Parametric analyses of anxiety in zebrafish scototaxis

Scototaxis, the preference for dark environments in detriment of bright ones, is an index of anxiety in zebrafish. In this work, we analyzed avoidance of the white compartment by analysis of the spatiotemporal pattern of exploratory behavior (time spent in the white compartment of the apparatus and shuttle frequency between compartments) and swimming ethogram (thigmotaxis, freezing and burst swimming in the white compartment) in four experiments. In Experiment 1, we demonstrate that spatiotemporal measures of white avoidance and locomotion do not habituate during a single 15-min session. In Experiments 2 and 3, we demonstrate that locomotor activity habituates to repeated exposures to the apparatus, regardless of whether inter-trial interval is 15-min or 24-h; however, no habituation of white avoidance was observed in either experiment. In Experiment 4, we confined animals for three 15-min sessions in the white compartment prior to recording spatiotemporal and ethogram measures in a standard preference test. After these forced exposures, white avoidance and locomotor activity showed no differences in relation to non-confined animals, but burst swimming, thigmotaxis and freezing in the white compartment were all decreased. These results suggest that neither avoidance of the white compartment nor approach to the black compartment account for the behavior of zebrafish in the scototaxis test. (C) 2010 Elsevier B.V. All rights reserved.

Genetic covariance between indices of body condition and immunocompetence in a passerine bird

Background: Condition-dependence is a ubiquitous feature of animal life histories and has important implications for both natural and sexual selection. Mate choice, for instance, is typically based on condition-dependent signals. Theory predicts that one reason why condition-dependent signals may be special is that they allow females to scan for genes that confer high parasite resistance. Such explanations require a genetic link between immunocompetence and body condition, but existing evidence is limited to phenotypic associations. It remains unknown, therefore, whether females selecting males with good body condition simply obtain a healthy mate, or if they acquire genes for their offspring that confer high immunocompetence. Results: Here we use a cross-foster experimental design to partition the phenotypic covariance in indices of body condition and immunocompetence into genetic, maternal and environmental effects in a passerine bird, the zebra finch Taeniopygia guttata. We show that there is significant positive additive genetic covariance between an index of body condition and an index of cell-mediated immune response. In this case, genetic variance in the index of immune response explained 56% of the additive genetic variance in the index of body condition. Conclusion: Our results suggest that, in the context of sexual selection, females that assess males on the basis of condition-dependent signals may gain genes that confer high immunocompetence for their offspring. More generally, a genetic correlation between indices of body condition and imuunocompetence supports the hypothesis that parasite resistance may be an important target of natural selection. Additional work is now required to test whether genetic covariance exists among other aspects of both condition and immunocompetence.

Conspecific and heterospecific alarm substance induces behavioral responses in piau fish Leporinus piau

In ostariophysan fish, the detection of alarm substance released from the skin of a conspecific or a sympatric heterospecific may elicit alarm reactions or antipredator behavioral responses. In this study, experiments were performed to characterize and quantify the behavioral response threshold of Leporinus piau, both individually and in schools, to growing dilutions of conspecific (CAS) and heterospecific skin extract (HAS). The predominant behavioral response to CAS stock stimulation was biphasic for fish held individually, with a brief initial period of rapid swimming followed by a longer period of immobility or reduced swimming activity. As the dilution of skin extract was increased, the occurrence and magnitude of the biphasic alarm response tended to decrease, replaced by a slowing of locomotion. Slowing was the most common antipredator behavior, observed in 62.5% of animals submitted to HAS stimulation. School cohesion, measured as proximity of fish to the center of the school, and swimming activity near the water surface significantly increased after exposure to CAS when compared with the control group exposed to distilled water. Histological analysis of the epidermis revealed the presence of Ostariophysi-like club cells. The presence of these cells and the behavioral responses to conspecific and heterospecific skin extract stimulation suggest the existence of a pheromone alarm system in L. piau similar to that in Ostariophysi, lending further support for the neural processing of chemosensory information in tropical freshwater fish.

The visual ecology of avian photoreceptors

The spectral sensitivities of avian retinal photoreceptors are examined with respect to microspectrophotometric measurements of single cells, spectrophotometric measurements of extracted or in vitro regenerated visual pigments, and molecular genetic analyses of visual pigment opsin protein sequences. Bird species from diverse orders are compared in relation to their evolution, their habitats and the multiplicity of visual tasks they must perform. Birds have five different types of visual pigment and seven different types of photo receptor-rods, double (uneven twin) cones and four types of single cone. The spectral locations of the wavelengths of maximum absorbance (lambda (max)) of the different visual pigments, and the spectral transmittance characteristics of the intraocular spectral filters (cone oil droplets) that also determine photoreceptor spectral sensitivity, vary according to both habitat and phylogenetic relatedness. The primary influence on avian retinal design appears to be the range of wavelengths available for vision, regardless of whether that range is determined by the spectral distribution of the natural illumination or the spectral transmittance of the ocular media (cornea, aqueous humour, lens, vitreous humour). Nevertheless, other variations in spectral sensitivity exist that reflect the variability and complexity of avian visual ecology. (C) 2001 Elsevier Science Ltd. All rights reserved.

Phylogenetic revision of Agapophytus Guerin (Diptera : Therevidae : Agapophytinae)

Morphologically diverse and species-rich, the endemic Australasian genus Agapophytus is revised. Eleven previously described species are redescribed and twenty-nine species are described for the first time: A. adonis, sp. nov., A. annamariae, sp. nov., A. antheliogynaion, sp. nov., A. asprolepidotos, sp. nov., A. atrilaticlavius, sp. nov., A. biluteus, sp. nov., A. borealis, sp. nov., A. caliginosus, sp. nov., A. cerrusus, sp. nov., A. chaetohypopion, sp. nov., A. chrysosisyrus, sp. nov., A. decorus, sp. nov., A. dieides, sp. nov., A. discolor, sp. nov., A. eli, sp. nov., A. fenestratum, sp. nov., A. galbicaudus, sp. nov., A. labifenestellus, sp. nov., A. laparoceles, sp. nov., A. lissohoplon, sp. nov., A. lyneborgi, sp. nov., A. notozophos, sp. nov., A. novaeguineae, sp. nov., A. pallidicrus, sp. nov., A. palmulus, sp. nov., A. paramonovi, sp. nov., A. septentrionalis, sp. nov., A. yeatesi, sp. nov. and A. zebra, sp. nov. All 40 species of Agapophytus were compared in a cladistic analysis with three species of Acupalpa Krober using 134 states across 58 adult morphological characters. The analysis resulted in 36 most parsimonious trees with a length of 240 steps. The phylogenetic relationships of the species of Agapophytus are discussed with three main clades recognised: A. dioctriaeformis clade, A. australasiae clade and A. queenslandi clade.

Sex allocation in the sexually monomorphic fairy martin

Offspring sex ratios were examined at the population and family level in the sexually monomorphic, socially monogamous fairy martin Petrochelidon ariel at five colony sites over a 4-year period (1993 1996). The sex of 465 nestlings from 169 broods % as determined using sex-specific PCR at the CHD locus. In accordance with predicted sex allocation patterns, population sex ratios at hatching and fledging did not differ from parity in an), year and the variance in brood sex ratios did not deviate from the binomial distribution, Further, brood sex ratio did not vary with hatching date during the season, brood number, brood size or colony size, The sex ratio or broods with extra-pair young did not differ from those without, while the sex ratio of broods fathered by males that gained extra-pair fertilizations did not differ from broods fathered by other males. Extra-pair chicks were as likely to be male as female. Neither the total number of feeding visits to the brood nor the relative feeding contribution by the sexes varied significantly with brood sex ratio. Brood sex ratios were also unrelated to paternal size, condition and breeding experience or maternal condition and breeding experience, However, contrary to our prediction, brood sex ratio was negatively correlated with maternal size. Generally, these results were consistent with our expectations that brood sex ratios would not vary with environmental factors or parental characteristics, and would not influence the level of parental provisioning. However, the finding that females with longer tarsi produced an excess of daughters is difficult to reconcile with our current understanding or fairy martin life history and breeding ecology.

Ultraviolet signals in birds are special

Recent behavioural experiments have shown that birds use ultraviolet (UV)-reflective and fluorescent plumage as cues in mate choice. It remains controversial, however, whether such UV signals play a special role in sexual communication, or whether they are part of general plumage coloration. We use a comparative approach to test for a general association between sexual signalling and either UV-reflective or fluorescent plumage. Among the species surveyed, 72% have UV colours and there is a significant positive association between UV reflectance and courtship displays. Among parrots (Psittaciformes), 68% of surveyed species have fluorescent plumage, and again there is a strong positive association between courtship displays and fluorescence. These associations are not artefacts of the plumage used in courtship displays, being generally more 'colourful' because there is no association between display and colours lacking UV reflectance or fluorescence. Equally, these associations are not phylogenetic artefacts because all results remain unchanged when families or genera, rather than species, are used as independent data points. We also find that, in parrots, fluorescent plumage is usually found adjacent to UV-reflective plumage. Using a simple visual model to examine one parrot, the budgerigar Melopsittacus undulatus, we show that the juxtaposition of UV-reflective and fluorescent plumage leads to a 25-fold increase in chromatic contrast to the budgerigar's visual system. Taken together, these results suggest that signals based on UV contrast are of special importance in the context of active sexual displays. We review briefly six hypotheses on why this may be the case: suitability for short-range signalling; high contrast with backgrounds; invisibility to predators; exploitation of pre-existing sensory biases; advertisement of feather structure; and amplification of behavioural signals.

Contribuição para o estudo dos efeitos de descargas orgânicas em peixes

O principal objectivo deste trabalho foi avaliar os efeitos resultantes de diferentes tipos de descargas orgânicas, em frentes de descarga e instantâneas, na espécie Danio rerio, um peixe de água doce geralmente utilizado para avaliações de toxicidade. Para a simulação das descargas, utilizou-se um efluente sintético, que reproduz, aproximadamente, a composição de uma água residual doméstica, e um canal hidráulico laboratorial. Determinou-se a CL50, através de testes semi-estáticos, sob condições laboratoriais controladas, ao longo de 96 horas, tendo-se obtido o valor de 309 mg O2.L-1 (CQO). Para além de ensaios de dispersão de um corante no canal hidráulico, para melhor percepção do avanço de uma descarga, efectuaram-se ensaios simulando frentes de descarga e descargas instantâneas. Estes ensaios foram realizados com o efluente sintético, em diferentes condições, observando-se a evolução de concentrações no canal e relacionando-as com o comportamento do peixe referido na presença do agente poluente. Os resultados permitem concluir, através do comportamento observado dos peixes, que a intensidade de resposta, por afastamento ao contacto com a descarga orgânica, varia directamente com a concentração desta no meio, tendo sido possível obter curvas aproximadas para descrição daquela resposta. Assim, tendo-se constatado que para baixas concentrações a resposta é pouco acentuada, pode inferir-se do risco que lhe poderá estar associado, levando os peixes a permanecerem em situações de contaminação potencial com eventuais efeitos acumulativos.

Implementation of a zebrafish health program in a research facility : A 4-year retrospective study

In the past two decades, zebrafish (Danio rerio)-based research has contributed to significant scientific advances. Still, husbandry and health programs did not evolve at the same pace, as evidenced by the absence of general guidelines. Health monitoring is essential to animal welfare, to permit animal exchanges across facilities, to contribute to robust experimental results, and for data reproducibility. In this study, we report a health program implemented in a zebrafish research facility to prevent, monitor, and control pathogen, and disease dissemination. This program includes quarantine, routine health screening of sentinels, and nonroutine screenings of retired animals and sick/moribund individuals. An extensive list of clinical signs, lesions, and pathogens was monitored based on: daily observation of fish, necropsy, histology, and bacterial culture. The results indicate that the combined analysis of sentinels with the evaluation of sick/moribund animals enables a comprehensive description not only of pathogen prevalence but also of clinical and histopathologic lesions of resident animals. The establishment of a quarantine program revealed to be effective in the reduction of Pseudoloma neurophilia frequency in the main aquaria room. Finally, characterization of the colony health status based on this multiapproach program shows a low prevalence of lesions and pathogens in the facility.

Usefulness of zebrafish model to assess glomerular and tubular alterations induced by tenofovir

Tenofovir (TFV) is one of the most used antiretroviral drugs. However, it is associated with tubular damage with mitochondria as a possible target. Tubulopathy precedes glomerular dysfunction, thus classic markers of renal function like the glomerular filtration rate (GFR) do not detect early TFV damage. Prediction and management of drug induced renal injury (DIRI) rely on the mechanisms of the drug insult and in optimal animal models to explore it. Zebrafish (Danio rerio) offers unique advantages for assessing DIRI, since the pronephros is structurally very similar to its human counterpart and is fully developed at 3.5 days postfertilization. The main aim of the present work was to evaluate the effects of TFV, as well as its pro-drug, tenofovir disoproxil fumarate (TDF), on the GFR and in mitochondria morphology in tubular cells of zebrafish larvae. Lethality curves were performed to understand the relationship between drug concentration and lethality. LC10 was selected to explore the renal function using the FITC-inulin assay and to analyze the mitochondrial toxicity by electron microscopy on larvae exposed to TDF, TFV, paracetamol and gentamicin (positive controls) or water (negative control). Lethality curves showed that gentamicin was the most lethal drug, followed by TDF, TFV and paracetamol. Gentamicin and paracetamol decreased the GFR, but no differences were found for either TDF or TFV, when compared to controls (%FITC Control = 33±8; %FITC TDF = 35±10; %FITC TFV = 30±10; %FITC Gentamicin = 46±17; %FITC Paracetamol = 83±14). Tubular mitochondria from treated larvae were notably different from non-treated larvae, showing swelling, irregular shapes, decreased mitochondria network, cristae disruption and loss of matrix granules. These results are in agreement with the effects of these drugs in humans and thus, demonstrate that zebrafish larvae can be a good model to assess the functional and structural damage associated with DIRI.

The role of arl13b in cilia length regulation and in zebrafish development

RESUMO: Arl13b é uma importante proteína ciliar, presente em cílios primários e cílios móveis. Ratinhos mutantes para Arl13b têm comprimento dos cílios reduzido e defeitos nos B-túbulos dos cílios. Como consequência destes fenótipos, deficiências na Arl13b originam, em modelos animais, várias doenças congénitas, incluindo problemas no estabelecimento do eixo esquerda-direita, malformações cerebrais e deformações corporais. Nos seres humanos, deficiências na Arl13b levam a uma doença crónica congénita chamada Síndrome de Joubert. Por outro lado, a sobreexpressão de Arl13b origina cílios mais longos, no entanto existe uma ausência da caracterização dos fenótipos celulares e durante o desenvolvimento embrionário. Neste trabalho, quisemos explorar o efeito da sobre-expressão de Arl13b em embriões de peixezebra. Descobrimos que, ao nível ciliar, a sobre-expressão de Arl13b nas células aumenta o comprimento ciliar em cílios primários e móveis, no entanto, a esses cílios falta adequada acetilação da alfa-tubulina no citoesqueleto feito por microtúbulos. Os nossos resultados mostraram que esse efeito é específico de Arl13b sobre-expressão e quando se manipularam as enzimas responsáveis pela acetilação (Mec17) e pela de-acetilação (HDAC6) encontrámos uma sinergia potencial com ambas. Testámos ainda, que o aumento no comprimento ciliar não estava causalmente relacionado com a falta de acetilação, ou seja, os cílios com menos acetilação não eram necessariamente os mais longos. Também mostrámos que a sobre-expressão de Arl13b é capaz de restaurar o comprimento dos cílios em mutantes com cílios curtos e como isso pode ser explorado para um futuro potencial papel terapêutico para Arl13b. Em seguida, foi avaliado o impacto do aumento da quantidade de Arl13b no desenvolvimento embrionário do peixe-zebra. Observou-se que a sobre-expressão de Arl13b apresentava fenótipos muito fracos, quando comparados com a perda de função dos mutantes de Arl13b. Focados no inesperado fenótipo leve no estabelecimento do eixo esquerda-direita abordámos a questão através do estabelecimento de uma colaboração com matemáticos, descobrimos que os cílios mais longos que potencialmente têm a capacidade de movimentar mais fluido são atenuados por amplitudes de batimento menores, e, como resultado, estes longos cílios não prejudicam o movimento do fluido e consequentemente não afetam o estabelecimento dos padrões de esquerda-direita. Sugerimos assim que a Arl13b é um regulador chave, do comprimento ciliar. Descobrimos uma nova interação com as enzimas de acetilação/de-acetilação e levantamos novas hipóteses quanto aos mecanismos moleculares da função da Arl13b. Propomos um novo modelo para o mecanismo molecular da Arl13b na regulação do comprimento dos cílios onde podemos integrar os nossos resultados com os relatados na literatura. Este trabalho adiciona mais conhecimento para o mecanismo de ação da Arl13b e, portanto, fornece uma importante contribuição para o campo da investigação em cílios.---------------------------------------------------------------------------------------------------------------------- ABSTRACT: Arl13b is an important ciliary protein, present in primary and motile cilia. arl13b-/- mouse mutants have reduced cilia length and cilia B-tubule defects. As a consequence of these phenotypes, Arl13b loss of function animal models suffer from several congenital disorders including left-right problems, brain malformations and body deformations. In humans Arl13b depletion leads to a congenital chronic disease called Joubert Syndrome. On the other hand, overexpressing Arl13b leads to longer cilia but the characterization of the cellular and developmental phenotypes was missing. In this work we explore the effect of Arl13b overexpression in zebrafish embryos. We found that, at the ciliary level, Arl13b overexpression from 1 cell stage produces longer primary and motile cilia, but these cilia lack proper alpha tubulin acetylation of their microtubule cytoskeleton. Our results showed that this effect is specific from Arl13b overexpression and when we manipulated the enzymes responsible for acetylation, Mec17, and de-acetylation, HDAC6, we found a potential synergy of both mec17 knockdown and HDAC6 activity with Arl13b overexpression. We tested that the ciliary increase in length was not causally related to the lack of acetylation, meaning the more de-acetylated cilia were not necessarily the longer ones. We also showed that Arl13b overexpression is able to restore cilia length in short cilia mutants and how that may be explored to a potential future therapeutic role for Arl13b. Next, we evaluated the impact of increasing the amount of Arl13b in zebrafish embryonic development. We observed that Arl13b overexpression presented very mild phenotypes when compared to the loss of function mutants. We focused on the unexpected left-right mild phenotype and by establishing a mathematical modeling collaboration, we found out that the longer cilia generated force was attenuated by smaller beating amplitudes, and as a result, these long cilia were not impairing the cilia generated flow and the establishment of left-right patterning. We suggest that Arl13b is one key cilia length regulator. We disclosed a novel interaction with the acetylation / de-acetylation enzymes and raised new hypothesis as to the mechanisms of Arl13b function. We propose a new model for the Arl13b molecular mechanism of cilia length regulation where we integrate our findings with those reported in the literature. This work adds more knowledge to the Arl13b mechanism of action and therefore provides an important contribution to the cilia research field.

Potential implications of ketamine anesthesia on zebrafish behavior

Dissertação de mestrado integrado em Psicologia

Nanotechnological approaches using curcumin and Withania somnifera: neuroprotection and antimicrobial activities

Tese de Doutoramento em Biologia de Plantas

Avian erythrocytes have functional mitochondria, opening novel perspectives for birds as animal models in the study of ageing.

BACKGROUND: In contrast to mammalian erythrocytes, which have lost their nucleus and mitochondria during maturation, the erythrocytes of almost all other vertebrate species are nucleated throughout their lifespan. Little research has been done however to test for the presence and functionality of mitochondria in these cells, especially for birds. Here, we investigated those two points in erythrocytes of one common avian model: the zebra finch (Taeniopygia guttata). RESULTS: Transmission electron microscopy showed the presence of mitochondria in erythrocytes of this small passerine bird, especially after removal of haemoglobin interferences. High-resolution respirometry revealed increased or decreased rates of oxygen consumption by erythrocytes in response to the addition of respiratory chain substrates or inhibitors, respectively. Fluorometric assays confirmed the production of mitochondrial superoxide by avian erythrocytes. Interestingly, measurements of plasmatic oxidative markers indicated lower oxidative stress in blood of the zebra finch compared to a size-matched mammalian model, the mouse. CONCLUSIONS: Altogether, those findings demonstrate that avian erythrocytes possess functional mitochondria in terms of respiratory activities and reactive oxygen species (ROS) production. Interestingly, since blood oxidative stress was lower for our avian model compared to a size-matched mammalian, our results also challenge the idea that mitochondrial ROS production could have been one actor leading to this loss during the course of evolution. Opportunities to assess mitochondrial functioning in avian erythrocytes open new perspectives in the use of birds as models for longitudinal studies of ageing via lifelong blood sampling of the same subjects.

ALDH1A3 loss of function causes bilateral anophthalmia/microphthalmia and hypoplasia of the optic nerve and optic chiasm.

The major active retinoid, all-trans retinoic acid, has long been recognized as critical for the development of several organs, including the eye. Mutations in STRA6, the gene encoding the cellular receptor for vitamin A, in patients with Matthew-Wood syndrome and anophthalmia/microphthalmia (A/M), have previously demonstrated the importance of retinol metabolism in human eye disease. We used homozygosity mapping combined with next-generation sequencing to interrogate patients with anophthalmia and microphthalmia for new causative genes. We used whole-exome and whole-genome sequencing to study a family with two affected brothers with bilateral A/M and a simplex case with bilateral anophthalmia and hypoplasia of the optic nerve and optic chiasm. Analysis of novel sequence variants revealed homozygosity for two nonsense mutations in ALDH1A3, c.568A>G, predicting p.Lys190*, in the familial cases, and c.1165A>T, predicting p.Lys389*, in the simplex case. Both mutations predict nonsense-mediated decay and complete loss of function. We performed antisense morpholino (MO) studies in Danio rerio to characterize the developmental effects of loss of Aldh1a3 function. MO-injected larvae showed a significant reduction in eye size, and aberrant axonal projections to the tectum were noted. We conclude that ALDH1A3 loss of function causes anophthalmia and aberrant eye development in humans and in animal model systems.