Clarín bifronte y el secreto de Unamuno : De la crítica como espacio de intimidad

En la España de fines del siglo XIX, la erudición se proyectó como espacio de controversia. Inmerso en plena crisis de confianza en el método cientificista, Unamuno creyó encontrar en Clarín un aliado. Sin intuir el respeto bifronte que Alas mantuvo con respecto a las posturas críticas enfrentadas por entonces -y que La Regenta ya había ilustrado- en vano buscó Unamuno su apoyo para erosionar el espacio de saber que veía personificado en Menéndez y Pelayo. En medio de esas tensiones oblicuas e irónicas, la intimidad se torna espacio de contienda crítica. Las cartas, los artículos burlescos, las lecturas recíprocas: la literatura misma se torna entonces instancia de auto legitimación

Light-modulated responses of growth and photosynthetic performance to ocean acidification in the model diatom Phaeodactylum tricornutum

Ocean acidification (OA) due to atmospheric CO2 rise is expected to influence marine primary productivity. In order to investigate the interactive effects of OA and light changes on diatoms, we grew Phaeodactylum tricornutum, under ambient (390 ppmv; LC) and elevated CO2 (1000 ppmv; HC) conditions for 80 generations, and measured its physiological performance under different light levels (60 µmol/m**2/s, LL; 200 µmol/m**2/s, ML; 460 µmol/m**2/s, HL) for another 25 generations. The specific growth rate of the HC-grown cells was higher (about 12-18%) than that of the LC-grown ones, with the highest under the ML level. With increasing light levels, the effective photochemical yield of PSII (Fv'/Fm') decreased, but was enhanced by the elevated CO2, especially under the HL level. The cells acclimated to the HC condition showed a higher recovery rate of their photochemical yield of PSII compared to the LC-grown cells. For the HC-grown cells, dissolved inorganic carbon or CO2 levels for half saturation of photosynthesis (K1/2 DIC or K1/2 CO2) increased by 11, 55 and 32%, under the LL, ML and HL levels, reflecting a light dependent down-regulation of carbon concentrating mechanisms (CCMs). The linkage between higher level of the CCMs down-regulation and higher growth rate at ML under OA supports the theory that the saved energy from CCMs down-regulation adds on to enhance the growth of the diatom.

Light availability determines susceptibility of reef building corals to ocean acidification

Elevated seawater pCO2, and in turn ocean acidification (OA), is now widely acknowledged to reduce calcification and growth of reef building corals. As with other environmental factors (e.g., temperature and nutrients), light availability fundamentally regulates calcification and is predicted to change for future reef environments alongside elevated pCO2 via altered physical processes (e.g., sea level rise and turbidity); however, any potential role of light in regulating the OA-induced reduction of calcification is still unknown. We employed a multifactorial growth experiment to determine how light intensity and pCO2 together modify calcification for model coral species from two key genera, Acropora horrida and Porites cylindrica, occupying similar ecological niches but with different physiologies. We show that elevated pCO2 (OA)-induced losses of calcification in the light (G L) but not darkness (G D) were greatest under low-light growth conditions, in particular for A. horrida. High-light growth conditions therefore dampened the impact of OA upon G L but not G D. Gross photosynthesis (P G) responded in a reciprocal manner to G L suggesting OA-relieved pCO2 limitation of P G under high-light growth conditions to effectively enhance G L. A multivariate analysis of past OA experiments was used to evaluate whether our test species responses were more widely applicable across their respective genera. Indeed, the light intensity for growth was identified as a significant factor influencing the OA-induced decline of calcification for species of Acropora but not Porites. Whereas low-light conditions can provide a refuge for hard corals from thermal and light stress, our study suggests that lower light availability will potentially increase the susceptibility of key coral species to OA.

Physiological responses of the marine diatom Thalassiosira pseudonana to increased pCO2 and seawater acidity

We studied the effects of elevated CO2 concentration and seawater acidity on inorganic carbon acquisition, photoinhibition and photoprotection as well as growth and respiration in the marine diatom Thalassiosira pseudonana. After having grown under the elevated CO2 level (1000 µatm, pH 7.83) at sub-saturating photosynthetically active radiation (PAR, 75 µmol photons/m**2/s) for 20 generations, photosynthesis and dark respiration of the alga increased by 25% (14.69 ± 2.55 fmol C/cell/h) and by 35% (4.42 ± 0.98 fmol O2/cell/h), respectively, compared to that grown under the ambient CO2 level (390 µatm, pH 8.16), leading to insignificant effects on growth (1.09 ± 0.08 (1/d))v 1.04 ± 0.07 (1/d)). The photosynthetic affinity for CO2 was lowered in the high-CO2 grown cells, reflecting a down-regulation of the CO2 concentrating mechanism (CCM). When exposed to an excessively high level of PAR, photochemical and non-photochemical quenching responded similarly in the low- and high-CO2 grown cells, reflecting that photoinhibition was not influenced by the enriched level of CO2. In T. pseudonana, it appeared that the energy saved due to the down-regulated CCM did not contribute to any additional light stress as previously found in another diatom Phaeodactylum tricornutum, indicating differential physiological responses to ocean acidification between these two diatom species.

Wave-induced changes in seaweed toughness entail plastic modifications in snail traits maintaining consumption efficacy, link to supplementary material

1. Environmental stress can influence species traits and performance considerably. Using a seaweed-snail system from NW (Nova Scotia) and NE (Helgoland) Atlantic rocky shores, we examined how physical stress (wave exposure) modulates traits in the seaweed Fucus vesiculosus and indirectly in its main consumer, the periwinkle Littorina obtusata. 2. In both regions, algal tissue toughness increased with wave exposure. Reciprocal-transplant experiments showed that tissue toughness adjusts plastically to the prevailing level of wave exposure. 3. Choice experiments tested the feeding preference of snails from sheltered, exposed, and very exposed habitats for algae from such wave exposures. Snails from exposed and very exposed habitats consumed algal tissues at similar rates irrespective of the exposure of origin of the algae. However, snails from sheltered habitats consumed less algal tissues from very exposed habitats than tissues from sheltered and exposed habitats. Choice assays using reconstituted algal food (triturated during preparation) identified high thallus toughness as the explanation for the low preference of snails from sheltered habitats for algae from very exposed habitats. 4. Ultrastructural analyses of radulae indicated that rachidian teeth were longest and the number of cusps in lateral teeth (grazing-relevant traits) was highest in snails from very exposed habitats, suggesting that radulae are best suited to rupture tough algal tissues in such snails. 5. No-choice feeding experiments revealed that these radular traits are also phenotypically plastic, as they adjust to the toughness of the algal food. 6. Synthesis. This study indicates that the observed plasticity in the feeding ability of snails is mediated by wave exposure through phenotypic plasticity in the tissue toughness of algae. Thus, plasticity in consumers and their resource species may reduce the potential effects of physical stress on their interaction.

Long-term dynamics of adaptive evolution in a globally important coccolithophore to ocean acidification

Recent evolution experiments have revealed that marine phytoplankton may adapt to global change, for example to ocean warming or acidification. Long-term adaptation to novel environments is a dynamic process and phenotypic change can take place thousands of generations after exposure to novel conditions. Using the longest evolution experiment performed in any marine species to date (4 yrs, = 2100 generations), we show that in the coccolithophore Emiliania huxleyi, long-term adaptation to ocean acidification is complex and initial phenotypic responses may revert for important traits. While fitness increased continuously, calcification was restored within the first 500 generations but later reduced in response to selection, enhancing physiological declines of calcification in response to ocean acidification. Interestingly, calcification was not constitutively reduced but revealed rates similar to control treatments when transferred back to present-day CO2 conditions. Growth rate increased with time in controls and adaptation treatments, although the effect size of adaptation assessed through reciprocal assay experiments varied. Several trait changes were associated with selection for higher cell division rates under laboratory conditions, such as reduced cell size and lower particulate organic carbon content per cell. Our results show that phytoplankton may evolve phenotypic plasticity that can affect biogeochemically important traits, such as calcification, in an unforeseen way under future ocean conditions.

Experiment: Adaptation of a globally important coccolithophore to ocean warming and acidification

Although oceanwarming and acidification are recognized as two major anthropogenic perturbations of today's oceanswe know very little about how marine phytoplankton may respond via evolutionary change.We tested for adaptation to ocean warming in combination with ocean acidification in the globally important phytoplankton species Emiliania huxleyi. Temperature adaptation occurred independently of ocean acidifcation levels. Exponential growth rates were were up to 16% higher in populations adapted for one year to warming when assayed at their upper thermal tolerance limit. Particulate inorganic (PIC) and organic (POC) carbon production was restored to values under present-day ocean conditions, owing to adaptive evolution, and were 101% and 55% higher under combined warming and acidification, respectively, than in non-adapted controls. Cells also evolved to a smaller size while they recovered their initial PIC:POC ratio even under elevated CO2. The observed changes in coccolithophore growth, calcite and biomass production, cell size and elemental composition demonstrate the importance of evolutionary processes for phytoplankton performance in a future ocean. At the end of a 1-yr temperature selection phase, we conducted a reciprocal assay experiment in which temperature-adapted asexual populations were compared to the respective non-adapted control populations under high temperature, and vice versa (1. Assay Data, Dataset #835336). Mean exponential growth rates ? in treatments subjected to high temperature increased rapidly under all high temperature-CO2 treatment combinations during the temperature selection phase (2. time series, Dataset #835339).

Normative Influences of "Special and Differential Treatment" on North-South RTAs

Recently, there has been a surge in bilateral and regional trade arrangements between developed and developing countries, which are known as North-South RTAs. Under the current legal system of the WTO, North-South RTAs are governed by Article XXIV of the GATT or Article V of the GATS, which means such RTAs must be reciprocal and must cover substantially all the trade. On the other hand, there is another category of rules on RTAs based on the so-called Enabling Clause, which, in exceptional circumstances, provides special and differential treatment (SDT) for RTAs among developing countries. This paper considers the applicability of the SDT concept to North-South RTAs by examining why rules on RTAs and the concept of SDT were incorporated into the GATT/WTO legal framework so as to permit the derogation of most-favored-nation (MFN) obligations.

Remote Effects of Hippocampal Sclerosis on Effective Connectivity during Working Memory Encoding: A Case of Connectional Diaschisis?

Accumulating evidence suggests a role for the medial temporal lobe (MTL) in working memory (WM). However, little is known concerning its functional interactions with other cortical regions in the distributed neural network subserving WM. To reveal these, we availed of subjects with MTL damage and characterized changes in effective connectivity while subjects engaged in WM task. Specifically, we compared dynamic causal models, extracted from magnetoencephalographic recordings during verbal WM encoding, in temporal lobe epilepsy patients (with left hippocampal sclerosis) and controls. Bayesian model comparison indicated that the best model (across subjects) evidenced bilateral, forward, and backward connections, coupling inferior temporal cortex (ITC), inferior frontal cortex (IFC), and MTL. MTL damage weakened backward connections from left MTL to left ITC, a decrease accompanied by strengthening of (bidirectional) connections between IFC and MTL in the contralesional hemisphere. These findings provide novel evidence concerning functional interactions between nodes of this fundamental cognitive network and sheds light on how these interactions are modified as a result of focal damage to MTL. The findings highlight that a reduced (top-down) influence of the MTL on ipsilateral language regions is accompanied by enhanced reciprocal coupling in the undamaged hemisphere providing a first demonstration of “connectional diaschisis.”

Resistance to malathion and lambda-cyhalothrin in Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

La mosca mediterránea de la fruta, Ceratitis capitata (Wiedemann, 1824) (Diptera: Tephritidae), es una de las plagas de mayor incidencia económica en cítricos y otros frutales a nivel mundial. En España las medidas de control de esta plaga en cítricos, desde mediados de los 90 hasta 2009, se basaron principalmente en el monitoreo de las poblaciones y en la aplicación de tratamientos aéreos y terrestres con malatión cebo. Sin embargo, desde la retirada en la Unión Europea en 2009 de los productos fitosanitarios que contienen malatión, los insecticidas más utilizados para el control de esta plaga han sido lambda-cihalotrina y spinosad. En 2004-2005 se detectaron poblaciones españolas de C. capitata resistentes a malatión. Esta resistencia se ha asociado a una mutación (G328A) en la acetilcolinesterasa (AChE), a una duplicación del gen de la AChE (Ccace2) (una de las copias lleva la mutación G328A) y a resistencia metabólica mediada por esterasas (posiblemente aliesterasas). Sin embargo, cuando se secuenció la aliesterasa CcE7 en individuos de una línea resistente a malatión, no se encontró ninguna de las mutaciones (G137D y/o W251L/S/G) asociadas a resistencia en otras especies, si bien se encontraron otras mutaciones al compararlos con individuos de una línea susceptible. Asimismo, mediante la selección en laboratorio de una línea resistente a malatión (W-4Km) con lambda-cihalotrina, se ha podido obtener una línea resistente a lambda-cihalotrina (W-1K). Finalmente, se ha demostrado la capacidad de esta especie para desarrollar resistencia a spinosad mediante selección en laboratorio. Los múltiples mecanismos de resistencia identificados evidencian el potencial de esta especie para desarrollar resistencia a insecticidas con diferentes modos de acción. Los objetivos de esta tesis doctoral son: 1) evaluar la susceptibilidad de poblaciones españolas de campo de C. capitata a lambda-cihalotrina y dilucidar los mecanismos de resistencia en la línea W-1Kλ; 2) comparar la herencia, el coste biológico y la estabilidad de la resistencia a malatión mediada por la mutación G328A y la duplicación del gen Ccace2 (una de las copias lleva la mutación G328A); y 3) investigar el papel de las mutaciones identificadas en la aliesterasa CcαE7 en la resistencia a malatión. Estos estudios son de utilidad para el desarrollo de estrategias de manejo de la resistencia que puedan prevenir o retrasar la aparición de resistencia y aumentar la sostenibilidad de los insecticidas disponibles para el control de esta plaga. Nuestros resultados indican que las poblaciones españolas de C. capitata analizadas han desarrollado resistencia a lambda-cihalotrina. Los valores de CL50 estimados para las poblaciones recogidas en la Comunidad Valenciana, Cataluña y Andalucía oscilaron entre 129 ppm y 287 ppm, igualando o sobrepasando la concentración recomendada para los tratamientos de campo (125 ppm). Estos resultados contrastan con los obtenidos con tres poblaciones de campo recogidas en Túnez, cuya susceptibilidad fue similar a la de la línea control (C). La línea resistente a lambda-cihalotrina W-1K se continuó seleccionando en el laboratorio alcanzándose unos niveles de resistencia de 205 veces con respecto a la línea C, siendo su CL50 (4224 ppm) más de 30 veces superior a la concentración recomendada para los tratamientos de campo. Esta línea resistente mostró altos niveles de resistencia cruzada a deltametrina (150 veces) y a etofenprox (240 veces), lo que sugiere que el desarrollo de resistencia a lambda-cihalotrina podría comprometer la eficacia de otros piretroides para el control de esta plaga. Hemos demostrado que la resistencia de la línea W-1K a lambda-cihalotrina fue casi completamente suprimida por el sinergista PBO, lo que indica que las enzimas P450 desempeñan un papel muy importante en la resistencia a este insecticida. Sin embargo, tanto las moscas de la línea susceptible C como las de la línea resistente W-1K perdieron inmediatamente la capacidad de caminar (efecto “knock-down”) al ser tratadas tópicamente con lambda-cihalotrina, lo que sugiere que la resistencia no está mediada por alteraciones en la molécula diana (resistencia tipo “kdr”). La resistencia metabólica mediada por P450 fue analizada comparando la expresión de 53 genes CYP (codifican enzimas P450) de las familias CYP4, CYP6, CYP9 y CYP12 en adultos de la línea resistente W-1K y de la línea susceptible C. Nuestros resultados muestran que el gen CYP6A51 (número de acceso GenBank XM_004534804) fue sobreexpresado (13-18 veces) en la línea W-1K. Por otra parte, la expresión del gen CYP6A51 fue inducida tanto en adultos de la línea W-1K como de la línea C al ser tratados con lambda-cihalotrina. Sin embargo, no se obtuvieron diferencias significativas entre la línea susceptible C y la línea resistente W-1K al comparar la cantidad de P450 y la actividad NADPH-citocromo c reductasa presente en fracciones microsomales obtenidas a partir de abdómenes. Asimismo, no hemos podido correlacionar el metabolismo de deltametrina, estimado in vitro mediante la incubación de este insecticida con fracciones microsomales, con el nivel de resistencia a este piretroide observado en los bioensayos con la línea W-1K. Por otro lado, no se encontró ninguna alteración en la región promotora 5'UTR del gen CYP6A51 (-500 pb desde el inicio de la traducción) que pudiera explicar su sobreexpresión en la línea W-1K. Los datos obtenidos sugieren que la resistencia a lambda-cihalotrina en la línea W-1K está mediada por P450 y que la sobreexpresión de CYP6A51 puede desempeñar un papel importante, aunque se necesitan más evidencias para establecer una asociación directa de la resistencia con este gen. Hemos estudiado la herencia, el coste biológico y la estabilidad de la resistencia a malatión mediada por la mutación G328A y la duplicación del gen Ccace2 (una de las copias lleva la mutación G328A). La línea susceptible C, donde no se encuentra la mutación G328A (genotipo S/S), se cruzó con dos isolíneas establecidas para representar genotipos únicos correspondientes a los dos mecanismos de resistencia asociados a la molécula diana: 1) la isolínea 267Y (genotipo R/R) establecida a partir de una pareja que portaba la mutación G328A en homocigosis; 2) la isolínea 306TY (genotipo RS/RS) establecida a partir de una pareja que portaba en homocigosis la duplicación del gen Ccace2. No se realizaron cruces recíprocos, ya que mediante experimentos de hibridación in situ en cromosomas politénicos se pudo comprobar que el locus de la AChE y la duplicación (probablemente en tándem) se localizan en el cromosoma autosómico 2L. La susceptibilidad al malatión de los parentales resistentes (R/R o RS/RS) y susceptibles (S/S), los cruces F1 (S/R, S/RS y R/RS) y los retrocruzamientos indican que la resistencia a malatión es semi-dominante en ambos casos. Sin embargo, nuestros resultados no fueron concluyentes con respecto a la naturaleza monogénica de la resistencia a malatión en estas isolíneas. Por lo tanto, no podemos descartar que otros genes que contribuyan a la resistencia, además de la mutación G328A (isolínea 267Y) y de la duplicación del gen Ccace2 (isolínea 306TY), puedan haber sido seleccionados durante el proceso de selección de 267Y y 306TY. Varios parámetros biológicos fueron evaluados para determinar si estos dos mecanismos de resistencia a malatión suponen un coste biológico para los genotipos resistentes. Individuos con genotipo R/R mostraron un retraso en el tiempo de desarrollo de huevo a pupa, un peso de pupa reducido y una menor longevidad de los adultos, en comparación con los individuos con genotipo S/S. Sin embargo, el peso de pupa de los individuos con genotipo RS/RS fue similar al de los individuos S/S, y su desarrollo de huevo a pupa intermedio entre S/S y R/R. Estas diferencias en el coste biológico pueden estar relacionadas con la reducción de la eficiencia catalítica de la AChE mutada en los individuos R/R, y al efecto compensatorio que la copia no mutada del gen tiene en los individuos RS/RS que portan la duplicación. La estabilidad de la resistencia a malatión mediada por la mutación G328A y la duplicación se analizó mediante el seguimiento de los caracteres de resistencia en la progenie de retrocruzamientos S/R x R/R y S/RS x RS/RS a lo largo de varias generaciones en ausencia de presión de selección con insecticidas. Nuestros resultados muestran que la frecuencia del alelo que porta la mutación G328A disminuyó desde 67,5% en la primera generación del retrocruzamiento S/R x R/R (75% esperado, asumiendo segregación mendeliana y que sólo hay dos alelos: uno mutado y otro no mutado) a 12% después de 10 generaciones. Por el contrario, la frecuencia de la duplicación sólo disminuyó desde 75% en en la primera generación del retrocruzamiento S/RS x RS/RS (75% esperado, asumiendo segregación Mendeliana y que la duplicación segrega como un único alelo) a 50% en el mismo período, lo que indica que la duplicación es más estable que la mutación. Asimismo, se analizó la presencia de la mutación y de la duplicación en poblaciones de campo recogidas en seis localidades en 2004-2007, cuando todavía se usaba el malatión, y se comparó con poblaciones recogidas en los mismos campos en 2010, un año después de la prohibición del malatión en la Unión Europea. La frecuencia media del genotipo susceptible (S/S) aumentó del 55,9% en el período 2004-2007 a 70,8% en 2010, mientras que la frecuencia de los genotipos portadores de la mutación en homocigosis o heterocigosis (R/R y S/R) disminuyó del 30,4 al 9,2%, los que llevan la duplicación en homocigosis o heterocigosis (RS/RS y S/RS) aumentaron levemente desde 12,8 hasta 13,3%, y los que llevan a la vez la mutación y la duplicación (R/RS) también aumentaron del 1 al 6,7%. Estos resultados son consistentes con que la duplicación del gen Ccace2 (con una copia con la mutación G328A y la otra copia no mutada) es más ventajosa que la mutación G328A por si sola, ya que la duplicación mantiene los niveles de resistencia a la vez que limita el coste biológico. Para investigar la asociación entre la resistencia a malatión y las mutaciones encontradas previamente en CcE7, hemos generado isolíneas con mutaciones específicas seleccionadas por su ubicación próxima a la entrada al centro activo de la enzima. La isolínea Sm2 (procedente de una hembra heterocigota para la mutación V96L y un macho homocigoto para el alelo no mutado) mantuvo altos niveles de resistencia a malatión, incluso después de 30 generaciones sin presión de selección. Por el contrario, la isolínea 267Y (compuesta por individuos homocigotos para la mutación L267Y) y la línea 306TY (compuesta por individuos homocigotos para la doble mutación R306T-N307Y) mostraron una reducción significativa en los niveles de resistencia. También hemos encontrado que la resistencia a malatión de la línea Sm2 fue parcialmente revertida por DEF y TPP, y que Sm2 mostró una reducción significativa en la actividad MTB, como se ha descrito en otras especies que muestran resistencia específica a malatión mediada por aliesterases. Además, fue posible asociar la presencia de la mutación V96L en individuos de la línea Sm2 con supervivencia a una concentración discriminante de malatión (5,000 ppm) y con una baja actividad MTB. Estos resultados sugieren una posible relación entre la mutación V96L en la aliesterasa CcE7 y la resistencia a malatión, aunque todavía no se puede concluir que la resistencia es causada por esta mutación, siendo necesarios más estudios para comprobar su contribución a la resistencia. En conclusión, se ha encontrado por primera vez resistencia a lambda-cihalotrina en poblaciones de campo de C. capitata, y nuestros resultados indican que las P450 son el principal mecanismo de resistencia en la línea W-1K. Esta situación se suma al caso previamente descrito de resistencia en campo a malatión asociada a la mutación G328A, a la duplicación del gen Ccace2 (una de las copias lleva la mutación G328A) y a resistencia metabólica mediada por esterasas. Nuestros resultados también indican que la alteración de la molécula diana AChE parece ser responsable de un cierto nivel de resistencia a malatión en C. capitata, que puede ser estimada como aproximadamente 25-40 veces para la mutación G328A y 40-60 veces para la duplicación; mientras que la resistencia mediada por esterasas y que ha sido asociada en este estudio con la mutación V96L en CcE7 puede conferir un efecto multiplicativo (por un factor de 5 a 10) aumentando la resistencia a malatión a 200-400 veces. Por otra parte, hemos demostrado que los insectos resistentes que llevan la duplicación tienen un coste biológico menor y muestran una estabilidad mayor que aquellos con la mutación G328A en ausencia de presión de selección con insecticidas. Esto representa un escenario en el que los genotipos con la duplicación permanecerán en el campo en frecuencias bajas a moderadas, pero podrían ser seleccionados rápidamente si se utilizan malatión u otros insecticidas que muestren resistencia cruzada. Estos resultados tienen importantes implicaciones para los programas de manejo de la resistencia, ya que el repertorio de insecticidas eficaces para el control de C. capitata es cada vez más limitado. Además, la coexistencia de múltiples mecanismos de resistencia en poblaciones de campo ofrece el potencial para desarrollar resistencia frente a otros insecticidas disponibles para el control de esta plaga. Estrategias para de manejo de la resistencia basadas en la alternancia de insecticidas con diferentes modos de acción, y su combinación con otros métodos de control, deben ser implementadas para evitar el desarrollo de resistencia en campo. ABSTRACT The Mediterranean fruit fly (Medfly), Ceratitis capitata (Wiedemann, 1824) (Diptera: Tephritidae), is one of the most economically damaging pests of citrus and other fruit crops worldwide. Control measures in citrus crops in Spain from the mid 90's to 2009 were mainly based on field monitoring of population levels and aerial and ground treatments with malathion bait sprays. However, since the withdrawal of phytosanitary products containing malathion in the European Union in 2009, lambda-cyhalothrin and spinosad have become the most widely used insecticides for the control of this pest. Resistance to malathion was found in Spanish field populations of C. capitata in 2004-2005. This resistance has been associated with a mutation G328A in the acetylcholinesterase (AChE), a duplication of the AChE gene (Ccace2) (one of the copies bearing the mutation G328A), and metabolic resistance mediated by esterases (probably aliesterases). However, when the gene of the aliesterase CcE7 was sequenced in individuals from a malathion resistant strain of C. capitata, none of the known G137D and/or W251L/S/G mutations associated to resistance in other species were found, though other mutations were detected when compared with individuals from a susceptible strain. Noteworthy, a lambda-cyhalothrin resistant strain (W-1K) was obtained by selecting a field-derived malathion resistant strain (W-4Km) with lambda-cyhalothrin. Moreover, it has also been demonstrated the capacity of this species to develop resistance to spinosad by laboratory selection. The multiple resistance mechanisms identified highlight the potential of this species to develop resistance to insecticides with different modes of action. The objectives of this PhD Thesis are: 1) to assess the susceptibility of Spanish field populations of C. capitata to lambda-cyhalothrin and to elucidate the resistance mechanisms in the W-1Kλ strain; 2) to compare the inheritance, fitness cost and stability of the malathion resistance mediated by the G328A mutation and the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A); and 3) to investigate the role of the aliesterase CcαE7 mutations in malathion resistance. All these studies will be of use for devising proactive resistance management strategies that could prevent or delay resistance development and would increase the sustainability of the insecticides available for Medfly control. Our results indicate that Spanish field populations of C. capitata have developed resistance to lambda-cyhalothrin. The LC50 values estimated for populations collected at Comunidad Valenciana, Cataluña and Andalucía ranged from 129 ppm to 287 ppm, equaling or overpassing the recommended concentration for field treatments (125 ppm). These results contrast with those obtained with three different Tunisian field populations, whose susceptibility was similar to that of the control (C) strain. The lambda-cyhalothrin resistant W-1K strain has been further selected to achieve a 205-fold resistance compared to the C strain, being its LC50 (4,224 ppm) more than 30 times higher than the recommended concentration for field applications. This resistant strain showed high levels of cross-resistance to deltamethrin (150-fold) and etofenprox (240-fold), suggesting that the development of resistance to lambda-cyhalothrin may compromise the effectiveness of other pyrethroids for the control of this species. We have shown that the resistance of the W-1K strain to lambda-cyhalothrin was almost completely suppressed by the synergist PBO, indicating that P450 enzymes play a very important role in resistance to this insecticide. However, both susceptible C and resistant W-1K flies were knocked down after topical treatment with lambda-cyhalothrin, suggesting that kdr resistance mediated by alterations of the target site is not playing a major role. Metabolic resistance mediated by P450 was further analyzed by comparing the expression of 53 genes of the families CYP4, CYP6, CYP9 and CYP12 in adults flies from the resistant W-1K and the susceptible C strains. We found that the gene CYP6A51 (GenBank accession number XM_004534804) was overexpressed (13-18-fold) in the W-1K strain. Moreover, the expression of the CYP6A51 gene was induced when adults of the W-1K and C strains were treated with lambda-cyhalothrin. However, no significant differences were obtained between susceptible C and resistant W-1K strains for the quantity of P450 and for the activity of NADPH- cytochrome c reductase measured in microsomal fractions obtained from abdomens. Moreover, we failed to correlate the metabolism of deltamethrin, analyzed in vitro by incubating this insecticide with microsomal fractions, with the resistance level against this pyrethroid observed in bioassays with W-1K. The sequencing of the 5´UTR region of the CYP6A51 gene failed in finding an alteration in the promoter region (-500 bp from translation start site) that could explain overexpression in the W-1K strain. All data obtained suggest that resistance to lambda-cyhalothrin in the W- 1K strain is mediated by P450 and that overexpression of CYP6A51 may play a major role, although further evidences are needed to establish a direct association of resistance with this gene. We have studied the inheritance, fitness cost and stability of the malathion resistance mediated by the G328A mutation and the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A). The malathion-susceptible C strain where the G328A mutation is not found (S/S genotype) was crossed with two isolines established to represent unique genotypes corresponding to the two target-site resistance mechanisms: 1) the 267Y isoline (genotype R/R) was established from a couple bearing the mutation G328A in homozygosis; and 2) the 306TY isoline (genotype RS/RS) was established from a couple being homozygous for the duplication of the Ccace2 gene. Reciprocal crosses have not been performed, since in situ hybridization on polythene chromosomes showed that the AChE locus and the duplication (most probably in tandem) are placed at the autosomal chromosome 2L. Mortality responses to malathion of resistant isolines (R/R or RS/RS) and susceptible (S/S) genotypes, F1 crosses (S/R, S/RS, and R/RS), and the back-crosses indicated that resistance to malathion is inherited as a semi-dominant trait in both cases. However, our results were not conclusive about the monogenic nature of the resistance to malathion in these isolines. Thus, we can not discard that other genes contributing to resistance, in addition to the mutation G328A (isoline 267Y) and the duplication of the Ccace2 gene (isoline 306TY), may have been selected during the selection process of 267Y and 306TY. Several biological parameters were evaluated to determine if these two malathion resistance mechanisms impose a fitness cost for resistant genotypes. Individuals with genotype R/R have a reduced fitness in terms of developmental time from egg to pupa, pupal weight and adult longevity, when compared to susceptible individuals (genotype S/S). Interestingly, the fitness cost was substantially diminished in individuals with genotype RS/RS. These differences in fitness may be related to the reduction of the catalytic efficiency of mutated AChE in individuals R/R, and the compensatory effect that the non-mutated copy of the gene has on individuals RS/RS bearing the duplication. The stability of malathion reistance associated with the mutation G328A or the duplication was analyzed by following these resistant traits in the progeny of the back-crosses S/RS x RS/RS and S/R x R/R over consecutive generations in the absence of insecticide selection pressure. Our results show that the frequency of the allele bearing the mutation G328A decreased from 67.5% at the first generation of the back-cross S/R x R/R (75% expected, assuming Mendelian segregation and that there are only two alleles: one mutated and the other non-mutated) to 12% after 10 generations. By contrast, the frequency of the duplication only declined from 75% at the first generation of the back-cross S/RS x RS/RS (75% expected, assuming Mendelian segregation and that the duplication segregates as an unique allele) to 50% in the same period, indicating that the duplication is more stable than the mutation. The presence of the mutation and the duplication was analyzed in field populations collected in six localities in 2004-2007, when malathion was still used, and compared to populations collected in the same fields in 2010, one year after the prohibition of malathion in the European Union. The average frequency of the susceptible genotype (S/S) increased from 55.9% in the period 2004-2007 to 70.8% in 2010, whereas the frequency of those genotypes carrying the mutation in homozygosis or heterozygosis (R/R and S/R) declined from 30.4 to 9.2%, those carrying the duplication in homozygosis or heterozygosis (RS/RS and S/RS) increased slightly from 12.8 to 13.3%, and those carrying both the mutation and the duplication (R/RS) also increased from 1 to 6.7%. These results are consistent with the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A and the other copy non-mutated) being more advantageous than the G328A mutation alone by maintaining resistance while restoring part of the fitness. In order to investigate the association of malathion resistance with mutations previously found in the aliesterase CcE7, we have generated isolines bearing specific mutations selected by their putative location near the upper part of the active site gorge of the enzyme. The isoline Sm2 (originating from a female heterozygous for the mutation V96L and a male homozygous for the non-mutated allele) kept high levels of resistance to malathion, even after 30 generations without selection pressure. On the contrary, the isoline 267Y (composed by individuals homozygous for the mutation L267Y) and the strain 306TY (composed by homozygous for the double mutation R306T-N307Y) showed a significant reduction in the levels of resistance. We have found also that resistance to malathion in the Sm2 isoline was partially reverted by DEF and TPP, and that Sm2 showed a significant reduction in MTB activity, as reported for other species showing malathion-specific resistance mediated by aliesterases. Besides, it was possible to associate the presence of the mutation V96L in individuals from the Sm2 isoline with both survival to a discriminating concentration of malathion (5,000 ppm) and low MTB activity. Our results point out to a possible connection betwen the mutation V96L in the aliesterase CcE7 and resistance to malathion, though we can not yet conclude that the resistance is caused by the mutation, being needed further work to understand its contribution to resistance. In conclusion, resistance to lambda-cyhalothrin has been found for the first time in field populations of C. capitata, and metabolic resistance mediated by P450 appears to be the main resistance mechanism in the resistant strain W-1K. These findings add to the previously reported case of field resistance to malathion, associated to the G328A mutation and the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A) and to metabolic resistance mediated by esterases. Our results also indicate that altered target site AChE appears to be responsible for a certain level of resistance to malathion in C. capitata, that can be estimated as about 25-40-fold for the mutation G328A and 40-60-fold for the duplication; whereas metabolic resistance mediated by esterases and associated in this study with the mutation V96L in CcE7 may confer a multiplicative effect (by a factor of 5 to10) increasing malathion resistance to 200-400-fold. Moreover, we have shown that resistant insects carrying the duplication have better fitness and exhibit a higher stability than those with the mutation G328A in the absence of insecticide pressure. This represents a scenario where genotypes with the duplication will remain in the field at low to moderate frequencies, but could be rapidly selected if malathion or other insecticides showing cross-resistance are used. These findings have important implications for resistance management programs, as the repertoire of effective insecticides for C. capitata control is becoming very limited. Besides, multiple resistance mechanisms coexisting in field populations provide the potential to develop resistance to other available insecticides for the control of this pest. Appropriate resistance management strategies based on the alternation of insecticides with different modes of action, and their combination with other control methods, must then be implemented to avoid the evolution of resistance in the field.

Unconformity-bounded stratigraphic units (UBSUs) in an italian alluvial-plain area: recognizing and dating

We studied the coastal zone of the Tavoliere di Puglia plain, (Puglia region, southern Italy) with the aim to recognize the main unconformities, and therefore, the unconformity-bounded stratigraphic units (UBSUs; Salvador 1987, 1994) forming its Quaternary sedimentary fill. Recognizing unconformities is particularly problematic in an alluvial plain, due to the difficulties in distinguishing the unconformities that bound the UBSUs. So far, the recognition of UBSUs in buried successions has been made mostly by using seismic profiles. Instead, in our case, the unavailability of the latter has prompted us to address the problem by developing a methodological protocol consisting of the following steps: I) geological survey in the field; II) draft of a preliminary geological setting based on the field-survey results; III) dating of 102 samples coming from a large number of boreholes and some outcropping sections by means of the amino acid racemization (AAR) method applied to ostracod shells and 14C dating, filtering of the ages and the selection of valid ages; IV) correction of the preliminary geological setting in the light of the numerical ages; definition of the final geological setting with UBSUs; identification of a ‘‘hypothetical’’ or ‘‘attributed time range’’ (HTR or ATR) for each UBSU, the former very wide and subject to a subsequent modification, the latter definitive; V) cross-checking between the numerical ages and/or other characteristics of the sedimentary bodies and/or the sea-level curves (with their effects on the sedimentary processes) in order to restrict also the hypothetical time ranges in the attributed time ranges. The successful application of AAR geochronology to ostracod shells relies on the fact that the ability of ostracods to colonize almost all environments constitutes a tool for correlation, and also allow the inclusion in the same unit of coeval sediments that differ lithologically and paleoenvironmentally. The treatment of the numerical ages obtained using the AAR method required special attention. The first filtering step was made by the laboratory (rejection criteria a and b). Then, the second filtering step was made by testing in the field the remaining ages. Among these, in fact, we never compared an age with a single preceding and/or following age; instead, we identified homogeneous groups of numerical ages consistent with their reciprocal stratigraphic position. This operation led to the rejection of further numerical ages that deviate erratically from a larger, homogeneous age population which fits well with its stratigraphic position (rejection criterion c). After all of the filtering steps, the valid ages that remained were used for the subdivision of the sedimentary sequences into UBSUs together with the lithological and paleoenvironmental criteria. The numerical ages allowed us, in the first instance, to recognize all of the age gaps between two consecutive samples. Next, we identified the level, in the sedimentary thickness that is between these two samples, that may represent the most suitable UBSU boundary based on its lithology and/or the paleoenvironment. The recognized units are: I) Coppa Nevigata sands (NEA), HTR: MIS 20–14, ATR: MIS 17–16; II) Argille subappennine (ASP), HTR: MIS 15–11, ATR: MIS 15–13; III) Coppa Nevigata synthem (NVI), HTR: MIS 13–8, ATR: MIS 12–11; IV) Sabbie di Torre Quarto (STQ), HTR: MIS 13–9.1, ATR: MIS 11; V) Amendola subsynthem (MLM1), HTR: MIS 12–10, ATR: MIS 11; VI) Undifferentiated continental unit (UCI), HTR: MIS 11–6.2, ATR: MIS 9.3–7.1; VII) Foggia synthem (TGF), ATR: MIS 6; VIII) Masseria Finamondo synthem (TPF), ATR: Upper Pleistocene; IX) Carapelle and Cervaro streams synthem (RPL), subdivided into: IXa) Incoronata subsynthem (RPL1), HTR: MIS 6–3; ATR: MIS 5–3; IXb) Marane La Pidocchiosa–Castello subsynthem (RPL3), ATR: Holocene; X) Masseria Inacquata synthem (NAQ), ATR: Holocene. The possibility of recognizing and dating Quaternary units in an alluvial plain to the scale of a marine isotope stage constitutes a clear step forward compared with similar studies regarding other alluvial-plain areas, where Quaternary units were dated almost exclusively using their stratigraphic position. As a result, they were generically associated with a geological sub-epoch. Instead, our method allowed a higher detail in the timing of the sedimentary processes: for example, MIS 11 and MIS 5.5 deposits have been recognized and characterized for the first time in the study area, highlighting their importance as phases of sedimentation.

A model of the spatially dependent mechanical properties of the axon during its growth

Neuronal growth is a complex process involving many intra- and extracellular mechanisms which are collaborating conjointly to participate to the development of the nervous system. More particularly, the early neocortical development involves the creation of a multilayered structure constituted by neuronal growth (driven by axonal or dendritic guidance cues) as well as cell migration. The underlying mechanisms of such structural lamination not only implies important biochemical changes at the intracellular level through axonal microtubule (de)polymerization and growth cone advance, but also through the directly dependent stress/stretch coupling mechanisms driving them. Efforts have recently focused on modeling approaches aimed at accounting for the effect of mechanical tension or compression on the axonal growth and subsequent soma migration. However, the reciprocal influence of the biochemical structural evolution on the mechanical properties has been mostly disregarded. We thus propose a new model aimed at providing the spatially dependent mechanical properties of the axon during its growth. Our in-house finite difference solver Neurite is used to describe the guanosine triphosphate (GTP) transport through the axon, its dephosphorylation in guanosine diphosphate (GDP), and thus the microtubules polymerization. The model is calibrated against experimental results and the tensile and bending mechanical stiffnesses are ultimately inferred from the spatially dependent microtubule occupancy. Such additional information is believed to be of drastic relevance in the growth cone vicinity, where biomechanical mechanisms are driving axonal growth and pathfinding. More specifically, the confirmation of a lower stiffness in the distal axon ultimately participates in explaining the controversy associated to the tensile role of the growth cone.

Dynamic gamma frequency feedback coupling between higher and lower order visual cortices underlies perceptual completion in humans

To perceive a coherent environment, incomplete or overlapping visual forms must be integrated into meaningful coherent percepts, a process referred to as ?Gestalt? formation or perceptual completion. Increasing evidence suggests that this process engages oscillatory neuronal activity in a distributed neuronal assembly. A separate line of evidence suggests that Gestalt formation requires top-down feedback from higher order brain regions to early visual cortex. Here we combine magnetoencephalography (MEG) and effective connectivity analysis in the frequency domain to specifically address the effective coupling between sources of oscillatory brain activity during Gestalt formation. We demonstrate that perceptual completion of two-tone ?Mooney? faces induces increased gamma frequency band power (55?71 Hz) in human early visual, fusiform and parietal cortices. Within this distributed neuronal assembly fusiform and parietal gamma oscillators are coupled by forward and backward connectivity during Mooney face perception, indicating reciprocal influences of gamma activity between these higher order visual brain regions. Critically, gamma band oscillations in early visual cortex are modulated by top-down feedback connectivity from both fusiform and parietal cortices. Thus, we provide a mechanistic account of Gestalt perception in which gamma oscillations in feature sensitive and spatial attention-relevant brain regions reciprocally drive one another and convey global stimulus aspects to local processing units at low levels of the sensory hierarchy by top-down feedback. Our data therefore support the notion of inverse hierarchical processing within the visual system underlying awareness of coherent percepts.