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Com o objetivo de obter informações básicas sobre a bioecologia e o comportamento de Sphenophorus levis Vaurie, foram realizados diferentes bioensaios para conhecer seu comportamento sexual e reprodutivo; obter e testar a atividade biológica de seu feromônio natural em condições de laboratório e campo; verificar a eficiência de captura do feromônio sintético de agregação em condições de campo; determinar o tipo de armadilha mais eficiente para a captura e manejo de adultos; e avaliar o raio de ação das atuais armadilhas. Os resultados mostraram que S. levis, acasala-se em qualquer hora do dia, tem múltiplas cópulas, e os casais entre 14 e 35 dias de idade apresentaram as maiores frequências de cópulas, com 76 a 88%, respectivamente. A duração média da primeira cópula foi de 5,75 ± 4,22 h, e o tempo total de cópula variou entre 1 a 17 h. O período de pré-oviposição em fêmeas entre 14 e 35 dias, foi de 5,52 ± 4,53 a 10,90 ± 5,37 dias. O número de ovos/fêmea foi de 0,25 a 7,80, com viabilidade entre 33 e 63%. Fêmeas de S. levis com idade avançada entre 85 e 210 foram receptivas ao acasalamento (66%), ovipositaram entre 4,50 a 7,09 ovos, com viabilidade entre 49 e 79%. Machos e fêmeas de S. levis aptos ao acasalamento, apresentaram uma sequência típica de comportamentos para efetivar a cópula. A duração da pré-cópula variou de 2 a 5 horas. A cópula foi longa, com uma duração entre 7 a 13 horas. A pós-cópula durou entre 1,31 a 1,41 horas. O comportamento sexual associado à cópula foi influenciado pelo contato visual entre os casais e físico com outros indivíduos da espécie. Bioensaios em olfatômetro Y demonstraram que os machos foram estimulados por voláteis de cana; cana + machos; e cana + fêmeas; enquanto as fêmeas apenas por cana + machos. Os machos responderam ao extrato natural de macho + cana; as fêmeas ao extrato de machos + cana, extrato de fêmeas e extrato de fêmeas + cana. O isômero (S)-2-metil-4-octanol gerou atividade comportamental de ambos os sexos de S. levis. No campo, a avaliação da atratividade dos extratos naturais e os compostos sintéticos (isômeros S; R e mistura racêmica S-R), mostraram diferença na captura de fêmeas e totais de adultos de S. levis entre o isômero R e o controle; no entanto, não diferiram dos demais tratamentos. A captura de adultos selvagens de S. levis nas diferentes armadilhas testadas, diferiu entre o controle (isca CTC) e as armadilhas testadas (modelos galão, funil, e moleque da bananeira). A recaptura de adultos marcados de S. levis foi baixa e correspondeu a apenas 2% dos insetos liberados, sendo maior a distância de 5 m (10 adultos). De forma geral, estes resultados contribuem para o conhecimento do comportamento sexual e reprodutivo de S. levis e são úteis para futuros programas de manejo integrado desta praga. / With the aim of obtaining basic information on bioecology and behavior of Sphenophorus levis Vaurie, different bioassays were realized in order to understand its sexual and reproductive behavior; obtain and test the biological activity of its natural pheromone under laboratory and field conditions; verify the capture efficiency of the synthetic aggregation pheromone under field conditions; determine the most efficient type of traps for capture and management of the adults; and evaluate the radius of action of the current traps. The results showed that S. levis mates any time of the day, has multiple mating and the mates between the ages of 14 and 35 days presented the highest mating frequencies of 76 to 88%, respectively. The mean duration for the first mating was 5.75 ± 4.22 h, and the total mating time varied between 1 to 17 h. The pre-oviposition period for females between 14 and 35 days, was 5.52 ± 4.53 to 10.90 ± 5.37 days. The number of eggs/female was 0.25 to 7.80, with viability of between 33 and 63%. Females of S. levis with advanced age between 85 and 210 days were receptive to mating (66%), oviposited between 4.50 to 7.09 eggs with viability between 49 and 79%. Males and females of S. levis with capacity to mate presented typical frequency behavior to effect mating. The pre-mating duration varied from 2 to 5 hours. Mating was long with duration of 7 to 13 hours. The post-mating duration lasted between 1.31 to 1.41 hours. Sexual behavior associated with mating was influenced by visual contact between the couples and the physical among coespecifics. Bioassays in a Y tube olfatometer demonstrated that the males were responsive to sugarcane volatiles; sugarcane + males; and sugarcane + females; while the females were stimulated at least by sugarcane + males. The males responded to the natural extract of male + sugarcane; while the females to the extract of males + sugarcane, extract of females and extract of females + sugarcane. The isomer (S)-2- methil-4-octanol generated behavioral activity of both sexes of S. levis. In the field, the evaluation of attractivity of natural extracts and the synthetic compounds (isomer S; R and racemic mixture of S-R), showed differences in the capture of females and totals of adults of S. levis between the isomer R and the control; however, it was not different in other treatments. The capture of wild S. levis adults in the different tested traps differed between the control (bait CTC) and the tested baits/traps (models galão, funil, and moleque da bananeira). The recapture of marked adults of S. levis was low and corresponded to 2% of the liberated insects within the longest distance of 5 m (10 adults). In general, these results add to the knowledgment of S. levis sexual and reproductive behavior and are useful to future programs of integrated management of this pest.

The Quaternary climate has known dramatic global variations oscillating between long glacial and shorter interglacial periods of approximately 100 000 and 20 000 years, respectively with the last succession as an example. During the glacial episodes the continental ice sheet expansion and sea level drop, in turn, have locally disturbed the environment (at least in the northern hemisphere). These climatic and environmental disturbances caused changes in the geographic distributions of animal and plant species. For each species, the changes possibly took the form of demographic events like population extinction or fragmentation associated with genetic bottlenecks (loss in genetic diversity) and, inversely, population expansions sometimes with inherent founder effects (stochastic sampling of the source genetic diversity) and/or contact between diverged groups (secondary contact zone) resulting in genetic diversity gradients through the geographic range of the species. We therefore understand that the demographic history of a species can be reconstruct through the investigation of genetic signature(s) it possibly left and that are still observed in the genome of that species. For European taxa, phylogeographic studies taking advantage of these signatures have mainly focused their attention on temperate species; pieces of knowledge for species adapted to cold environments are too scarce although their response to climate change could not only simply follow an inverse tendency compare to temperate climate species.<p><p>As a whole, this thesis project intended to study the evolutionary history of four sister species of cold-adapted leaf beetles investigating their response to past global climatic changes. The four species share many traits (life cycle, dispersal capacity, morphology, feeding behavior.) but their geographic distributions differ, further calling for interest in the factors that shaped them. Furthermore, leaf beetles are specialist insect herbivores each feeding only on one or a few different plant species. This host plant specialization offers an additional dimension to the study of climatic change impacts to understand the evolution of the insect-host relationship. The study of this species complex thus also aimed at understanding processes like speciation possibly driven through diet specialization. The project connects three main axes briefly described here after.<p><p>The first axe of the project allowed us to gain sufficient knowledge of the four species sub-genus Goniomena (Chrysomelidae, Gonioctena). We have defined some important barriers separating each four species among them. The analysis of five independent molecular markers sequences obtained for many individuals sampled through the entire species ranges allowed us to characterize four genetically distinct groups corresponding to the four species, to precisely identify their host plant(s) and their geographic distribution. The second axe was realized on the same multi-locus dataset and aimed at exploiting an array of methods to reconstruct the demographic history of the four species; the methods consist of some commonly used and some promising ones used in synergy hoping to strengthen our interpretations on the species history. For this purpose, we combined the species distribution modeling (SDM) techniques to infer current and past geographic range for the leaf beetles and for their host(s) in order to generate the most realistic historical hypotheses. Subsequently, the different hypotheses were evaluated with two different complementary approaches, Approximate Bayesian Computation (ABC) and a spatial coalescence simulation-based method, allowing for outputs comparison. Finally, the third axe focus on the study of a commonly used program designed for demographic parameters inference (including divergence times, effective populations sizes, migration rates). We created non-empiric datasets obtained with three largely used simulation programs to investigate the inference performances of the program.<p><p>We hope this project will help to better understand the way species currently present in cold environments in Europe responded to climatic changes. It certainly demonstrated and allowed to isolate some specific character of such respons while suggesting certain common patterns. Our findings are rich and varied; the current distribution of genetic diversity in the four sister species of leaf beetles involves processes like introgression and hybridization, competition and invasion, allopatric and possibly sympatric speciation, dispersal limitation and response differentiation against climatic changes. In the light of our results, further investigations are encouraged; the mechanisms driving or underlying the different speciation settings, the host plant specialization, the niche differentiation and the hybridization in secondary contact zones are planned to be investigated with biological material and analytic resources we already own. Practically, we explored promising analyses procedures using the resources of our multilocus multispecies dataset. All along, we emphasize on the need to work with multispecies empiric datasets at least equivalent to the one in this project (number of molecular markers investigated and sample sizes) if the aim is to study the evolutionary history of a species. We also caution on certain methodological limitations that need to be considered to enlighten a study project from experimental design to result interpretation.<p>/Durant le Quaternaire, le climat de la Terre entière a été marqué par des oscillations importantes entre périodes glaciaires relativement longues et interglaciaires plus courtes d’environ 100 000 et 20 000 ans, respectivement, si l’on prend l’exemple de la dernière succession. Pendant les épisodes glaciaires, la calotte glaciaire continentale et la baisse du niveau des mers et des océans ont, à leur tour, affecté localement les conditions environnementales, au moins en ce qui concerne l’hémisphère nord. Ces perturbations climatiques et environnementales ont provoqué des changements dans la distribution géographique des espèces animales et végétales. Spécifiquement, ces changements ont pris la forme d’événements démographiques tels que l’extinction ou la fragmentation des populations associées à des goulots d’étranglements (réduction de la diversité génétique) ou, à l’inverse, des expansions de populations parfois accompagnées d’un effet fondateur (échantillonnage aléatoire à partir de la diversité génétique source) et/ou de la rencontre subséquente entre des groupes génétiquement différenciés (zone de contact secondaire) résultant en des gradients de diversité génétique à travers toute la distribution actuelle de l’espèce. On comprend dès lors que l’histoire démographique d’une espèce peut être reconstruite en étudiant les signatures qu’une telle histoire a pu laisser dans son génome. En ce qui concerne les taxa européens, les études phylogéographiques, qui utilisent ces signatures, se sont principalement intéressées aux espèces des régions tempérées; les connaissances acquises dans le domaine pour les espèces adaptées aux environnements plus froids sont plus rares bien que leur réponse envers les changements climatiques pourrait ne pas simplement avoir une tendance inverse par rapport à celle des espèces tempérées.<p><p>Dans son ensemble, l’objectif du présent projet de thèse est d’étudier l’histoire évolutive d’un groupe de quatre espèces sœurs de chrysomèles adaptées à un environnement froid dans le but de comprendre leur réponse face aux changements climatiques passés. Les quatre espèces partagent de nombreux traits en commun (cycle de vie, capacité de dispersion, morphologie générale) mais présentent des distributions géographiques qui se différencient incitant encore plus à s’intéresser aux facteurs qui ont pu les structurer. De plus, les chrysomèles sont des insectes herbivores très spécialisés, chaque espèce d’insecte ne se nourrissant que d’une ou quelques espèces de plantes bien précises. Cette spécialisation ajoute une dimension supplémentaire à l’étude de l’impact des changements climatiques pour comprendre l’évolution de la relation insecte-plante hôte. L’étude de ce complexe d’espèces avait donc également pour but de comprendre des processus tels que ceux de spéciation qui a pu être induite via une spécialisation alimentaire ou de séparation géographique suite à un changement des distributions. Le projet s’articule autour de trois axes décrits ci-dessous.<p><p>Le premier axe du projet a permis d’acquérir une connaissance plus fine des quatre espèces de chrysomèle appartenant au sous-genre Goniomena (Chrysomelidae, Gonioctena). Nous avons défini les barrières importantes séparant les quatre espèces. L’analyse des séquences d’ADN de cinq marqueurs moléculaires indépendants obtenues pour un grand nombre d’individus échantillonnés sur toute l’aire de répartition de chacune des quatre espèces nous a permis de définir quatre groupes génétiquement distincts, d’identifier précisément leur(s) espèce(s) de plante(s) hôte(s) ainsi que leur distribution géographique. Le second axe a été réalisé sur le même jeu de données multilocus et avait pour but d’exploiter un éventail de méthodes communément utilisées ainsi que d’autres prometteuses en étude phylogéographique dont on exploite la synergie afin de renforcer les interprétations concernant l’histoire de chaque espèce. Pour cela, nous avons combiné la modélisation des distributions passées et présentes des quatre espèces et de leur(s) plante(s) hôte(s) pour générer des hypothèses les plus réalistes possibles. Ensuite, les différentes hypothèses historiques ont été évaluées via deux approches différentes, une méthode d’approximation de calcul bayésien approché (Approximate Bayesian Computation, ABC) et une autre basée sur des simulations spatiales de coalescence, permettant d’ensuite comparer les résultats. Enfin, avec le troisième axe, nous nous sommes intéressés à un programme d’inférence de paramètres démographiques (temps de divergence, taille effective des populations, taux de migration) communément utilisé. Nous avons créés des jeux de données non empiriques, construits avec trois simulateurs également très répandus dans les analyses de génétique des populations, pour explorer les performances d’inférence du programme.<p><p>Nous espérons que ce projet va aider à mieux comprendre la façon dont les espèces adaptées à un climat froid en Europe répondent aux changements climatiques. Il a démontré et permis d’isoler des traits spécifiques dans ces réponses tout en suggérant néanmoins certains schémas communs. Nos résultats sont riches et variés; la distribution contemporaine de la diversité génétique chez les quatre espèces soeurs de chrysomèle implique des processus tels que l’introgression et l’hybridation, la compétition et l’invasion, la spéciation allopatrique et sympatrique, le potentiel de dispersion et la différentiation des réponses aux changements climatiques. Au vu de ces résultats, d’autres recherches sont envisagées; les mécanismes donnant lieu ou sous-jacents aux différents types de spéciation, à la spécialisation alimentaire, à la différentiation de niche et à l’hybridation dans les zones de contact secondaire seront explorés à l’aide de matériel biologique et de ressources analytiques déjà acquises. En pratique, nous avons réalisé notre étude en explorant des procédures d’analyses prometteuses tout en exploitant les ressources d’un jeu de données multi-locus et multi-espèces. Tout au long du projet, nous mettons l’accent sur la nécessité de travailler avec des jeux de données empiriques multi-espèces au moins équivalents à celui de ce projet (en termes de nombre de marqueurs, de taille d’échantillons) si on vise à réaliser une telle étude sur l’histoire évolutive d’une espèce. Nous mettons également en garde sur certaines limites méthodologiques qui doivent être considérées pour la conception d’un projet d’étude allant de la mise en œuvre expérimentale jusqu’à l’interprétation des résultats.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

All previous taxonomic studies on the Ectemnorhinus group of weevils have been based primarily on morphological data. While these studies are invaluable, some questions can only be addressed adequately through molecular studies. This is especially true when studying the genetic relationships and phylogeograpic patterns of taxa endemic to the South Indian Ocean Province (SIP) biotas that have long been controversial. The Ectemnorhinus group of genera is a monophyletic unit of weevils endemic to the region. The present study focused mainly on the Ectemnorhinus group of weevils found on the Prince Edward Islands archipelago (PEIA). The mitochondrial cytochrome oxidase I gene was targeted when investigating relationships among members of this weevil group. On the PEIA, it is important to note that Marion Island (MI) and Prince Edward Island (PEI) differ in terms of alien invasive species, such as the introduced house mouse Mus musculus and in conservation management strategies. Since emergence, a series of volcanic and glaciation events have occurred on Marion Island, whilst Prince Edward Island has remained largely unaffected by glaciation. Phylogenetic analyses revealed the presence of two genetically and morphometrically distinct species of Ectemnorhinus weevils on PEI, whilst evidence for a single species, comprising diverse genetically discrete populations was found on MI. Based on these results, the species unique to PEI has been designated E. kuscheli n. sp., whilst the present study confirmed the synonymy between E. similis and E. marioni, the two species originally described from MI. Ectemnorhinus kucheli appears to be restricted to PEI, whereas E. similis occurs on both MI and PEI. When investigating the population dynamics of the Ectemnorhinus weevils on the PEIA, the data indicated that PEI was the first of the two islands of the PEIA to be colonized by Ectemnorhinus weevils, at an estimated time of coalescence of approximately 0.3116 million years ago (MYA). The PEI population then acted as the source population for the colonization of MI by Ectemnorhinus weevils some time before the last glaciation, approximately 10 000 to 35 000 years ago. The separation by distance of the PEI Ectemnorhinus weevils from those on MI then gave rise to two species by allopatric speciation on MI. During the last glaciations, MI was extensively glaciated with only the southwestern corner of the island being free of ice. This extensive glaciation of MI would have resulted in the eradication of all E. similis on MI except for those occurring on the ice-free southwestern corner of the island. At the end of the last glacial maximum, when the ice started to melt, the coastal areas of MI emerged first from beneath the ice and were available for re-colonization by weevils. The movement of weevils that were isolated in the south-western corner of MI, along the coastal areas of the island, was assisted by strong, frequent south-western winds. Subsequent, post-glacial volcanism during the Holocene was then responsible for the fragmentation of the new migrants, resulting in small population pockets surrounded by fresh, uninhabitable lava and subsequent divergence of each populations. When the Holocene black lava became re-colonizeable, the weevils from the different isolated populations migrated to the remainder of the island. Currently, members of the different genetically-identified populations occur in sympatry and in some cases even on the same plant, but no noticeable geneflow was detected between them. It is thus suggested that the time of isolation, before the post-glacial black lava during Holocene became hospitable, was sufficiently long and the populations sufficiently small that a number of genetically-discrete populations arose. Consequently, the present study recognises two genetically discrete populations of E. kucheli on PEI and seven discrete E. similis populations on MI that are morphologically indistinct. When examining the relationships among 13 species from five different islands within the South Indian Ocean Province (SIP) that are representative of 22 populations within the genera Palirhoeus, Bothrometopus and Ectemnorhinus, there was little support for separating the genus Palirhoeus from Bothrometopus, and no support for the morphologically-delineated species groups currently recognized within Bothrometopus. The present study shows that colonization of the Prince Edward Islands is likely to have occurred repeatedly from other islands within the SIP and that Bothrometopus parvulus on the PEIA comprises two species that are not sister taxa. The second novel con-generic species was therefore designated Bothrometopus huntleyi n. sp. and examination of the genetically identified specimens resulted in the indentification of distinguishing morphological characteristics. The analyses indicated that B. huntleyi arose approximately 0.5 million years ago from a high-altitude population that is still present on MI. The first major intra- and inter-island dispersal event occurred ~0.338 MYA, coinciding with the glaciation-free second volcanic stage on MI. Apart from this early inter-island colonisation, only one other between-island dispersal event, corresponding with the glaciation-free seventh volcanic stage, was detected. Genetically discrete weevil complexes on each of the islands of the PEIA together with the low levels of inter-island gene flow reaffirm the need to control alien invasive mice, which are restricted to MI, and which prey on these weevil species. / Thesis (PhD)--University of Pretoria, 2012. / Zoology and Entomology / unrestricted

Los insectos saproxílicos, que dependen de la presencia de madera muerta y/o en descomposición, de hongos de la madera, o bien de la presencia de otros organismos saproxílicos, en algún momento de su ciclo vital, desempeñan una función clave para el correcto funcionamiento de los ecosistemas de bosque, actuando como recicladores de los nutrientes contenidos en la madera en descomposición. La diversidad de insectos saproxílicos, y más concretamente de coleópteros, así como la estructura de los ensambles que conforman, se encuentran condicionadas por una serie de variables de carácter físico, químico y biótico. Comprender cómo este amplio conjunto de variables afecta a los patrones espacio-temporales de las comunidades, resulta esencial a la hora de elaborar estrategias de manejo de los ecosistemas, que permitan preservar este tipo de fauna, en muchas ocasiones, amenazada. Con este marco de fondo, el objetivo general de esta tesis doctoral reside en analizar el conjunto de factores ambientales que afectan a la diversidad y a la distribución espacio-temporal de los ensambles de coleópteros saproxílicos que habitan los bosques mediterráneos de roble melojo ubicados en el oeste de la península ibérica para, de este modo, poder proponer una serie de directrices a seguir, a la hora de manejar este tipo de ecosistemas. Este objetivo general se divide en una serie de objetivos específicos, cada uno de los cuales ha derivado en la redacción de un artículo científico y que pueden ser consultados de manera individualizada en los capítulos II, III y IV. En el capítulo II se evaluó la influencia de un total de 24 variables ambientales a nivel de paisaje y a nivel de árbol, muchas de ellas consecuencia del manejo tradicional del bosque en el oeste ibérico por parte del hombre, analizando la riqueza observada de especies y empleando los estimadores de diversidad verdadera. El estudio permitió demostrar que, son muchas las variables que deben ser tenidas en cuenta a la hora de aplicar estrategias de manejo de los ecosistemas forestales y que no todos los métricos empleados para evaluar la diversidad de los ensambles saproxílicos responden de la misma manera frente a esas variables derivadas del manejo. De acuerdo con los resultados obtenidos, es posible establecer una serie de recomendaciones para la gestión forestal de áreas sometidas a técnicas tradicionales de manejo: favorecer en cierta medida la apertura del medio, reducir la carga ganadera y la explotación de madera a nivel vecinal, mantener en el paisaje un elevado volumen de madera en avanzado estado de descomposición, realizar un trasmoche selectivo de cierta cantidad de árboles para acelerar la aparición de oquedades y conservar los denominados “árboles hábitat”, que presenten microhábitats potenciales para este tipo de fauna, ya sean oquedades o tumores. El capítulo III se centró en analizar la posible influencia de los factores abióticos de temperatura y humedad relativa, en los patrones intranuales mostrados por los ensambles de coleópteros saproxílicos que habitan los bosques de roble melojo en la península ibérica, tanto en términos de riqueza como de composición de especies. Además, se abordó el estudio del tamaño corporal, como un rasgo que puede afectar tanto a la fisiología como a la eficacia biológica (fitness) de las especies y que podría explicar, al menos en parte, la dinámica temporal de estos ensambles. Este estudio permitió demostrar que la dinámica temporal de los ensambles de coleópteros saproxílicos que habitan los bosques de roble en el oeste ibérico está condicionada, al menos en gran medida, por las fluctuaciones anuales de las variables abióticas de temperatura y humedad relativa. Considerando el marco actual de calentamiento global, el hecho de conocer cómo ambas variables abióticas afectan a la estructura y diversidad de estos ensambles, permitirá predecir algunas de las consecuencias que el cambio climático puede tener en la distribución temporal de estas comunidades faunísticas. Además, se observó que no todas las especies de coleópteros saproxílicos muestran la misma plasticidad a la hora de adaptarse a esas fluctuaciones de temperatura y que muchas de ellas pueden permanecer activas únicamente bajo un cierto rango térmico. En este sentido, se recomienda profundizar en el estudio de los rangos térmicos de las especies, ya que proporcionará información muy valiosa a la hora de tratar de entender los patrones temporales mostrados por estos ensambles. Finalmente, en el capítulo IV, se estudió la influencia que pueden tener las emisiones de compuestos orgánicos volátiles (COVs) por parte del roble melojo, en los ensambles de coleópteros saproxílicos que emplean esta especie arbórea como planta hospedadora. Para ello, se partió de la premisa de que los árboles añosos (generalmente de mayor tamaño y en un estado de descomposición avanzado) son más visitados por los insectos saproxílicos, que en el caso de los árboles más jóvenes (generalmente de menor porte y en un estado de descomposición nulo o poco avanzado); las emisiones de COVs podrían explicar, al menos en parte, ciertos aspectos de la etología de este grupo de insectos. A pesar de la ausencia de significación estadística, los resultados mostraron una tendencia clara, que indicó que los árboles de mayor porte (árboles añosos, en un estado de descomposición avanzado) emiten este tipo de compuestos con una mayor intensidad que los árboles de menor tamaño. Si tenemos en cuenta, además, que este estudio demostró que los árboles añosos, de mayor perímetro (generalmente en un estado de senescencia más avanzado) albergaron una mayor riqueza de especies de coleópteros saproxílicos, se llega a la conclusión de que la emisión de COVs debe ser tenida en cuenta como una variable más (dentro del amplio conjunto de las ya comentadas en apartados previos), que puede estar condicionando la dinámica espacio-temporal de los ensambles de coleópteros saproxílicos que habitan los bosques de roble melojo en la península ibérica. Este estudio abre una novedosa vía de investigación que permitirá profundizar en el estudio de la relación directa que puede existir entre los compuestos químicos emitidos por los árboles y la fauna saproxílica circundante.

Digestion of proteins in the midgut of lepidopteran larvae relies on different types of peptidases, among the trypsins and chymotrypsins. In this work four chymotrypsinlike peptidases (MsCTLP1–4) were identified from the larval midgut of M. sexta, which are distantly related to another chymotrypsin (MsCT), a previously described peptidase present in the larval midgut of M. sexta. MsCTLP1–4 fit perfectly into a novel subgroup of insect CTLPs by sequence similarity and by the replacement of GP by SA in the highly conserved GDSGGP motif. Examination of MsCTLP expression in different tissues showed that most of the peptidases were predominantly expressed in the anterior and median midgut, while some were found in the Malpighian tubules. Expression analysis of MsCTLPs at different physiological states revealed that the mRNA amounts did not differ considerably in feeding and starving larvae except for MsCTLP2, whose mRNA dropped significantly upon starvation. During molting, however, the mRNA amounts of all MsCTLPs dropped significantly. Immunological determination of MsCTLP1 amounts showed that the mature peptidase was only detectable in the gut lumen of feeding and re-fed larvae, but not in that of starving or molting larvae, suggesting that MsCTLP1 secretion is suspended during starvation or molt. Differential regulation of transcript levels as well as their partial expression in Malpighian tubules might point to a role, which is distinct from digestion for at least some MsCTLPs. In line with this assumption, MsCTLP1 was shown to interact with the chitin synthase 2 (MsCHS2), necessary for chitin synthesis in the course of peritrophic matrix formation in the midgut of M. sexta. The occurrence of this interaction in vivo is supported by colocalization and co-immunoprecipitation. The data suggest that chitin synthesis is controlled by an intestinal proteolytic signaling cascade linking chitin synthase activity to the nutritional state of the larvae. As MsCTLP1 appears to be involved in such signaling cascades, other midgut peptidases could have other targets and may therefore regulate different activities. To gain more insight into the functions of CTLPs, the gene family encoding these peptidases in the genome of the red flour beetle, T. castaneum, was analyzed. Using an extended search pattern, 14 TcCTLP genes were identified that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. Analysis of the expression patterns of seven TcCTLP genes at various developmental stages revealed that some TcCTLP genes were exclusively expressed in feeding larval and adult stages (TcCTLP-5A/B, TcCTLP-6A). Others were also detected in non-feeding embryonic (TcCTLP-5C, TcCTLP-6D) and pupal stages (TcCTLP-5C, TcCTLP- 6C/D/E). TcCTLP genes were expressed predominantly in the midgut where they presumably function in digestion. However, TcCTLP-5C and TcCTLP-6C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, western blots were performed using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6C suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, systemic RNAi experiments were performed. While injections of dsRNAs to TcCTLP-5A/B and TcCTLP-6A/D/E into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects. Recombinant expressed TcCTLP-5C2 was moreover activated by trypsin and was able to hydrolyze AAPF, hence making TcCTLP-5C the first described chymotrypsin-like peptidase ever to be involved in molting.

Algunas plagas causan daños importantes en productos y granos almacenados, lo cual conlleva consecuentemente a pérdidas de producción y calidad en estos productos.Las principales plagas del arroz almacenado en España son, Sitophilus oryzae L. (Coleoptera Curculionidae), Rhyzopertha dominica Fabricius (Coleoptera: Bostrichidae), y Cryptolestes pusillus Schönherr (Coleoptera: Cucujidae). Las dos primeras son plagas primarias que atacan directamente el grano y resultan bastante destructivas debido a que sus larvas se alimentan y desarrollan dentro de él. C.pusillus es, sin embargo, una plaga secundaria que se beneficia de granos que ya están dañados y rotos.Actualmente, el uso de fumigantes e insecticidas de síntesis sigue siendo el principal método de lucha para controlar las plagas de almacén, si bien recientemente (dadas las continuas restricciones al uso de agroquímicos) existe un gran interés en la utilización de otras alternativas tales como el control biológico, el almacenamiento a bajas temperaturas, o los tratamientos con calor entre otros.Igualmente muchos productos obtenidos principalmente de plantas y que derivan del metabolismo secundario de las mismas ofrecen una fuente de bioinsecticidas que podrían representar una alternativa ecológica frente a los insecticidas de síntesis ya que su uso masivo e indiscriminado ha ocasionado problemas tales como la aparición de resistencias en determinadas especies de insectos frente a diferentes materias activas, desequilibrios ecológicos y problemas medioambientales sin olvidar el riesgo que entrañan para la salud humana.Con este trabajo se ha pretendido estudiar la actividad plaguicida de los aceites esenciales extraídos de tres plantas: Coriandrum sativum L. (Umbelliferae), Carum carvii L. (Umbelliferae) y Ocimum basilicum L. (Labiatae) y su posterior fraccionamiento para identificar dentro de estos aceites los compuestos químicos responsables de esta actividad insecticida sobre tres plagas de almacén de arroz (S. oryzae, R.dominica y C.pusillus).Del estudio de estos monoterpenoides, linalol, S-carvona y estragol resultaron tener una alta actividad insecticida sobre estas plagas. Sin embargo el E-anetol fue más selectivo para R.dominica y C.pusillus, así como el limoneno, &#947;-terpineno, geraniol y eucaliptol sólo resultaron activos frente a C.pusillus.Algunos monoterpenoides podrían actuar de sinergistas potenciando la actividad de otros, como podría ser el caso del alcanfor, acetato de geranilo y E-anetol con linalol en R.dominica y C.pusillus, o el caso del metoxicinamaldehido, p-anisaldehido y linalol que pueden tener efecto sinergista sobre el estragol.Este trabajo también abarcó el estudio de un posible modo de acción de estos monoterpenoides, la inhibición de la acetilcolinesterasa, para alcanzar un mayor entendimiento del comportamiento de estas sustancias en el interior del insecto.Así se pudo observar como la mayoría de monoterpenoides estudiados inhibían en cierta medida esta enzima, siendo fenchona, S-carvona y linalol los monoterpenoides que mayor inhibición originaron.Del mismo modo se observó como fenchona, &#947;-terpineno, geraniol y linalol inhibían competitivamente la acetilcolinesterasa, mientras que S-carvona, estragol y alcanfor producían una inhibición mixta para esta enzima.Sin embargo no se observó inhibición de la acetilcolinesterasa por parte del E-anetol a las concentraciones de monoterpenoides ensayadas.Para completar este trabajo se examinó de igual forma la capacidad que tenían estos bioinsecticidas de generar resistencia en estas tres plagas así como el mecanismo de resistencia implicado en el desarrollo de este proceso. Para ello se fueron seleccionando las poblaciones de insectos mediante la aplicación de los diferentes monoterpenoides a dosis crecientes durante 7 generaciones. De este modo se pudo calcular el factor de resistencia en cada una de las plagas y para cada uno de los monoterpenoides comparando las concentraciones letales 50 de las poblaciones seleccionadas con las poblaciones iniciales (sensibles).A continuación se analizaron tres posibles sistemas de detoxificación enzimáticos gracias al uso de sinergistas para estudiar el mecanismo de resistencia que podría estar involucrado.De tal forma se observó como estos monoterpenoides inducían lentamente resistencias resultando ventajosos en un futuro para el control de estas plagas.Esta Tesis ha englobado un estudio íntegro y profundo de estos insecticidas ecológicos desde la extracción de los aceites esenciales, seguido de la identificación de compuestos puros (CG-EM) con actividad insecticida junto con el estudio de un modo de acción de estos insecticidas, la selección de resistencia en las poblaciones de insectos y los posibles mecanismos de resistencia que pudieran estar implicados en este proceso. / Some pests cause serious damage to stored grains and stored products and consequently production and quality losses in these products.The rice weevil, Sitophilus oryzae L. (Coleoptera: Curculionidae), the lesser grain borer, Rhyzopertha dominica Fabricius (Coleoptera: Bostrichidae) and Cryptolestes pusillus Schönherr (Coleoptera: Cucujidae) were the main damaging pests found in stored rice in Spain.S.oryzae and R.dominica are primary pests attacking directly the intact grain and are quite destructive because their larvi feed and develop inside the grain whereas C.pusillus is a secondary pest which benefits from grains previously damaged.At the present time, organic synthetic pesticides are still the main method to control stored grain pests, however, recently (due to restriction in agrochemicals use) there is a great interest in using other altenatives such as biological control, storage at low-temperatures, or heat treatment.Likewise, many products obtained mainly from plants and derived from secondary metabolism have insecticidal activity against insects, such as monoterpenoids, which present a broad variety of bioinsecticide products which could turn out to be an ecologic alternative to synthetic pesticides since the majority of alternative products are not harmful for the human healthy and they become less environmentally damaging, exhibiting a low impact on the environment.In addition, it cannot be ignored the different difficulties related to resistance due to several active compounds from organic pesticides found in some species of insects.With this work, we have considered remarkable to study the insecticide activity of essential oils extracted from three plants: Coriandrum sativum L. (Umbelliferae), Carum carvii L. (Umbelliferae) and Ocimum basilicum L. (Labiatae) and carry out a bioassay-guided fractionation of their essential oils to identify which compounds were responsible for the volatile toxicity shown on three stored rice pests (S. oryzae, R. dominica and C. pusillus).Linalool, S-carvone and estragole turned out to have a high insecticide activity on these pests. Nevertheless E-anetol was more selective to R. dominica and C. pusillus, being only active on C. pusillus limonene, &#947;-terpinene, geraniol and eucalyptol.Some monoterpenoids were found as synergists, increasing the activity of the other ones, like for instance, camphor, geranyl acetate and E-anethole with linalool in R.dominica and C.pusillus or metoxycinnamaldehyde, p-anysaldehyde and linalool which could activate to estragole.In this work, the inhibition of acetylcholinesterase as a posible mode of action was studied as well, to reach a clear understanding about the action of these products inside the insects.The majority of monoterpenoids inhibited the enzyme acetylcholinesterase being fenchone, S-carvone and linalool the monoterpenoids that produced a higher inhibition.Furthermore, it was observed how fenchone, &#947;-terpinene, geraniol and linalool showed a competitive inhibition whereas S-carvone, estragole and camphor produced a mixed inhibition for this enzyme. However the enzyme acetylcholinesterase was not inhibited by E-anethole.To finish up this work, the selection for monoterpenoid resistance on these pests as well as the metabolic mechanisms implicated was studied.The resistant strains were selected from susceptible insect populations and survivors were reared separately for each monoterpenoid and successive generations were treated with higher concentrations. These populations were selected until seven times.As a result we could calculate the resistance factor on each pest (comparing lethal concentration 50 values of susceptible and resistant strains).Next, three enzymatic systems detoxifying these monoterpenoids were analysed to study the metabolic mechanism implicated.In this way we could realize that all monoterpenoids induced resistance slowly, concluding that these pesticides will be appropriated to control these pests in the future.This Thesis has concerned a study in depth about ecological insecticides from extraction of essential oils, identification of compounds (GC-MS) with insecticide activity, mode of action and study of resistance and mechanism of insecticide resistance involved in this process.

Pour des raisons patrimoniales autant que pour les services qu'elle rend à l'humanité, il apparaît nécessaire de soutenir la diversité des organismes vivants et, pour cela, de connaître leur utilisation des milieux dans les paysages ruraux. En particulier, des travaux récents soutiennent un modèle de colonisation cyclique qui énonce qu'une partie des espèces circulant dans les milieux cultivés en été trouvent un abri propice à l'hivernation dans les milieux semi-naturels. Notre but était ici de mettre au jour les facteurs locaux et paysagers déterminant la répartition spatiale et la structure des assemblages de Carabidae dans un paysage rural des coteaux de Gascogne. Les patrons de répartition des Carabidae, obtenus à deux échelles spatiales différentes, celle du paysage et celle de la parcelle, et à deux moments clés de la vie de ces insectes, la période durant laquelle ils sont actifs (circulants) et la période d'hivernation, ont été interprétés en termes de processus écologiques grâce à la prise en compte des traits biologiques et écologiques des espèces de Carabidae. Les résultats montrent que le type de milieu a un effet structurant majeur sur les assemblages de Carabidae circulants comme hivernants, comparable aux effets des conditions environnementales locales et paysagères réunies. Par ailleurs, les effets de lisière sur les Carabidae circulants, au niveau des interfaces entre les bois et les cultures, sont limités à quelques mètres ou quelques dizaines de mètres. Concernant les Carabidae hivernants, nous avons noté de manière surprenante que leur densité était de deux à six fois plus élevée dans les marges (cultivées) des cultures que dans les milieux semi-naturels. De plus, nous n'avons noté aucun contraste de répartition spatiale entre les Carabidae hivernants et les Carabidae circulants, quels que soient les espèces ou les groupes fonctionnels considérés. Ceci indique donc que les marges des cultures sont des milieux d’hivernation très importants dans le contexte considéré et que si un mouvement de colonisation cyclique existe pour les Carabidae des cultures, il doit se dérouler entre leur zone intérieure et leur marge. L'ensemble de nos résultats soutient l'idée que si l'agencement spatial des milieux seminaturels dans le paysage est important pour promouvoir les populations de Carabidae auxiliaires dans les cultures, la gestion des cultures elles-mêmes, et notamment de leurs marges, est également de première importance. / Supporting biodiversity is an ethical as well as a practical issue since it provides numerous ecosystem services. In that purpose, it is necessary to determine how organisms use the different habitats in rural landscapes. In this view, recent studies suggest that a number of arthropods cyclically colonise cultures, where they feed and reproduce, and semi-natural areas, where they overwinter. We aimed to determine the local and landscape determinants of spatial distribution and structure of ground beetle (Coleoptera: Carabidae) assemblages in a rural landscape of south-western France. We studied spatial distribution patterns of ground beetles at landscape and habitat scales, and at two key periods of their life, that when they are active, and winter. We then considered biological and ecological traits to deduce from these spatial patterns ecological processes affecting ground beetles. Our results show that habitat type has a major effect on species assemblages of active (circulating) as well as overwintering ground beetles, equivalent to that of local conditions and landscape context combined. Edge effects on circulating ground beetles at the woodlot-filed interface were limited to a few meters or a few dozen of meters in both habitats. Amazingly, we found that the density of overwintering ground beetles was two-to six-fold higher in crop margins than in semi-natural habitats. Moreover, we failed to show any difference between the spatial distribution of overwintering ground beetles and circulating ground beetles, whatever the species or functional group considered. Crop margins are thus major overwintering sites in the context studied and cyclic colonisation of ground beetles in crops, if real, may take place between the inner zone and the margins of crops. These results suggest that, in addition to spatial arrangement of semi-natural habitats, crop management, and in particular management of crop margins, is important to promote ground beetles and their beneficial effects on crop protection in agro-forested landscapes.

Interações planta-animal, tais como polinização, são a chave de processos ecológicos in muitas comunidades terrestres. O estudo de quem interage com quem é uma importante ferramenta para se entender os processos ecológicos e evolucionários. Em algumas comunidades tropicais, mais de um quarto de todas as espécies de planta pode ser polinizado por besouros. Eles são um grupo de inseto muito antigo e diversificado e eles interagem com angiospermas desde o período de suas origens e princípio da diversificação. Adicionalmente, a interação entre besouros e recursos florais provém singular oportunidade para se avaliar a complexidade de interações e a possibilidade de generalização como a tendência para plantas para usar uma enorme proporção da fauna de besouros visitantes como polinizadores, ou especialização com plantas usando uma proporção relativamente pequena da fauna disponível de visitantes como polinizadores. A distribuição espacial de espécies vegetais tem sido considerada um importante componente na determinação de interações planta-animal, sendo esperado que muitos padrões observados nestes sistemas resultem de variações na distribuição de recursos vegetais. A disponibilidade de recursos florais apresenta variações espaço-temporais que podem influenciar a eficiência dos polinizadores do ponto de vista quantitativo, pela freqüência de ocorrência da interação, ou qualitativo pela contribuição dada ao sucesso reprodutivo das espécies. Os principais objetivos da presente tese foram: 1) descrever a história natural e interação flores besouros em espécies de Cerrado em relação à heterogeneidade espaço-temporal da distribuição de recursos entre fitofisionomias localizadas na Estação Ecológica de Itirapina (EEI) e em área de Cerradão pertencente ao Instituto Arruda Botelho (22º12- 22º10S e 47º55- 47º57W, respectivamente) durante dois anos consecutivos; 2) verificar o padrão local de distribuição de flores e besouros em quatro fitofisionomias de cerrado estudadas; 3) caracterizar a nível de comunidade os padrões de interações observados na comunidade composta por besouros associados a flores; 4) finalmente, compreender detalhadamente um dos diversos sistemas de interação besouros-plantas registrados na área de estudo. Escolhemos o sistema de interação de D. furfuracea-besouros com o objetivo de descrever sua fenologia de floração e frutificação e seu sistema reprodutivo, verificando se há limitação polínica e de recursos. Também foi avaliada a função e o efeito dos visitantes florais sobre o sistema reprodutivo. Uma alta proporção de espécies de plantas tem suas flores visitadas por besouros em todas quatro fitofisionomias de Cerrado estudadas (12-40%), indicando que os dados disponíveis até o momento subestimam a ocorrência de cantarofilia para áreas de Cerrado. Este consiste no primeiro estudo focando toda uma comunidade de besouros associados a flores. Não houve variação temporal entre anos tanto para as redes de visitantes-flores ou para redes de polinizadores-plantas. Redes de polinizadores-plantas tiveram espécies de besouros altamente especialistas. Attalea geraensis¸ C. pubescens, D. furfuracea>, D. hispida, K. coriacea, S. petrea, T. formosa e X. aromatica consistem em espécies de planta envolvidas em muitas interações e foram consideradas espécies centrais. Tanto as redes de visitantes-flores, como a rede de polinizadores-plantas evidenciaram uma estrutura composta combinando estrutura de rede aninhada com compartimentada, mas com predomínio do padrão compartimentado. Estes compartimentos são resultantes tanto das muitas interações espécie-específica entre espécies de besouros e plantas, como daquelas espécies de planta que interagem com várias espécies especialistas de besouros. De modo geral, focando atenção nos besouros, ambas as redes, visitantes-flores e polinizadores-plantas foram definidas como altamente especializadas já que visitaram flores poucas espécies de planta tanto para comparações entre anos, como entre fitofisionomias. O presente estudo mostrou que o Cerrado apresenta sistemas de interação entre besouros e flores de espécies de planta com alta especificidade. Dentre as várias espécies de planta compreendidas nestas redes de interação de alta especificidade, merece desta D. furfuracea que apresenta uma fauna composta por várias espécies de besouros polinizadores altamente especialistas. Duguetia furfuracea é uma espécie auto-incompatível, cuja população estudada apresentou limitação polínica e de recursos no solo. Há duas guildas especializadas de besouros interagindo com esta espécie de planta. A primeira é composta por uma única espécie de curculionídeo (Plasilia sp.) que visitou suas flores em baixa abundância (média de 0,55 besouros por flor), mas com alta freqüência de ocorrência (44,9% das flores) e suas larvas consomem as sementes dos frutos que se desenvolvem. A segunda guilda é composta por várias espécies de besouros nitidulídeos, principalmente Colopterus sp.3 que visitaram as flores em grande abundância (média de 99 besouros por flor) e também com alta ocorrência de visitação (92% das flores) e que efetivamente promoveram a polinização. O sistema de polinização de D. furfuracea consiste no primeiro caso de polinização mutualística obrigatória envolvendo diferentes guildas de visitantes florais. Este também é o primeiro sistema de polinização combinando polinizadores previsíveis e confiáveis, consumo de sementes, limitação polínica e limitação por recursos, o que resulta em um complexo e eficiente mecanismo para regulação da população do visitante floral consumidor de sementes e para otimizar o sucesso reprodutivo da planta. / Plant-animal interactions, such as pollination, are a key element in many terrestrial communities. The study of who interacts with whom is an important approach for understanding ecological and evolutionary processes. In some tropical communities, up to one quarter of all plant species may be pollinated by beetles. They are an ancient and much diversified insect group and they interact with angiosperms since the time of their origin and early diversification. Additionally, the interaction between beetle and floral resource provide unique opportunity to evaluate the complexity of interactions and the possibility of generalization as the tendency for plants to use a large proportion of the available beetle-visiting fauna as pollinators, or specialization with plants using a relatively small proportion of the available beetle visiting fauna as pollinators. The spatial distributions of plant species have been considered an important component in the determination of plant-animal interactions, and it is expected that many patterns observed in these systems resulting from variations in the resource distribution of plants. The availability of floral resources presents spatio-temporal variations that may affect the pollinator efficiency in its quantitative traits through frequency of occurrence of the interaction, or qualitative by its contribution to the fitness plant. Thus, the main objectives of this present thesis were: 1) describe the natural history and interactions of beetle with flowers in Cerrado species focusing the spatio-temporal heterogeneity of the resource distribution among phytophysiognomies located in the Itirapina Ecological Station and in the Cerradão area belongs to the Arruda Botelho Institute (22º12- 22º10S and 47º55- 47º57W, respectively) during two consecutive years.; 2) verify the local pattern of flowers and beetle distribution in the four cerrado phytophysiognomies studied; 3) characterize in the community level the interaction patterns observed in the community composed by beetles associated to flowers; 4) finally, comprehend deeply one of the several beetle-flower systems recorded to this studied area. We chose the D. furfuracea-beetle interaction system with aim to describe its flowering and fruit phenology and its reproductive biology verifying if there are pollen and resource limitation. Also was evaluated the role and effect of floral visitors in the fitness plant. A high number of flowering species was visited by beetles (12-40%) indicating that the data available up to now underestimate the representation of this interaction in Cerrado areas. This work represents the first study focusing in the entire beetle community associated to flowers. Was found no temporal variation in the interaction between beetles and flowers. Pollinator-plant webs had beetle species highly specialist. A. geraensis¸ C. pubescens, D. furfuracea, D. hispida, K. coriacea, S. petrea, T. formosa and X. aromatica , involved in many interactions were considered as core species. Both the visitor-flower and the pollinator-plant webs showed a compound structures mixing nested and compartmented networks structure, but predominating the compartmented pattern. These compartments are resultant from both of the many species-specific interactions between beetle and plant species and of the plant species that interact with several specialist beetle species. Both visitor-flower and pollinator-plant webs may be denominated as highly specialist from the beetle perspective whereas that these beetle fauna visited flowers of very few plant taxa over time and space. The present study showed that the Cerrado presents interaction systems between beetles and plants species with high specificity. Among the several plant species comprised in these interaction webs with high specificity, D. furfuracea presents a very interesting pollinator system, presenting a fauna composed by several pollinator beetle species highly specialized. D. furfuracea is a self-incompatible species, which studied population presented pollen and resource limitation. There are two specialized beetle guilds interacting with this plant species. The first guild is composed just by one curculionid species (Plasilia sp.) that visited flowers with low abundance (median of 0.55 beetles per flower), but presenting high frequency of occurrence (44.9% of the flowers) and their larvae consumed seed of that fruits that developed. The second guild is composed by several nitidulid beetles, principally Colopterus sp.3 that visited flowers in large abundance (median of 99 beetles per flower) and also presented high visitation occurrence (92% of flowers) and that effectively promoted the pollination. The pollination system of D. furfuracea consists is the first case of obligate pollination mutualism case involving different guilds of floral visitors. This also constitutes the first pollination system combining predictable and reliable pollinators, seed consumption, pollen and resource limitation resulting in a complex and efficient mechanism to regulate the seed consuming by floral visitor`s population and to optimize the plant fitness

No description available.