Identification of novel HIV restriction factors

To efficiently replicate within mammalian cells, viruses have to manoeuvre through complex host mechanisms, hijacking a network of host proteins to achieve successful propagation. To prevent this invasion, cells have evolved over time to efficiently block the incursing pathogen by direct or indirect targeting. Human immunodeficiency virus (HIV) is a retrovirus of major global public health issue. In the last decade, extensive focus on innate immune proteins has been given, and particularly restriction factors, proteins inhibiting HIV replication by affecting various stages of the viral cycle. Because of the importance of developing new HIV therapies that are associated with reduced side effects and resistances, there is an urge to understand the antiviral response against HIV. Using common features of known restriction factors as a signature to identify new anti-HIV factors, candidates were identified. Particularly multiple members of the apolipoproteins L (APOL) family were found. Cotransfection experiments confirmed very potent inhibitory effects on HIV-1 expression. Further characterization of APOL6, the best candidate, was carried out. APOL6 was not able to inhibit HIV specifically but rather inhibited any gene-encoded DNA that was cotransfected and therefore APOL6 does not classify as a bona fide restriction factor. In addition, we were able to map the activity of APOL6 to the MAD domain and mainly to residue 174. We also found that other members of the family identified in the screen, APOL1 and 3, could have similar mechanism of action as APOL6. Finally, although the complete mechanism of action of APOL6 has yet to be elucidated, it might be blocked during transfections, potentially improving transfection of primary cells. -- Pour se répliquer efficacement dans les cellules de mammifères, les virus doivent manoeuvrer à travers des mécanismes cellulaires complexes et détourner un réseau de protéines de l'hôte. Pour empêcher cette invasion, les gènes de l'hôte ont évolué dans le temps pour cibler efficacement, directement ou indirectement, l'agent pathogène. Le virus de l'immunodéficience humaine (VIH) est un rétrovirus de problème majeur de santé publique mondiale, mais le faible risque de transmission du virus pourrait être expliqué par la présence d'un système antiviral de l'hôte qui, en cas d'échec, conduit à une infection productive. Durant la dernière décennie, il y a eu un intérêt spécial porté sur les protéines immunitaires innées appelé facteurs de restriction présentant des effets inhibiteurs puissants sur la réplication du VIH en affectant différentes étapes du cycle viral. En raison de l'importance de la recherche de nouvelles thérapies anti-VIH associées à des effets secondaires et des résistances réduites comparé aux traitements actuels, il existe un besoin de comprendre la réponse antivirale innée contre le VIH. Basé sur des caractéristiques communes des facteurs de restriction connus, nous avons proposé d'identifier de nouveaux facteurs anti-VIH. Nous avons trouvé une famille de protéines, les apolipoprotéines L (APOL) montrant les effets inhibiteurs très puissants contre l'expression du VIH-1 dans des expériences de co-transfection. Nous avons décidé d'approfondir le rôle de ces protéines dans l'immunité innée et de se concentrer sur le meilleur candidat APOL6. Nous avons en outre établi qu'APOL6 n'a pas d'activité anti-virale spécifique et donc pas classé comme un facteur de bonne foi de restriction. Par ailleurs, APOL6 est capable d'inhiber fortement l'expression de tout Plasmide cotransfecté. En outre, nous avons été en mesure de cartographier l'activité d'APOL6 au domaine MAD et principalement au résidu 174. Nous avons également constaté que d'autres membres de la famille identifiés dans l'étude, APOL1 et 3, pourraient avoir le même mécanisme d'action qu'APOL6. Enfin, bien que le mécanisme d'action complet d'APOL6 reste à être élucidé, il pourrait être d'une importance biotechnologique car il pourrait potentiellement faciliter la transfection de cellules primaires après l'inhibition d'APOL6.

Caloric restriction induces energy-sparing alterations in skeletal muscle contraction, fiber composition and local thyroid hormone metabolism that persist during catch-up fat upon refeeding.

Weight regain after caloric restriction results in accelerated fat storage in adipose tissue. This catch-up fat phenomenon is postulated to result partly from suppressed skeletal muscle thermogenesis, but the underlying mechanisms are elusive. We investigated whether the reduced rate of skeletal muscle contraction-relaxation cycle that occurs after caloric restriction persists during weight recovery and could contribute to catch-up fat. Using a rat model of semistarvation-refeeding, in which fat recovery is driven by suppressed thermogenesis, we show that contraction and relaxation of leg muscles are slower after both semistarvation and refeeding. These effects are associated with (i) higher expression of muscle deiodinase type 3 (DIO3), which inactivates tri-iodothyronine (T3), and lower expression of T3-activating enzyme, deiodinase type 2 (DIO2), (ii) slower net formation of T3 from its T4 precursor in muscles, and (iii) accumulation of slow fibers at the expense of fast fibers. These semistarvation-induced changes persisted during recovery and correlated with impaired expression of transcription factors involved in slow-twitch muscle development. We conclude that diminished muscle thermogenesis following caloric restriction results from reduced muscle T3 levels, alteration in muscle-specific transcription factors, and fast-to-slow fiber shift causing slower contractility. These energy-sparing effects persist during weight recovery and contribute to catch-up fat.

Post-transcriptional regulation of circadian gene expression by miRNAs

La grande majorité des organismes vivants ont développé un système d'horloges biologiques internes, appelées aussi horloges circadiennes, contrôlant l'expression de gênes impliqués dans de nombreux processus moléculaires et comportementaux. Au cours de la dernière décennie, des analyses « microarray » et séquençages à haut débit sur divers tissus de mammifères, indiquent que jusqu'à 20% du transcriptome serait sous contrôle circadien. Il était jusqu'à présent admis que la majorité des ARNm ayant une accumulation rythmique était générée par une transcription qui était elle-même rythmique. Toutefois, de récentes études ont suggéré qu'une proportion considérable des ARNm cycliques serait en fait générée par des mécanismes post-transcriptionnelles, incluant une régulation par micro-ARN (miARN). Lorsque j'ai débuté mon travail de thèse, l'influence des miARN sur l'expression des gènes circadiens, au niveau pangénomique, était encore méconnue. Par l'utilisation d'un modèle murin, dont la biogenèse des miARN a été spécifiquement désactivée au niveau des cellules hépatiques (knockout conditionnel pour Dicer), je me suis donc intéressée au rôle que jouaient ces molécules régulatrices sur la rythmicité de l'expression génique dans le foie. Des séquençages sur l'ensemble du transcriptome révèlent que l'horloge interne du foie est étonnement résistante à la perte totale des miARN. Nous avons cependant trouvé que les miARN agissent de façon importante sur la régulation de l'expression des gènes contrôlés par l'horloge moléculaire. La corégulation par les miARN, affectant jusqu'à 30% des gènes transcrits de façon rythmiques, conduit ainsi à une modulation de phase et d'amplitude du rythme de l'abondance des ARNm. En revanche, seuls peu de transcrits dépendent uniquement des miARN pour la rythmicité de leur accumulation. Enfin, mon travail met en évidence plusieurs miARN spécifiques, qui semblent préférentiellement moduler l'expression des gènes cycliques et permet l'identification de voies hépatiques particulièrement sujettes à une double régulation par les miARN et l'horloge biologique interne. La première masse d'analyses a essentiellement porté sur le rôle que jouent les miARN au niveau de l'expression des gènes contrôlés par l'horloge interne. Dans deux études de suivi, je me suis penchée sur deux aspects supplémentaires et complémentaires de la manière dont les miARN et l'oscillation de l'expression des gènes interagissent. Dans les hépatocytes murins, spécifiquement privés de Dicer, je me suis demandée si un phénotype horloge avait pu être masqué, dû à un entraînement stable de l'horloge du foie par l'horloge maîtresse du cerveau. J'ai donc commencé une série d'expériences ambitieuses (impliquant la mesure de la rythmicité du foie in vivo, chez l'animal vivant) afin de déséquilibrer l'entrainement de l'horloge hépatique via l'utilisation d'un protocole nutritionnel spécifique. Les premiers résultats suggèrent que dans des conditions où l'animal subit une restriction alimentaire pendant la journée, les miARN sont importants dans la cinétique d'adaptation des organes périphériques à un nouvel horaire de sustentation. Dans une deuxième ligne de recherche, j'ai plus profondément étudié quels seraient les miARN responsables des rythmes post-transcriptionnels des ARNm, en utilisant le séquençage de « small » ARN sur 24h. L'analyse est en cours et se poursuivra après l'obtention de mon diplôme. De façon générale, mon travail révèle d'importants et nouveaux rôles des miARN dans la modulation de l'expression circadienne des gènes hépatiques. De plus, le set de données générées dans l'étude déjà publiée, peut dorénavant servir de ressource valable pour de prochaines investigations sur le rôle physiologique que les miARN jouent au niveau du foie. -- Most living organisms have developed internal timing systems, called circadian clocks, to drive the rhythmic expression of genes involved in many molecular and behavioral processes. Over the last decade, microarray analyses and high- throughput sequencing from various mammalian tissues have indicated that up to 20% of the transcriptome are under circadian control. It was generally assumed that the majority of rhythmic mRNA accumulation is generated by rhythmic transcription. However, recent studies have suggested that a considerable proportion of mRNA cycling may actually be generated by post-transcriptional mechanisms, including by microRNAs. When I started my thesis work, it was still unknown how miRNAs influence circadian gene expression in a genome-wide fashion. Using a mouse model in which miRNA biogenesis can be inactivated in hepatocytes (conditional Dicer knockout mouse), I have thus addressed the role that these regulatory molecules play in rhythmic gene expression in the liver. Whole transcriptome sequencing revealed that the hepatic core clock was surprisingly resilient to total miRNA loss. However, we found that miRNAs acted as important regulators of clock-controlled gene expression. Co- regulation by miRNAs, which affected up to 30% of rhythmically transcribed genes, thus led to the modulation of phases and amplitudes of mRNA abundance rhythms. By contrast, only very few transcripts were strictly dependent on miRNAs for their rhythmic accumulation. Finally, my work highlights several specific miRNAs that appear to preferentially modulate cyclic gene expression, and identifies pathways in the liver that are particularly prone to dual regulation through miRNAs and the clock. The first bulk of analyses mainly dealt with the role that miRNAs play at the level of rhythmic clock output gene expression. In two follow-up studies I further delved into two additional, complementary aspects of how miRNAs and gene expression oscillations interact. First, I addressed whether a core clock phenotype in the hepatocyte-specific Dicer knockout could have been masked due to the stable entrainment of the liver clock by the animals' master clock in the brain. I thus started a series of ambitious experiments (involving the in vivo recording of liver rhythms in live animals) to bring the stable entrainment of the liver clock out of equilibrium using specific feeding protocols. My first results suggest that under conditions when animals are challenged by food restriction to daytime, miRNAs are important for the kinetics of adapting to unusual mealtime in peripheral tissue. In a second line of research, I have more carefully investigated which miRNAs are responsible for post- transcriptional mRNA rhythms using small RNA sequencing around-the-clock. The analyses are ongoing and will be continued after my graduation. Overall, my work uncovered important and novel roles of miRNA activity in shaping hepatic circadian gene expression; moreover, the datasets collect in the published studies can serve as a valuable resource for further investigations into the physiological roles that miRNAs play in liver. -- L'alternance du jour et de la nuit dirige depuis longtemps la vie quotidienne des êtres humains et de la plupart des organismes sur terre. Ce cycle de 24 heures façonne beaucoup de changements comportementaux et physiologiques tels que la vigilance, la température corporelle et le sommeil. Les rythmes journaliers, appelés rythmes circadiens, sont dirigés par des horloges biologiques tournant dans presque chaque cellule du corps. Une structure dans le cerveau agit en tant qu'horloge maitresse pour synchroniser les horloges internes entre elles et en fonction des signaux de jour/nuit extérieurs. Dans les cellules "les gènes de l'horloge" sont activés et désactivés une fois par jour ce qui déclenche des cycles dans lesquels des protéines sont produites de manière circadienne. Ces rythmes protéiques sont spécialisés pour chaque tissu ou organe et peuvent les aider à réaliser leurs tâches quotidiennes. Les rythmes circadiens peuvent être générés d'autres manières n'impliquant pas directement les composants des gènes de l'horloge. Les ARN messagers (ARNm) sont des molécules intermédiaires dans la production de protéines à partir d'ADN. Dans le foie des souris jusqu'à 20% des molécules d'ARNm sont produites suivant des rythmes circadiens. Le foie réalise des tâches essentielles dans le contrôle du métabolisme incluant celui des hydrates de carbone, des graisses et du cholestérol. Un timing précis est important afin de traiter les substances nutritives correctement lors des repas il en résulte une variation des quantités de certains ARNm et protéines coïncidant avec les repas. Les microARNs constituent une autre classe de molécules ARN de très petite taille qui régulent l'efficacité de traduction des ARNm en protéines et la stabilité des ARNm. Lors de mon travail de thèse, j'ai exploré de manière approfondie l'influence de ces petits régulateurs sur les rythmes circadiens du foie de souris. Ces expériences qui impliquaient le "Knock-out" d'un gène essentiel à la production de microARNs montrent qu'au lieu de générer les rythmes des ARNm, les microARNs les ajustent pour répondre aux besoins spécifiques du foie comme assurer leur pic au bon moment de la journée. Le ciblage de microARNs spécifiques peut révéler de nouvelles stratégies pour rectifier ces rythmes lorsque par exemple les fonctions métaboliques ne fonctionnent plus normalement. -- The rising and setting of the sun have long driven the daily schedules of humans and most organisms on the earth. This 24-hr cycle shapes many behavioural and physiological changes, such as alertness, body temperature, and sleep. These daily rhythms, which are called circadian rhythms, are dictated by biological clocks that are ticking in almost every single cell of the body. A region in the brain acts as a master clock to synchronize the internal clocks with each other and with the outside light/dark cycles. In cells, "core clock genes" are turned on and off once per day, which triggers cycles that cause some proteins to be produced in a circadian manner. The protein rhythms are specialized to a particular tissue or organ, and may help them to carry out their designated daily tasks. However, circadian rhythms might also be produced by other ways that do not involve these core clock components. Messenger RNAs (mRNAs) are intermediate molecules in the production of proteins from DNA. In the mouse liver, up to 20% of mRNA molecules are produced in circadian cycles. The liver performs essential tasks that control metabolism-including that of carbohydrates, fats, and cholesterol. Precisely timing when certain mRNAs and proteins reach peaks and troughs in their activities to coincide with mealtimes is important for nutrients to be properly processed. Other RNA molecules called microRNAs, i.e. RNAs of very small size, regulate at which rate mRNA molecules are translated into proteins. In my thesis work, I have explored at the influence of these small regulators on circadian rhythms in the mouse liver in greater detail. These experiments, which involved "knocking out" a gene that is essential for the production of microRNAs, show that rather than generating the mRNA rhythms, the microRNAs appear to adjust them to meet the specific needs of the liver, such as ensuring that they peak at the right time-of-day. Targeting specific microRNA molecules may reveal new strategies to tweak these rhythms, which could help to improve conditions when metabolic functions go wrong.

Feeding habits of Ophichthus rufus (Anguilliformes, Ophichthidae) in the western Mediterranean

Ophichthus Rufus is a Mediterranean benthic fish with nocturnal habits. It lives in sand or mud bottoms at depths between 50 and 150m. Until now, nothing about its feeding habits was known. Stomach contents of 689 individuals, collected between 1985 and 1987, were analysed. The diet is basically composed of benthic organisms, among which the decapods processa canaliculata and Alpheus glaber and the teleost fish Callionymus maculatus are prominent. Young of both sexes and adult males have a euryphagic carnivorous diet, whereas adult females are closer to a stenophagic piscivorous diet

Observations on the feeding habits of the narrow mouthed cat shark Schroederichthys bivius (Chondrichthyes, Scyliorhinidae) in the Beagle Channel

The study of the stomach contents of 97 specimens of the narrow mouthed cat shark Schroederichthys bivius shows that their basic food consists of crabs, mainly Munida subrugosa. No significant variations were observed between size classes. The strongly developed sexual dimorphism in Touch morphology and mouth shape was not correlated with the diet of males and females

Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver.

Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light-dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5'-Terminal Oligo Pyrimidine tract (5'-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5'-UTR (TISU) motif. The increased translation efficiency of 5'-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation.

A genotypic mutation system measuring mutations in restriction recognition sequences.

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of the human c-Ha-ras1 gene from a large excess of wild-type DNA. For this purpose we constructed a 272 bp fragment with 2 mutations in the PvuII recognition sequence 1727-1732 and studied the rescue by RFLP/PCR of a few copies of this 'PvuII mutant standard'. Following amplification with Taq-polymerase and cloning into lambda gt10, plaques containing wild-type sequence, PvuII mutant standard or Taq-polymerase induced bp changes were quantitated by hybridization with specific oligonucleotide probes. Our results indicate that 10 PvuII mutant standard copies can be rescued from 10(8) to 10(9) wild-type sequences. Taq polymerase errors originating from unrestricted, residual wild-type DNA were sequence dependent and consisted mostly of transversions originating at G.C bp. In contrast to a doubly mutated 'standard' the capacity to rescue single bp mutations by RFLP/PCR is limited by Taq-polymerase errors. Therefore, we assessed the capacity of our protocol to isolate a G to T transversion mutation at base pair 1698 of the MspI-site 1695-1698 of the c-Ha-ras1 gene from excess wild-type ras1 DNA. We found that 100 copies of the mutated ras1 fragment could be readily rescued from 10(8) copies of wild-type DNA.

Dietary calcium restriction enhances cadmium-induced metallothionein synthesis in rats.

Experiments were conducted with adult male rats to investigate the effects of dietary calcium (Ca) restriction upon intake and tissue distribution of cadmium (Cd), and Cd-metallothionein (Mt) synthesis. Four groups of animals were fed either a low-Ca, semisynthetic diet (0.1% Ca) or the same diet supplemented with 0.8% Ca (normal diet). The caloric intake was similar in all groups. Two groups (low-Ca and normal diet) were used as controls, and two groups (low-Ca and normal diet) received 100 mg/l Cd (as CdCl2) in drinking water. Cd levels in liver, kidney, spleen and red cells were measured in all animals after 8 weeks of treatment. Concomitantly, Mt levels in plasma, liver and kidney were evaluated by radioimmunoassay. Ca deficiency entailed marked and significant increases in accumulation of Cd and synthesis of Mt in all assayed tissues. It is concluded that dietary Ca restriction, independent of caloric intake, enhances Cd intestinal absorption and tissue accumulation, which is followed by increased tissue Mt synthesis.

Defensive Metabolites from Antarctic Invertebrates: Does Energetic Content Interfere with Feeding Repellence?

Many bioactive products from benthic invertebrates mediating ecological interactions have proved to reduce predation, but their mechanisms of action, and their molecular identities, are usually unknown. It was suggested, yet scarcely investigated, that nutritional quality interferes with defensive metabolites. This means that antifeedants would be less effective when combined with energetically rich prey, and that higher amounts of defensive compounds would be needed for predator avoidance. We evaluated the effects of five types of repellents obtained from Antarctic invertebrates, in combination with diets of different energetic values. The compounds came from soft corals, ascidians and hexactinellid sponges; they included wax esters, alkaloids, a meroterpenoid, a steroid, and the recently described organic acid, glassponsine. Feeding repellency was tested through preference assays by preparing diets (alginate pearls) combining different energetic content and inorganic material. Experimental diets contained various concentrations of each repellent product, and were offered along with control compound-free pearls, to the Antarctic omnivore amphipod Cheirimedon femoratus. Meridianin alkaloids were the most active repellents, and wax esters were the least active when combined with foods of distinct energetic content. Our data show that levels of repellency vary for each compound, and that they perform differently when mixed with distinct assay foods. The natural products that interacted the most with energetic content were those occurring in nature at higher concentrations. The bioactivity of the remaining metabolites tested was found to depend on a threshold concentration, enough to elicit feeding repellence, independently from nutritional quality.

Altérations dans le cordon ombilical de nouveau-nés avec une restriction de la croissance intra-utérine

Introduction: Le retard de croissance intra-utérin (RCIU) est défini comme une incapacité du foetus à atteindre son plein potentiel de croissance. C'est une complication fréquente (affectant ~8% des grossesses), associée à un risque accru de mortalité et morbidité périnatales et de maladies chroniques à l'âge adulte, telles que les maladies coronariennes, l'hypertension, ou le diabète. Une croissance foetale adéquate est déterminée principalement par la disponibilité en oxygène et nutriments apportés au foetus par la circulation ombilico-placentaire. Chez l'homme, le tonus vasculaire ombilical est régulé majoritairement par la voie du monoxyde d'azote (NO)/GMPc. Nous avons émis l'hypothèse que le RCIU pourrait être associé à des altérations dans la régulation de la circulation ombilicale, en particulier dans la voie du NO/GMPc. Méthodes: Cette étude a été conçue pour identifier dans des cordons ombilicaux, les changements structurels, fonctionnels et moléculaires survenant en cas de RCIU, en particulier dans la veine om-bilicale. Résultats: De façon générale, le diamètre du cordon ombilical était significativement réduit chez les nouveau-nés avec RCIU par rapport aux contrôles. Les mesures histomorphométriques ont mis en évidence une diminution significative de la surface transversale totale ainsi que de muscle lisse dans la veine ombilicale en cas de RCIU. Les études pharmacologiques effectuées sur des anneaux vasculaires de veines ombilicales ont montré une diminution de la tension maximale induite par des vasoconstricteurs chez les garçons avec RCIU, et une réduction significative de la relaxation induite par le NO chez les filles avec RCIU. Cette altération de la relaxation s'accompagne de modifications de plusieurs composants de la voie du NO/GMPc au niveau du muscle lisse de la veine ombilicale des filles avec RCIU. Enfin l'addition d'un inhibiteur non-spécifique des phosphodiesterases (PDEs) a permis d'améliorer la réponse au NO dans tous les groupes et surtout de compenser la réduction de la relaxation induite par le NO chez les filles avec RCIU. Conclusion: Cette étude a permis de mettre en évidence des modifications structurelles dans le cordon ombilical de nouveau-nés présentant un RCIU, ainsi que des changements fonctionnels et moléculaires dans la veine ombilicale, en particulier dans la voie du NO/GMPc, qui pourraient contribuer au développement du RCIU. L'effet bénéfique de l'inhibition des PDEs sur la relaxation suggère qu'elles pourraient constituer des cibles thérapeutiques potentielles.

Concerted changes in N and C primary metabolism in alfalfa (Medicago sativa) under water restriction

Although the mechanisms of nodule N2 fixation in legumes are now well documented, some uncertainty remains on the metabolic consequences of water deficit. In most cases, little consideration is given to other organs and, therefore, the coordinated changes in metabolism in leaves, roots, and nodules are not well known. Here, the effect of water restriction on exclusively N2-fixing alfalfa (Medicago sativa L.) plants was investigated, and proteomic, metabolomic, and physiological analyses were carried out. It is shown that the inhibition of nitrogenase activity caused by water restriction was accompanied by concerted alterations in metabolic pathways in nodules, leaves, and roots. The data suggest that nodule metabolism and metabolic exchange between plant organs nearly reached homeostasis in asparagine synthesis and partitioning, as well as the N demand from leaves. Typically, there was (i) a stimulation of the anaplerotic pathway to sustain the provision of C skeletons for amino acid (e.g. glutamate and proline) synthesis; (ii) re-allocation of glycolytic products to alanine and serine/glycine; and (iii) subtle changes in redox metabolites suggesting the implication of a slight oxidative stress. Furthermore, water restriction caused little change in both photosynthetic efficiency and respiratory cost of N2 fixation by nodules. In other words, the results suggest that under water stress, nodule metabolism follows a compromise between physiological imperatives (N demand, oxidative stress) and the lower input to sustain catabolism.

Feeding ecology and movements of the Barolo shearwater Puffinus baroli baroli in the Azores, NE Atlantic

Trophic ecology and movements are critical issues for understanding the role of marine predators in food webs and for facing the challenges of their conservation. Seabird foraging ecology has been increasingly studied, but small elusive species, such as those forming the"little shearwater" complex, remain poorly known. We present the first study on the movements and feeding ecology of the Barolo shearwater Puffinus baroli baroli in a colony from the Azores archipelago (NE Atlantic), combining global location-sensing units, stable isotope analyses of feathers (δ13C and δ15N), stomach flushings and data from maximum depth gauges. During the chick-rearing period, parents visited their nests most nights, foraged mainly south of the colony and fed at lower trophic levels than during the non-breeding period. Squid was the most diverse prey (6 families and at least 10 different taxa), but species composition varied considerably between years. Two squid families, Onychoteuthidae and Argonautidae, and the fish family Phycidae accounted for 82.3% of ingested prey by number. On average, maximum dive depths per foraging trip reached 14.8 m (range: 7.9 to 23.1 m). After the breeding period, birds dispersed offshore in all directions and up to 2500 km from the breeding colony, and fed at higher trophic levels. Overall, our results indicate that the Barolo shearwater is a non-migratory shearwater feeding at the lowest trophic level among Macaronesian seabirds, showing both diurnal and nocturnal activity and feeding deeper in the water column, principally on small schooling squid and fish. These traits contrast with those of 3 other Azorean Procellariiformes (Cory"s shearwater Calonectris diomedea, the Madeiran storm-petrel Oceanodroma castro and Monteiro"s storm-petrel O. monteiroi), indicating ecological segregation within the Azorean seabird community.

Nutritional and feeding ecology in the Cory"s shearwater (Calonectris diomedea).

In birds, parents adjust their feeding behaviour according to breeding duties, which ultimately may lead to seasonal adjustments in nutritional physiology and hematology over the breeding season. Although avian physiology has been widely investigated in captivity, few studies have integrated individual changes in feeding and physiological ecology throughout the breeding season in wild birds. To study relationships between feeding ecology and nutritional ecophysiology in Cory"s shearwater Calonectris diomedea, we weighed and took blood samples from 28 males and 19 females during the pre-laying, egg-laying, incubation, hatching and chick-rearing periods of the breeding season. In addition, we fitted 6 birds with geolocators to track their foraging movements throughout the reproductive period. Thus, we examined individual changes in (1) nutritional condition (biochemistry metabolites); (2) oxygen carrying capacity (hematology); and (3) feeding areas and foraging effort (stable isotopes and foraging movements). Geolocators revealed a latitudinal shift in main feeding areas towards more southern and more neritic waters throughout the breeding season, which is consistent with the steady increase in δ13C signatures in the blood. Geolocators also showed a decrease in foraging effort from egg-laying to hatching, reflecting the activity decrease associated with incubation duties. Plasma metabolites, body mass and oxygen carrying capacity were associated with temporal changes in nutritional state and foraging effort in relation to recovery after migration, egg formation, fasting shifts during incubation and chick provisioning. This study shows that combining physiological and ecological approaches can help us understand the influence of breeding duties on feeding ecology and nutritional physiology in wild birds.