Developmental plasticity reveals hidden fish phenotypes and enables morphospace diversification.

The establishment of a given phenotype is only one expression from a range of hidden developmental possibilities. Developmental plasticity at hidden reaction norms might elicit phenotypic diversification under new developmental environments. Current discussion benefits from empirical analyses that integrate multiple environmental stimuli to evaluate how plastic responses may shape phenotypic variation. We raised Megaleporinus macrocephalus fish in different environmental settings to address contributions of developmental plasticity for emergence of new phenotypes and subsequent morphospace diversification. Plastic morphotypes were evaluated at two complementary scales, the M. macrocephalus morphospace and the higher taxonomic level of Anostomidae family. Morphospace analyses demonstrated that developmental plasticity quickly releases distinct head morphotypes that were hidden in the parental monomorphic population. Plastic morphotypes occupied discrete and previously unfilled morphospace regions, a result obtained from comparisons with a control population and in analyses including several Anostomidae species. Plastic responses involved adjustments in shape and relative position of head bonesets, and fish raised under specific environmental combinations rescued phenotypic patterns described for different genera. Therefore, developmental plasticity possibly contributes to adaptive radiation in Anostomidae. Results illustrate how plastic responses enable morphospace diversification and contribute to evolution.

Combined microscopy and spectroscopy techniques to characterize a fossilized feather with minimal damage to the specimen.

The study of fossil feathers has been revitalized in the last few decades and has contributed significantly to paleontological studies of dinosaurs and birds. Specific morphological and physicochemical characteristics of the microscale structures of feathers and the protein keratin are key targets when preserved during the fossilization process. Keratin is a fibrous protein that composes some hard tissues such as hair, nails and feathers. It is part of the so called intermediate filaments inside keratinocyte cells and is rich in sulfur containing amino acid cysteine. To date, different microscopy and analytical methods have been used for the analysis and detailed characterization and classification of feathers. However, in this work we showed that analytical optical and electron microscopies can be quick and precise methods with minimal effects on the sample during analysis. This association of different approaches on the same sample results in correlative data albeit in different length scales. Intracellular bodies called melanosomes originally present in melanocyte cells were identified with Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), and had well-defined orientation and a mean aspect ratio comparable to melanosomes extant in dark feathers. The detection of sulphur in melanosomes via Energy Dispersive Spectroscopy both in SEM and TEM shows that, along the fossilization process, sulphur from the degraded keratin matrix could have been trapped inside the melanosomes. Chemical groups that make up keratin and melanin in the fossil sample were detected via FT-IR Spectroscopy and Confocal Laser Scanning Microscopy (CLSM). The use of combined analytical microscopy techniques can contribute significantly to the study of fossils generating precise results with minimum damage to the original sample.

Quantitative characterization and comparative study of feather melanosome internal morphology using surface analysis.

A successful feather development implies in a precise orchestration of cells in the follicle, which culminates in one of the most complex epidermal structures in nature. Melanocytes contribute to the final structure by delivering melanosomes to the barb and barbule cells. Disturbance to the tissue during the feather growth can damage the final structure. Here, melanosomes seen in an unusual outgrowth on the barb cortex of a flight feather are reported and compared to commonly observed melanosomes embedded in the cortex. Transmission Electron Microscopy in scanning-transmission mode (STEM) generated images coupled with secondary electron detection. The two classes of melanosomes were registered on images combining transmitted and secondary electron signals. Image processing allowed surface analyses of roughness and texture of the internal morphology of these organelles. Results showed that the two classes of melanosomes are significantly distinct internally, indicating that different physiological processes up to feather maturation could have occurred. Surface analysis methods are not regularly used in cell biology studies, but here it is shown that it has great potential for microscopic image analysis, which could add robust information to studies of cell biology events.

Interrelations between the parasitophorous vacuole of Toxoplasma gondii and host cell organelles.

Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.