Incipient species formation in salamanders of the Ensatina complex

The Ensatina eschscholtzii complex of plethodontid salamanders, a well-known “ring species,” is thought to illustrate stages in the speciation process. Early research, based on morphology and coloration, has been extended by the incorporation of studies of protein variation and mitochondrial DNA sequences. The new data show that the complex includes a number of geographically and genetically distinct components that are at or near the species level. The complex is old and apparently has undergone instances of range contraction, isolation, differentiation, and then expansion and secondary contact. While the hypothesis that speciation is retarded by gene flow around the ring is not supported by molecular data, the general biogeographical hypothesis is supported. There is evidence of a north to south range expansion along two axes, with secondary contact and completion of the ring in southern California. Current research targets regions once thought to show primary intergradation, but which molecular markers reveal to be zones of secondary contact. Here emphasis is on the subspecies E. e. xanthoptica, which is involved in four distinct secondary contacts in central California. There is evidence of renewed genetic interactions upon recontact, with greater genetic differentiation within xanthoptica than between it and some of the interacting populations. The complex presents a full array of intermediate conditions between well-marked species and geographically variable populations. Geographically differentiated segments represent a diversity of depths of time of isolation and admixture, reflecting the complicated geomorphological history of California. Ensatina illustrates the continuing difficulty in making taxonomic assignments in complexes studied during species formation.

Suppression of O-Methyltransferase Gene by Homologous Sense Transgene in Quaking Aspen Causes Red-Brown Wood Phenotypes1

Homologous sense suppression of a gene encoding lignin pathway caffeic acid O-methyltransferase (CAOMT) in the xylem of quaking aspen (Populus tremuloides Michx.) resulted in transgenic plants exhibiting novel phenotypes with either mottled or complete red-brown coloration in their woody stems. These phenotypes appeared in all independent transgenic lines regenerated with a sense CAOMT construct but were absent from all plants produced with antisense CAOMT. The CAOMT sense transgene expression was undetectable, and the endogenous CAOMT transcript levels and enzyme activity were reduced in the xylem of some transgenic lines. In contrast, the sense transgene conferred overexpression of CAOMT and significant CAOMT activity in all of the transgenic plants' leaves and sclerenchyma, where normally the expression of the endogenous CAOMT gene is negligible. Thus, our results support the notion that the occurrence of sense cosuppression depends on the degree of sequence homology and endogene expression. Furthermore, the suppression of CAOMT in the xylem resulted in the incorporation of a higher amount of coniferyl aldehyde residues into the lignin in the wood of the sense plants. Characterization of the lignins isolated from these transgenic plants revealed that a high amount of coniferyl aldehyde is the origin of the red-brown coloration—a phenotype correlated with CAOMT-deficient maize (Zea mays L.) brown-midrib mutants.

Female mating preference for bold males in the guppy, Poecilia reticulata.

Although females prefer to mate with brightly colored males in numerous species, the benefits accruing to such females are virtually unknown. According to one hypothesis of sexual selection theory, if the expression of costly preferred traits in males (such as conspicuous colors) is proportional to the male's overall quality or reveals his quality, a well-developed trait should indicate good condition and/or viability for example. A female choosing such a male would therefore stand to gain direct or indirect fitness benefits, or both. Among potential phenotypic indicators of an individual's quality are the amount and brightness of its carotenoid-based colors and its boldness, as measured by its willingness to risk approaching predators without being killed. Here, we show experimentally that in the Trinidadian guppy (Poecilia reticulata) the visual conspicuousness of the color pattern of males correlates positively with boldness toward, and with escape distance from, a cichlid fish predator. Bold individuals are thus more informed about nearby predators and more likely to survive encounters with them. Mate-choice experiments showed that females prefer colorful males as mates, but prefer bolder males irrespective of their coloration when given the opportunity to observe their behavior toward a potential fish predator. By preferentially mating with colorful males, female guppies are thus choosing on average, relatively bold, and perhaps more viable, individuals. In doing so, and to the extent that viability is heritable, they potentially gain indirect fitness benefits by producing more viable offspring than otherwise.

Innovative analytical strategies for the development of sensor devices and mass spectrometry methods

Il lavoro presentato in questa tesi di Dottorato è incentrato sullo sviluppo di strategie analitiche innovative basate sulla sensoristica e su tecniche di spettrometria di massa in ambito biologico e della sicurezza alimentare. Il primo capitolo tratta lo studio di aspetti metodologici ed applicativi di procedure sensoristiche per l’identificazione e la determinazione di biomarkers associati alla malattia celiaca. In tale ambito, sono stati sviluppati due immunosensori, uno a trasduzione piezoelettrica e uno a trasduzione amperometrica, per la rivelazione di anticorpi anti-transglutaminasi tissutale associati a questa malattia. L’innovazione di questi dispositivi riguarda l’immobilizzazione dell’enzima tTG nella conformazione aperta (Open-tTG), che è stato dimostrato essere quella principalmente coinvolta nella patogenesi. Sulla base dei risultati ottenuti, entrambi i sistemi sviluppati si sono dimostrati una valida alternativa ai test di screening attualmente in uso per la diagnosi della celiachia. Rimanendo sempre nel contesto della malattia celiaca, ulteriore ricerca oggetto di questa tesi di Dottorato, ha riguardato lo sviluppo di metodi affidabili per il controllo di prodotti “gluten-free”. Il secondo capitolo tratta lo sviluppo di un metodo di spettrometria di massa e di un immunosensore competitivo per la rivelazione di prolammine in alimenti “gluten-free”. E’ stato sviluppato un metodo LC-ESI-MS/MS basato su un’analisi target con modalità di acquisizione del segnale selected reaction monitoring per l’identificazione di glutine in diversi cereali potenzialmente tossici per i celiaci. Inoltre ci si è focalizzati su un immunosensore competitivo per la rivelazione di gliadina, come metodo di screening rapido di farine. Entrambi i sistemi sono stati ottimizzati impiegando miscele di farina di riso addizionata di gliadina, avenine, ordeine e secaline nel caso del sistema LC-MS/MS e con sola gliadina nel caso del sensore. Infine i sistemi analitici sono stati validati analizzando sia materie prime (farine) che alimenti (biscotti, pasta, pane, etc.). L’approccio sviluppato in spettrometria di massa apre la strada alla possibilità di sviluppare un test di screening multiplo per la valutazione della sicurezza di prodotti dichiarati “gluten-free”, mentre ulteriori studi dovranno essere svolti per ricercare condizioni di estrazione compatibili con l’immunosaggio competitivo, per ora applicabile solo all’analisi di farine estratte con etanolo. Terzo capitolo di questa tesi riguarda lo sviluppo di nuovi metodi per la rivelazione di HPV, Chlamydia e Gonorrhoeae in fluidi biologici. Si è scelto un substrato costituito da strips di carta in quanto possono costituire una valida piattaforma di rivelazione, offrendo vantaggi grazie al basso costo, alla possibilità di generare dispositivi portatili e di poter visualizzare il risultato visivamente senza la necessità di strumentazioni. La metodologia sviluppata è molto semplice, non prevede l’uso di strumentazione complessa e si basa sull’uso della isothermal rolling-circle amplification per l’amplificazione del target. Inoltre, di fondamentale importanza, è l’utilizzo di nanoparticelle colorate che, essendo state funzionalizzate con una sequenza di DNA complementare al target amplificato derivante dalla RCA, ne permettono la rivelazione a occhio nudo mediante l’uso di filtri di carta. Queste strips sono state testate su campioni reali permettendo una discriminazione tra campioni positivi e negativi in tempi rapidi (10-15 minuti), aprendo una nuova via verso nuovi test altamente competitivi con quelli attualmente sul mercato.

Preparação e caracterização de nanopartículas de prata para aplicação no desenvolvimento de imunoensaio para imunoglobulina G humana

Neste trabalho, anticorpos anti-IgGh foram conjugados às nanopartículas de prata (NPAg) para detectar imunoglobulina G humana (IgGh). Um imunoensaio colorimétrico baseado na diminuição da agregação devido ao aumento da repulsão eletrostática após a interação ligante-alvo. A agregação é induzida pela variação da força iônica e uma mudança da coloração da suspensão coloidal de amarelo para vermelho pode ser observada. Na presença de IgGh, a agregação é inibida e a coloração da suspensão coloidal não se altera. As nanopartículas foram obtidas por meio de cinco procedimentos diferentes e caracterizadas por espectroscopia UV-Vis, espalhamento dinâmico de luz, difração de raios-X e microscopia eletrônica. Glicose e borohidreto de sódio foram utilizados como agentes redutores, enquanto CTAB e β-ciclodextrina foram utilizados como estabilizantes. Citrato de sódio foi utilizado como agente redutor e/ou estabilizante. Nanoesferas de carbono foram obtidas por tratamento hidrotérmico de uma solução aquosa de glicose e também foram utilizadas no preparo das nanopartículas. As nanopartículas foram funcionalizadas com ácido mercaptossuccínico e a conjugação ocorreu devido à interação entre grupos aminas e grupos carboxílicos ionizados, presentes no anticorpo e agente de acoplamento, respectivamente. A estabilidade dos conjugados e o efeito da adição de IgGh foram avaliados para todos os sistemas preparados. As nanopartículas de prata preparadas com borohidreto de sódio e citrato de sódio foram selecionadas para serem aplicadas no desenvolvimento do imunoensaio e as condições experimentais foram avaliadas. Em condições ótimas, observou-se uma correlação linear entre a diminuição da agregação do sistema (NPAg-anti-IgGh) e a concentração de IgGh (0 a 200 ng mL-1). O limite de detecção foi estimado em 25 ng mL-1. O método colorimétrico apresentou boa seletividade para a detecção de IgGh. Além disso, foi obtido um resultado satisfatório ao aplicar o método para determinação do fator IX de coagulação. Foi desenvolvido também um método para determinação de ATP baseado na agregação de nanopartículas de ouro. Aptâmeros foram utilizados como elemento de reconhecimento. Em princípio, o método pode ser aplicável à determinação de outros analitos, por meio da substituição do aptâmero utilizado neste trabalho pelo oligonucleotídeo específico para o alvo de interesse.

Estudo das táticas reprodutivas em duas espécies de Coenagrionidae (Odonata: Zygoptera)

Nos estudos sobre a teoria da Seleção Sexual, as libélulas têm sido amplamente estudadas devido à grande variedade de padrões comportamentais, de coloração e táticas reprodutivas. Como forma de demonstrar táticas reprodutivas adotadas por duas espécies de libélulas, esta dissertação teve como objetivos principais: i) investigar o papel de traços secundários como a coloração corporal na competição intra-sexual de uma espécie territorial e ii) analisar se os traços corporais como tamanho e morfologia das asas predizem a tática de acasalamento adotada por machos de uma espécie nãoterritorial. Sugere-se que a coloração corporal pode predizer o resultado de lutas e também se correlacionar positivamente com a condição física dos machos territoriais de Tigriagrion aurantinigrum. Ademais, traços corporais como o tamanho e a morfologia das asas influenciam na tática reprodutiva utilizada por machos não-territoriais de Epipleoneura williamsoni. Portanto, com os resultados obtidos, conclui-se que a variação nos sinais visuais exerce um papel essencial na comunicação animal e na resolução de conflitos, indicando ainda que pode haver uma sinalização da condição física dos machos. Além disso, os resultados mostraram evidências em como diferentes táticas reprodutivas se relacionam com traços corporais como agilidade e tamanho corporal, os quais podem influenciar no sucesso reprodutivo dos indivíduos.

Potentiostatic Reversible Photoelectrochromism: An Effect Appearing in Nanoporous TiO2/Ni(OH)2 Thin Films

In the field of energy saving, finding composite materials with the ability of coloring upon both illumination and change of the applied electrode potential keeps on being an important goal. In this context, chemical bath deposition of Ni(OH)2 into nanoporous TiO2 thin films supported on conducting glass leads to electrodes showing both conventional electrochromic behavior (from colorless to dark brown and vice versa) together with photochromism at constant applied potential. The latter phenomenon, reported here for the first time, is characterized by fast and reversible coloration upon UV illumination. The bleaching kinetics shows first order behavior with respect to the NiIII centers in the film, and an order 1.2 with respect to electrons in the TiO2 film. From a more applied point of view, this study opens up the possibility of having two-mode smart windows showing not only conventional electrochromism but also reversible darkening upon illumination.

Further analyses on Micronesian banana, taro, breadfruit and other foods for provitamin A carotenoids and minerals

Few Micronesian foods have been analyzed for nutrient content. Information is needed on locally grown, culturally acceptable foods that could be promoted to alleviate, vitamin A deficiency in the Federated States of Micronesia. Using an ethnographic approach that included key informant interviews and observation, Micronesian cultivars with potential for high-carotenoid content according to their coloration were identified. These cultivars of banana, giant swamp taro, breadfruit and other foods were analyzed for alpha- and beta-carotene using high-performance liquid chromatography (HPLC) and for nine minerals using inductively coupled plasma (ICP). A wide range of provitamin A carotenoid levels was found in banana, taro, and breadfruit cultivars, some containing very high levels (beta-carotene content from 515 to 6360 mug/100 g in banana, 260 to 1651 mug/100 g in taro, and 295 to 868 mug/100 g in breadfruit, edible portion). Other cultivars contained moderate levels, but as they can be eaten in large quantities, they may contribute significantly to vitamin A status. The taro samples contained very high levels of zinc (mean 5.9 mg/100 g) and significant levels of other minerals (mean content of calcium was 120 mg/100 g). These staples with cultural acceptability and high availability potentially could play a role in vitamin A, micronutrient, and chronic disease programs in the Pacific. (C) 2003 Elsevier Science Ltd. All rights reserved.

Provitamin A carotenoid content of different cultivars of edible pandanus fruit

As part of an overall study to identify vitamin A-rich foods, a study was carried out in the Federated States of Micronesia (FSM) to provide information on production, acquisition, consumption and cultural acceptability of edible pandanus cultivars, Pandanus teetorius, and to identify their carotenoid content. Samples of five pandanus cultivars were collected and analyzed for alpha- and beta-carotene by HPLC. The results showed that the two cultivars with yellow fruit coloration contained low levels of carotenoids, while the orange fruits, which were also well liked as a food in the community, contained higher levels at maxima of 190 mug/100 g and 393 mug/100 g for alpha- and beta-carotene, respectively. Common patterns of intake when the fruit is available show that pandanus can provide a large proportion of estimated requirements of retinol equivalents. Local people were generally unaware that pandanus had health benefits, although the food was very popular. Nevertheless, key informants report that production had greatly decreased in recent years. To reverse this trend, those acceptable cultivars high in carotenoid content should be promoted both for their general enjoyment and their health benefits. (C) 2003 Elsevier Science Ltd. All rights reserved.

Micronesian banana, taro, and other foods: newly recognized sources of provitamin A and other carotenoids

Vitamin A deficiency (VAD) and chronic diseases are serious problems in the Federated States of Micronesia and other Pacific island countries. Nutrition education programs to address these in Micronesia have had limited success, partly due to lack of information on nutrient content in local foods. The study objective was to identify local plant foods rich in provitamin A and other carotenoids that have high levels of cultural acceptability. Food cultivars likely to be carotenoid-rich (suggested by coloration) were identified using an ethnographic approach including key informant interviews. Raw and cooked samples (mostly cultivars previously not analyzed) of 12 banana, 13 giant swamp taro, 10 breadfruit cultivars and four other local foods were analyzed by high-performance liquid chromatography. Many banana and taro cultivars were found with significant levels of beta- and alpha-carotene; the beta-carotene levels ranged from 30 to 2780 mug/100 g (banana) and. 50 to 2040 mug/100 g (taro). The results highlight the potential significance of cultivar differences in human nutrition, important for evaluation of the diet, establishment of locally relevant dietary guidelines, and research on the relationship between diet, health, and disease. These highly acceptable food cultivars could play an important role in VAD and chronic disease prevention programs in the Pacific. (C) 2003 Elsevier Science Ltd. All rights reserved.

Chic chicks: the evolution of chick ornamentation in rails

Competition over access to food has led to the evolution of a variety of exaggerated visual and vocal displays in altricial nestling birds. Precocial chicks that are fed by their parents also vary widely in appearance ranging from those with inconspicuous coloration to those with brightly colored bills, fleshy parts, and plumes. These ornaments are lost by the end of the period of parental dependence, suggesting they function in competition over parental care. We use a comparative approach to evaluate which ecological or life-history variables may have favored the evolution of conspicuous ornamentation in precocial chicks. We compiled data on chick morphology, ecology, and social organization of species in the Family Rallidae, a group with highly variable downy chicks. Chick ornamentation in the form of brightly colored bills, fleshy patches, or plumes is observed in 36 of 97 species for which downy chicks are described. Phylogenetic reconstructions suggest that nonornamentation is the ancestral state. Chick ornamentation has evolved multiple times within the Rallidae and is significantly associated with large clutch sizes and polygamous mating systems. Chick ornamentation was also weakly associated with adult ornamentation and adult dimorphism. We argue that these results support the hypothesis that lineages with higher levels of sibling competition are more likely to evolve ornamented chicks.

Experience overrides colour in lizard contests

We examined the role of conspicuous coloration in male-male contests for two species of Australian dragon lizards, Ctenophortis decresii and C. vadnappa, in which conspicuous coloration has a demonstrated predation cost. We conducted contests in which the overall conspicuousness of male coloration was manipulated using paints that matched the spectral reflectance of the lizards, as well as natural (control) contests. There was little evidence for an influence of colour on contest outcome or aggression levels for either species when all experiments were considered. However, we found a significant effect of trial order and experience on contest outcome and aggression levels (the same pair of males was used for both types of contest), despite a 2-3 week interval between contests. When we examined only the first trial between unfamiliar males, we found that male C. vadnappa that had been painted to appear more conspicuous consistently won. Comparison with the natural trials suggests that the aspect of colour manipulation that was responsible for this result was the 'hue' of the throat: males with yellower throats consistently beat males with bluer throats in both natural and painted trials. The difference in coloration of flank markings also predicted the difference in aggression scores between contestants in the natural trials. These results suggest that although colour is important in opponent assessment and in determining contest outcome in C vadnappa, previous agonistic experience can override the effects of colour and have a long-lasting influence on aggressive behaviour.

Can non-directional male mating preferences facilitate honest female ornamentation?

Recent studies have demonstrated male mate choice for female ornaments in species without sex-role reversal. Despite these empirical findings, little is known about the adaptive dynamics of female signalling, in particular the evolution of male mating preferences. The evolution of traits that signal mate quality is more complex in females than in males because females usually provide the bulk of resources for the developing offspring. Here, we investigate the evolution of male mating preferences using a mathematical model which: (i) specifically accounts for the fact that females must trade-off resources invested in ornaments with reproduction; and (ii) allows male mating preferences to evolve a non-directional shape. The optimal adaptive strategy for males is to develop stabilizing mating preferences for female display traits to avoid females that either invests too many or too few resources in ornamentation. However, the evolutionary stability of this prediction is dependent upon the level of error made by females when allocating resources to either signal or fecundity.

Estimating nutrient intake in comparative studies of animals: an example using dietary carotenoid content in birds

Many different methods of reporting animal diets have been used in ecological research. These vary greatly in level of accuracy and precision and therefore complicate attempts to measure and compare diets, and quantitites of nutrients in those diets, across a wide range of taxa. For most birds, the carotenoid content of the diet has not been directly measured. Here, therefore, I use an avian example to show how different methods of measuring the quantities of various foods in the diet affect the relative rankings of higher taxa (families, subfamilies, and tribes), and species within these taxa, with regard to the carotenoid contents of their diets. This is a timely example, as much recent avian literature has focused on the way dietary carotenoids may be traded off among aspects of survival, fitness and signalling. I assessed the mean dietary carotenoid contents of representatives of thirty higher taxa of birds using four different carotenoid intake indices varying in precision, including trophic levels, a coarse-scale and a fine-scale categorical index, and quantitative estimates of dietary carotenoids. This last method was used as the benchmark. For comparisons among taxa, all but the trophic level index were significantly correlated with each other. However, for comparisons of species within taxa, the fine-scale index outperformed the coarse-scale index, which in turn outperformed the trophic level index. In addition, each method has advantages and disadvantages, as well as underlying assumptions that must be considered. Examination and comparison of several possible methods of diet assessment appears to highlight these so that the best possible index is used given available data, and it is recommended that such a step be taken prior to the inclusion of estimated nutrient intake in any statistical analysis. Although applied to avian carotenoids here, this method could readily be applied to other taxa and types of nutrients.

Are corals colorful?

Using in situ spectrometry data and visual system modeling, we investigate whether the colors conferred to the reef-building corals by GFP-like proteins would look colorful not only to humans, but also to fish occupying different ecological niches on the reef. Some GFP-like proteins, most notably fluorescent greens and nonfluorescent chromoproteins, indeed generate intense color signals. An unexpected finding was that fluorescent proteins might also make corals appear less colorful to fish, counterbalancing the effect of absorption by the photosynthetic pigments of the endosymbiotic algae, which might be a form of protection against herbivores. We conclude that GFP-determined coloration of corals may be an important factor in visual ecology of the reef fishes.