.l. prevalence and genospecies composition was highly variable for the same sites sampled in subsequent years (Chapter 2). This suggests that dynamic variables such as reservoir host densities and deer should be measured as well as more static habitat and climatic factors to understand the drivers of B. burgdorferi s.l. infection in ticks. Heterogeneity in parasite loads amongst hosts is a common finding which has implications for disease ecology and management. Using a 17-year data set for tick infestations in a wild bird community in Scotland, different effects of age and sex on tick burdens were found among four species of passerine bird (Chapter 5). There were also different rates of decline in tick burdens among bird species in response to a long term decrease in questing tick pressure over the study. Species specific patterns may be driven by differences in behaviour and immunity and highlight the importance of comparative approaches. Combining whole genome sequencing (WGS) and population genetics approaches offers a novel approach to identify ecological drivers of pathogen populations. An initial analysis of WGS from B. burgdorferi s.s. isolates sampled 16 years apart suggests that there is a signal of measurable evolution (Chapter 6). This suggests demographic analyses may be applied to understand ecological and evolutionary processes of these bacteria. This work shows how host communities, habitat and climatic factors can affect the local transmission dynamics of B. burgdorferi s.l. and the potential risk of infection to humans. Spatial and temporal heterogeneity in pathogen dynamics poses challenges for the prediction of risk. New tools such as WGS of the pathogen (Chapter 6) and blood meal analysis techniques will add power to future studies on the ecology and evolution of B. burgdorferi s.l.

Why is Asari (= Manila) clam Ruditapes philippinarum fitness poor in Arcachon Bay: a meta-analysis to answer?

Asari (= Manila) clam, Ruditapes philippinarum, is the second bivalve mollusc in terms of production in the world and, in many coastal areas, can beget important socio-economic issues. In Europe, this species was introduced after 1973. In Arcachon Bay, after a decade of aquaculture attempt, Asari clam rapidly constituted neo-naturalized population which is now fished. However, recent studies emphasized the decline of population and individual performances. In the framework of a national project (REPAMEP), some elements of fitness, stressors and responses in Arcachon bay were measured and compared to international data (41 publications, 9 countries). The condition index (CI=flesh weight/shell weight) was the lowest among all compared sites. Variation in average Chla concentration explained 30% of variation of CI among different areas. Among potential diseases, perkinsosis was particularly prevalent in Arcachon Bay, with high abundance, and Asari clams underwent Brown Muscle Disease, a pathology strictly restricted to this lagoon. Overall element contamination was relatively low, although arsenic, cobalt, nickel and chromium displayed higher values than in other ecosystems where Asari clam is exploited. Finally, total hemocyte count (THC) of Asari clam in Arcachon Bay, related to the immune system activity, exhibited values that were also under what is generally observed elsewhere. In conclusion, this study, with all reserves due to heterogeneity of available data, suggest that the particularly low fitness of Asari clam in Arcachon Bay is due to poor trophic condition, high prevalence and intensity of a disease (perkinsosis), moderate inorganic contamination, and poor efficiency of the immune system.

Long term repeated fire disturbance alters soil bacterial diversity but not the abundance in an Australian wet sclerophyll forest

Effects of fire on biogeochemical cycling in terrestrial ecosystem are widely acknowledged, while few studies have focused on the bacterial community under the disturbance of long-term frequent prescribed fire. In this study, three treatments (burning every two years (B2), burning every four years (B4) and no burning (B0)) were applied for 38 years in an Australian wet sclerophyll forest. Results showed that bacterial alpha diversity (i.e. bacterial OTU) in the top soil (0-10 cm) was significantly higher in the B2 treatment compared with the B0 and B4 treatments. Non-metric multidimensional analysis (NMDS) of bacterial community showed clear separation of the soil bacterial community structure among different fire frequency regimes and between the depths. Different frequency fire did not have a substantial effect on bacterial composition at phylum level or bacterial 16S rRNA gene abundance. Soil pH and C:N ratio were the major drivers for bacterial community structure in the most frequent fire treatment (B2), while other factors (EC, DOC, DON, MBC, NH 4 +, TC and TN) were significant in the less frequent burning and no burning treatments (B4 and B0). This study suggested that burning had a dramatic impact on bacterial diversity but not abundance with more frequent fire.

Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)

Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure.

Real-time PCR quantification and diversity analysis of the functional genes aprA and dsrA of sulfate-reducing prokaryotes in marine sediments of the Peru continental margin and the Black Sea

Sulfate-reducing prokaryotes (SRP) are ubiquitous and quantitatively important members in many ecosystems, especially in marine sediments. However their abundance and diversity in subsurface marine sediments is poorly understood. In this study, the abundance and diversity of the functional genes for the enzymes adenosine 5'-phosphosulfate reductase (aprA) and dissimilatory sulfite reductase (dsrA) of SRP in marine sediments of the Peru continental margin and the Black Sea were analyzed, including samples from the deep biosphere (ODP site 1227). For aprA quantification a Q-PCR assay was designed and evaluated. Depth profiles of the aprA and dsrA copy numbers were almost equal for all sites. Gene copy numbers decreased concomitantly with depth from around 10(8)/g sediment close to the sediment surface to less than 10(5)/g sediment at 5 mbsf. The 16S rRNA gene copy numbers of total bacteria were much higher than those of the functional genes at all sediment depths and used to calculate the proportion of SRP to the total Bacteria. The aprA and dsrA copy numbers comprised in average 0.5-1% of the 16S rRNA gene copy numbers of total bacteria in the sediments up to a depth of ca. 40 mbsf. In the zone without detectable sulfate in the pore water from about 40-121 mbsf (Peru margin ODP site 1227), only dsrA (but not aprA) was detected with copy numbers of less than 10(4)/g sediment, comprising ca. 14% of the 16S rRNA gene copy numbers of total bacteria. In this zone, sulfate might be provided for SRP by anaerobic sulfide oxidation. Clone libraries of aprA showed that all isolated sequences originate from SRP showing a close relationship to aprA of characterized species or form a new cluster with only distant relation to aprA of isolated SRP. For dsrA a high diversity was detected, even up to 121 m sediment depth in the deep biosphere.

Explaining dinophysis cf. acuminata abundance in Antifer (Normandy, France) using dynamic linear regression

Classical regression analysis can be used to model time series. However, the assumption that model parameters are constant over time is not necessarily adapted to the data. In phytoplankton ecology, the relevance of time-varying parameter values has been shown using a dynamic linear regression model (DLRM). DLRMs, belonging to the class of Bayesian dynamic models, assume the existence of a non-observable time series of model parameters, which are estimated on-line, i.e. after each observation. The aim of this paper was to show how DLRM results could be used to explain variation of a time series of phytoplankton abundance. We applied DLRM to daily concentrations of Dinophysis cf. acuminata, determined in Antifer harbour (French coast of the English Channel), along with physical and chemical covariates (e.g. wind velocity, nutrient concentrations). A single model was built using 1989 and 1990 data, and then applied separately to each year. Equivalent static regression models were investigated for the purpose of comparison. Results showed that most of the Dinophysis cf. acuminata concentration variability was explained by the configuration of the sampling site, the wind regime and tide residual flow. Moreover, the relationships of these factors with the concentration of the microalga varied with time, a fact that could not be detected with static regression. Application of dynamic models to phytoplankton time series, especially in a monitoring context, is discussed.

Development of Imaging Mass Spectrometry Analysis of Lipids in Biological and Clinically Relevant Applications

La spectrométrie de masse mesure la masse des ions selon leur rapport masse sur charge. Cette technique est employée dans plusieurs domaines et peut analyser des mélanges complexes. L’imagerie par spectrométrie de masse (Imaging Mass Spectrometry en anglais, IMS), une branche de la spectrométrie de masse, permet l’analyse des ions sur une surface, tout en conservant l’organisation spatiale des ions détectés. Jusqu’à présent, les échantillons les plus étudiés en IMS sont des sections tissulaires végétales ou animales. Parmi les molécules couramment analysées par l’IMS, les lipides ont suscité beaucoup d'intérêt. Les lipides sont impliqués dans les maladies et le fonctionnement normal des cellules; ils forment la membrane cellulaire et ont plusieurs rôles, comme celui de réguler des événements cellulaires. Considérant l’implication des lipides dans la biologie et la capacité du MALDI IMS à les analyser, nous avons développé des stratégies analytiques pour la manipulation des échantillons et l’analyse de larges ensembles de données lipidiques. La dégradation des lipides est très importante dans l’industrie alimentaire. De la même façon, les lipides des sections tissulaires risquent de se dégrader. Leurs produits de dégradation peuvent donc introduire des artefacts dans l’analyse IMS ainsi que la perte d’espèces lipidiques pouvant nuire à la précision des mesures d’abondance. Puisque les lipides oxydés sont aussi des médiateurs importants dans le développement de plusieurs maladies, leur réelle préservation devient donc critique. Dans les études multi-institutionnelles où les échantillons sont souvent transportés d’un emplacement à l’autre, des protocoles adaptés et validés, et des mesures de dégradation sont nécessaires. Nos principaux résultats sont les suivants : un accroissement en fonction du temps des phospholipides oxydés et des lysophospholipides dans des conditions ambiantes, une diminution de la présence des lipides ayant des acides gras insaturés et un effet inhibitoire sur ses phénomènes de la conservation des sections au froid sous N2. A température et atmosphère ambiantes, les phospholipides sont oxydés sur une échelle de temps typique d’une préparation IMS normale (~30 minutes). Les phospholipides sont aussi décomposés en lysophospholipides sur une échelle de temps de plusieurs jours. La validation d’une méthode de manipulation d’échantillon est d’autant plus importante lorsqu’il s’agit d’analyser un plus grand nombre d’échantillons. L’athérosclérose est une maladie cardiovasculaire induite par l’accumulation de matériel cellulaire sur la paroi artérielle. Puisque l’athérosclérose est un phénomène en trois dimension (3D), l'IMS 3D en série devient donc utile, d'une part, car elle a la capacité à localiser les molécules sur la longueur totale d’une plaque athéromateuse et, d'autre part, car elle peut identifier des mécanismes moléculaires du développement ou de la rupture des plaques. l'IMS 3D en série fait face à certains défis spécifiques, dont beaucoup se rapportent simplement à la reconstruction en 3D et à l’interprétation de la reconstruction moléculaire en temps réel. En tenant compte de ces objectifs et en utilisant l’IMS des lipides pour l’étude des plaques d’athérosclérose d’une carotide humaine et d’un modèle murin d’athérosclérose, nous avons élaboré des méthodes «open-source» pour la reconstruction des données de l’IMS en 3D. Notre méthodologie fournit un moyen d’obtenir des visualisations de haute qualité et démontre une stratégie pour l’interprétation rapide des données de l’IMS 3D par la segmentation multivariée. L’analyse d’aortes d’un modèle murin a été le point de départ pour le développement des méthodes car ce sont des échantillons mieux contrôlés. En corrélant les données acquises en mode d’ionisation positive et négative, l’IMS en 3D a permis de démontrer une accumulation des phospholipides dans les sinus aortiques. De plus, l’IMS par AgLDI a mis en évidence une localisation différentielle des acides gras libres, du cholestérol, des esters du cholestérol et des triglycérides. La segmentation multivariée des signaux lipidiques suite à l’analyse par IMS d’une carotide humaine démontre une histologie moléculaire corrélée avec le degré de sténose de l’artère. Ces recherches aident à mieux comprendre la complexité biologique de l’athérosclérose et peuvent possiblement prédire le développement de certains cas cliniques. La métastase au foie du cancer colorectal (Colorectal cancer liver metastasis en anglais, CRCLM) est la maladie métastatique du cancer colorectal primaire, un des cancers le plus fréquent au monde. L’évaluation et le pronostic des tumeurs CRCLM sont effectués avec l’histopathologie avec une marge d’erreur. Nous avons utilisé l’IMS des lipides pour identifier les compartiments histologiques du CRCLM et extraire leurs signatures lipidiques. En exploitant ces signatures moléculaires, nous avons pu déterminer un score histopathologique quantitatif et objectif et qui corrèle avec le pronostic. De plus, par la dissection des signatures lipidiques, nous avons identifié des espèces lipidiques individuelles qui sont discriminants des différentes histologies du CRCLM et qui peuvent potentiellement être utilisées comme des biomarqueurs pour la détermination de la réponse à la thérapie. Plus spécifiquement, nous avons trouvé une série de plasmalogènes et sphingolipides qui permettent de distinguer deux différents types de nécrose (infarct-like necrosis et usual necrosis en anglais, ILN et UN, respectivement). L’ILN est associé avec la réponse aux traitements chimiothérapiques, alors que l’UN est associé au fonctionnement normal de la tumeur.

GC-MS Analysis, Antimicrobial and Antioxidant Activities of Extracts of the Aerial Parts of Conyza sumatrensis

Phytochemical analyses as well as antimicrobial and antioxidant activities of the extracts of C. sumatrensis aerial parts were investigated in this study. METHODS: The aerial parts of C. sumatrensis were air dried, weighed and exhaustively extracted with hexane, ethyl acetate and methanol successively. The crude extracts were screened for metabolites. These extracts of the plant were evaluated for antimicrobial and antioxidant activities using agar diffusion and DPPH method respectively. The extracts were also analysed using Gas chromatography – Mass spectrometry, and the chromatogram coupled with mass spectra of the compounds were matched with a standard library. RESULTS: Preliminary phytochemical investigation of crude n-hexane, ethyl acetate and methanol extracts of the aerial parts of Conyza sumatrensis revealed the presence of anthraquinones, flavonoids, terpenoids, phenolics, tannin, glycosides and carbohydrate. All the crude extracts gave a clear zone of inhibition against the growth of the test bacteria ( Staphylococcus aureus , Escherichia coli , Bacillus subtilis , Pseudomona aeruginosa, Salmonella typhi , Klebsiellae pneumonae ) at moderate to high concentrations, as well as test fungi ( Candida albicans , Aspergillus niger , penicillium notatum and Rhizopus stolonifer ) at high concentration. Methanolic extract exhibited significant radical scavenging property with IC50 of 17.08 μg/mL while n-hexane and ethyl acetate extracts showed no significant antioxidant activity. GC-MS of N-hexane extract showed a total number of eleven chemical constituents with α-Farnesene and spathulenol being the most abundance compounds constituting 20.27 and 22.28% of the extract respectively. Ethyl acetate extract revealed thirteen compounds with two most abundant compounds, cis-β-farnesene (16.64 %) and cis-pinane (21.09 %). While methanolic extract affords seventeen compounds with Ephytol being the most abundant compound (19.36 %).

Aerial line transect survey to estimate abundance of marsh deer (Blastocerus dichotomus) (Illiger, 1815)

A população de cervo-do-pantanal (Blastocerus dichotomus) está drasticamente reduzida no Brasil. O nosso objetivo foi o de estimar a abundância do cervo-do-pantanal na bacia do Rio Paraná e discutir a metodologia aplicada. Os resultados darão suporte para uma análise do impacto do enchimento da represa de Porto Primavera sobre essa população. Sessenta e nove animais foram registrados através de sobrevôo utilizando-se a metodologia de transecção linear com amostragem das distâncias. Os dados não corrigidos resultaram em uma densidade estimada de 0,0035ind/ha e uma população de 636 indivíduos. A correção de g para os animais que não foram vistos apresentou uma densidade de 0,0049 ind/ha e uma abundância de 896 (CV=0,27) indivíduos. A metodologia foi aplicada com sucesso na estimativa de cervo-do-pantanal. Esse resultado é importante para avaliarmos a população do cervo-do-pantanal na área e para futuramente analisarmos o impacto do enchimento da represa.

Abundance of Lutzomyia longipalpis in urban households as risk factor of transmission of visceral leishmaniasis

Urban occurrence of human and canine visceral leishmaniasis (VL) is linked to households with characteristics conducive to the presence of sand flies. This study proposes an ad hoc classification of households according to the environmental characteristics of receptivity to phlebotominae and an entomological study to validate the proposal. Here we describe the phlebotominae population found in intra- and peridomiciliary environments and analyse the spatiotemporal distribution of the VL vector Lutzomyia longipalpis of households receptive to VL. In the region, 153 households were classified into levels of receptivity to VL followed by entomological surveys in 40 of those properties. Kruskal-Wallis verified the relationship between the households’ classification and sand fly abundance and Kernel analysis evaluated L. longipalpis spatial distribution: of the 740 sand flies were captured, 91% were L. longipalpis; 82% were found peridomiciliary whilst the remaining 18% were found intradomiciliary. No statistically significant association was found between sandflies and households levels. L. longipalpis counts were concentrated in areas of high vulnerability and some specific households were responsible for the persistence of the infestation. L. longipalpis prevails over other sand fly species for urban VL transmission. The entomological study may help target the surveillance and vector control strategies to domiciles initiating and/or maintaining VL outbreaks.

Adaptation of motor control to training and disuse : contribution of muscle synergy analysis and movement variability

The well-known degrees of freedom problem originally introduced by Nikolai Bernstein (1967) results from the high abundance of degrees of freedom in the musculoskeletal system. Such abundance in motor control have two sides: i) because it is unlikely that the Central Nervous System controls each degree of freedom independently, the complexity of the control needs to be reduced, and ii) because there are many options to perform a movement, a repetition of a given movement is never the same. It leads to two main topics in motor control and biomechanics: motor coordination and motor variability. The present thesis aimed to understand how motor systems behave and adapt under specific conditions. This thesis comprises three studies that focused on three topics of major interest in the field of sports sciences and medicine: expertise, injury risk and fatigue. The first study (expertise) has focused on the muscle coordination topic to further investigate the effect of expertise on the muscle synergistic organization, which ultimately may represent the underlying neural strategies. Studies 2 (excessive medial knee displacement) and 3 (fatigue) both aimed to better understand its impact on the dynamic local stability. The main findings of the present thesis suggest: 1) there is a great robustness in muscle synergistic organization between swimmers at different levels of expertise (study 1, chapter II), which ultimately indicate that differences in muscle coordination is mainly explained by peripheral adaptations; 2) injury risk factors such as excessive medial knee displacement (study 2, chapter III) and fatigue (study 3, chapter IV) alter the dynamic local stability of the neuromuscular system towards a more unstable state. This change in dynamic local stability represents a loss of adaptability in the neuromuscular system reducing the flexibility to adapt to a perturbation.

Occurrence and abundance models of threatened plant species: Applications to mitigate the impact of hydroelectric power dams

Species occurrence and abundance models are important tools that can be used in biodiversity conservation, and can be applied to predict or plan actions needed to mitigate the environmental impacts of hydropower dams. In this study our objectives were: (i) to model the occurrence and abundance of threatened plant species, (ii) to verify the relationship between predicted occurrence and true abundance, and (iii) to assess whether models based on abundance are more effective in predicting species occurrence than those based on presence–absence data. Individual representatives of nine species were counted within 388 randomly georeferenced plots (10 m × 50 m) around the Barra Grande hydropower dam reservoir in southern Brazil. We modelled their relationship with 15 environmental variables using both occurrence (Generalised Linear Models) and abundance data (Hurdle and Zero-Inflated models). Overall, occurrence models were more accurate than abundance models. For all species, observed abundance was significantly, although not strongly, correlated with the probability of occurrence. This correlation lost significance when zero-abundance (absence) sites were excluded from analysis, but only when this entailed a substantial drop in sample size. The same occurred when analysing relationships between abundance and probability of occurrence from previously published studies on a range of different species, suggesting that future studies could potentially use probability of occurrence as an approximate indicator of abundance when the latter is not possible to obtain. This possibility might, however, depend on life history traits of the species in question, with some traits favouring a relationship between occurrence and abundance. Reconstructing species abundance patterns from occurrence could be an important tool for conservation planning and the management of threatened species, allowing scientists to indicate the best areas for collection and reintroduction of plant germplasm or choose conservation areas most likely to maintain viable populations.

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

Variation in myogenic differentiation 1 mRNA abundance is associated with beef tenderness in Nelore cattle.

The myogenic differentiation 1 gene (MYOD1) has a key role in skeletal muscle differentiation and composition through its regulation of the expression of several muscle-specific genes. We first used a general linear mixed model approach to evaluate the association of MYOD1 expression levels on individual beef tenderness phenotypes. MYOD1 mRNA levels measured by quantitative polymerase chain reactions in 136 Nelore steers were significantly associated (P ? 0.01) with Warner?Bratzler shear force, measured on the longissimus dorsi muscle after 7 and 14 days of beef aging. Transcript abundance for the muscle regulatory gene MYOD1 was lower in animals with more tender beef. We also performed a coexpression network analysis using whole transcriptome sequence data generated from 30 samples of longissimus muscle tissue to identify genes that are potentially regulated by MYOD1. The effect of MYOD1 gene expression on beef tenderness may emerge from its function as an activator of muscle-specific gene transcription such as for the serum response factor (C-fos serum response element-binding transcription factor) gene (SRF), which determines muscle tissue development, composition, growth and maturation.

Multifactorial analysis and food safety issues of edible marine gastropods intended for human consumption

The present thesis aims to evaluate a method to assess the viability; estimate the bacterial and viral (Hepatitis A and Norovirus) contamination; describe how some parameters change during a week in refrigerated condition and after 24 hours of immersion; estimate indole-producing bacteria and biogenic amines; evaluate the presence of saxitoxin and tetrodotoxin. The method to assess the viability using sea salt is easy to apply. Marine gastropods did not accumulate fecal contaminants, but vibrios due to their feeding. The Vibrio spp. load was even higher than the one registered on Ruditapes philippinarum belonging to the same area For what to concern the evaluation during a week in refrigerated condition and after 24 hours of immersion, non-re-immersed gastropods exceeded the acceptable mortality (10%) after three days in refrigerated conditions, but the Vibrio spp. load did not show a significant increase within three days. The TVC was already high from the beginning and its major part consisted of SSOs, which could be explained by gastropods’ feed, such as the Pseudomonas spp. load and the abundance of IPB. The BAs amount was also correlated with viability and had a statistically significant difference within a week on refrigerated conditions, principally because putrescine, tyramine, spermidine, and cadaverine rise in non-re-immersed samples. It also should be noted that the BAs amount was higher on average than the recommendation of literature. Moreover, re-immersed batches showed acceptable viability even after 3 days, and the Vibrio spp. load, TVC, SSOs, and biogenic amines remained almost constant within a week contrary to non-re-immersed samples. Finally, T. mutabilis and B. brandaris did not accumulate NoVs and TTX. We obtained only one positivity of the HAV sample and traces of STX (not at levels toxic to humans). Our results contribute to identifying food-borne hazards for T. mutabilis and B. brandaris.