Evaluation of standard imaging techniques and volumetric preservation of nervous tissue in genetically identical offspring of the crayfish Procambarus fallax cf. virginalis (Marmorkrebs)

In the field of comparative neuroanatomy, a meaningful interspecific comparison demands quantitative data referring to method-specific artifacts. For evaluating the potential of state-of-the-art imaging techniques in arthropod neuroanatomy, micro-computed X-ray microscopy (μCT) and two different approaches using confocal laser-scanning microscopy (cLSM) were applied to obtain volumetric data of the brain and selected neuropils in Procambarus fallax forma virginalis (Crustacea, Malacostraca, Decapoda). The marbled crayfish P. fallax cf. virginalis features a parthogenetic reproduction generating genetically identical offspring from unfertilized eggs. Therefore, the studied organism provides ideal conditions for the comparative analysis of neuroanatomical imaging techniques and the effect of preceding sample preparations of nervous tissue. We found that wet scanning of whole animals conducted with μCT turned out to be the least disruptive method. However, in an additional experiment it was discovered that fixation in Bouin’s solution, required for μCT scans, resulted in an average tissue shrinkage of 24% compared to freshly dissected and unfixed brains. The complete sample preparation using fixation in half-strength Karnovsky’s solution of dissected brains led to an additional volume decrease of 12.5%, whereas the preparation using zinc-formaldehyde as fixative resulted in a shrinkage of 5% in comparison to the volumes obtained by μCT. By minimizing individual variability, at least for aquatic arthropods, this pioneer study aims for the inference of method-based conversion factors in the future, providing a valuable tool for reducing quantitative neuroanatomical data already published to a common denominator. However, volumetric deviations could be shown for all experimental protocols due to methodological noise and/or phenotypic plasticity among genetically identical individuals. MicroCT using undried tissue is an appropriate non-disruptive technique for allometry of arthropod brains since spatial organ relationships are conserved and tissue shrinkage is minimized. Collecting tissue-based shrinkage factors according to specific sample preparations might allow a better comparability of volumetric data from the literature, even if another technique was applied

An atlas of larval organogenesis in the European shore crab Carcinus maenas L. (Decapoda, Brachyura, Portunidae)

Abstract Background The life history stages of brachyuran crustaceans include pelagic larvae of the Zoea type which grow by a series of moults from one instar to the next. Zoeae actively feed and possess a wide range of organ systems necessary for autonomously developing in the plankton. They also display a rich behavioural repertoire that allows for responses to variations in environmental key factors such as light, hydrostatic pressure, tidal currents, and temperature. Brachyuran larvae have served as distinguished models in the field of Ecological Developmental Biology fostering our understanding of diverse ecophysiological aspects such as phenotypic plasticity, carry-over effects on life-history traits, and adaptive mechanisms that enhance tolerance to fluctuations in environmental abiotic factors. In order to link such studies to the level of tissues and organs, this report analyses the internal anatomy of laboratory-reared larvae of the European shore crab Carcinus maenas. This species has a native distribution extending across most European waters and has attracted attention because it has invaded five temperate geographic regions outside of its native range and therefore can serve as a model to analyse thermal tolerance of species affected by rising sea temperatures as an effect of climate change. Results Here, we used X-ray micro-computed tomography combined with 3D reconstruction to describe organogenesis in brachyuran larvae. We provide a detailed atlas of the larval internal organization to complement existing descriptions of its external morphology. In a multimethodological approach, we also used cuticular autofluorescence and classical histology to analyse the anatomy of selected organ systems. Conclusions Much of our fascination for the anatomy of brachyuran larvae stems from the opportunity to observe a complex organism on a single microscopic slide and the realization that the entire decapod crustacean bauplan unfolds from organ anlagen compressed into a miniature organism in the sub-millimetre range. The combination of imaging techniques used in the present study provides novel insights into the bewildering diversity of organ systems that brachyuran larvae possess. Our analysis may serve as a basis for future studies bridging the fields of evolutionary developmental biology and ecological developmental biology