Ecological conditions shape (adaptive) responses at the molecular, anatomical, and behavioral levels. Understanding these responses is key to predict the outcomes of intra- and inter-specific competitions and the evolutionary trajectory of populations. Recent technological advances have enabled large-scale molecular (e.g., RNAseq) and behavioral (e.g., computer vision) studies, but the study of anatomical responses to ecological conditions has lagged behind. Here, we highlight the role of X-ray micro-computed tomography (micro-CT) in generating in vivo and ex vivo 3D imaging of anatomical structures, which can enable insights into adaptive anatomical responses to ecological environments. To demonstrate the application of this method, we manipulated the larval density of Drosophila melanogaster Meigen flies and applied micro-CT to investigate the anatomical responses of the male reproductive organs to varying intraspecific competition levels during development. Our data is suggestive of two classes of anatomical responses which broadly agree with sexual selection theory: increasing larval density led to testes and ejaculatory duct to be overall larger (in volume), while the volume of accessory glands and, to a lesser extent, ejaculatory duct decreased. These two distinct classes of anatomical responses might reflect shared developmental regulation of the structures of the male reproductive system. Overall, we show that micro-CT can be an important tool to advance the study of anatomical (adaptive) responses to ecological environments.
Amphibians are models for studying applied ecological issues such as habitat loss, pollution, disease, and global climate change due to their sensitivity and vulnerability to changes in the environment. Developmental series of amphibians are informative about their biology, and X-ray based 3D reconstruction holds promise for quantifying morphological changes during growth—some with a direct impact on the possibility of an experimental investigation on several of the ecological topics listed above. However, 3D resolution and discrimination of their soft tissues have been difficult with traditional X-ray computed tomography, without time-consuming contrast staining. Tomographic data were initially performed (pre-processing and reconstruction) using the open-source software tool SYRMEP Tomo Project. Data processing and analysis of the reconstructed tomography volumes were conducted using the segmentation semi-automatic settings of the software Avizo Fire 8, which provide information about each investigated tissues, organs or bone elements. Hence, volumetric analyses were carried out to quantify the development of structures in different tadpole developmental stages. Our work shows that synchrotron X-ray microtomography using phase-contrast mode resolves the edges of the internal tissues (as well as overall tadpole morphology), facilitating the segmentation of the investigated tissues. Reconstruction algorithms and segmentation software played an important role in the qualitative and quantitative analysis of each target structure of the Thoropa miliaris tadpole at different stages of development, providing information on volume, shape and length. The use of the synchrotron X-ray microtomography setup of the SYRMEP beamline of Elettra Synchrotron, in phase-contrast mode, allows access to volumetric data for bone formation, eye development, nervous system and notochordal changes during the development (ontogeny) of tadpoles of a cycloramphid frog Thoropa miliaris. As key elements in the normal development of these and any other frog tadpole, the application of such a comparative ontogenetic study, may hold interest to researchers in experimental and environmental disciplines.
Selection favour males that anticipate intraspecific competition levels and develop appropriate competitive traits. In holometabolous insects, larval density can act as an ecological cue of males future intraspecific competition levels while also limiting access to food allocated to sexually selected traits (e.g., testes). To date, however, most studies have been conducted in larval densities that may have little or no relevance to species ecology. Here, we manipulated Drosophila melanogaster larval density based on the natural history of the species and used 3D micro-CT imaging to show a strong modulation of testes volume based on larval density: males from high larval densities generally had larger testes. Conversely, males from high densities tended (albeit not statistically significantly) to have the smallest accessory glands and ejaculatory bulb. Overall, we used micro-CT to measure male reproductive morphology in an ecologically relevant design for which the findings broadly support predictions from sexual selection theory.
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