Classification of target tissues of Eisenia fetida using sequential multimodal chemical analysis and machine learning.

Acquiring comprehensive knowledge about the uptake of pollutants, impact on tissue integrity and the effects at the molecular level in organisms is of increasing interest due to the environmental exposure to numerous contaminants. The analysis of tissues can be performed by histological examination, which is still time-consuming and restricted to target-specific staining methods. The histological approaches can be complemented with chemical imaging analysis. Chemical imaging of tissue sections is typically performed using a single imaging approach. However, for toxicological testing of environmental pollutants, a multimodal approach combined with improved data acquisition and evaluation is desirable, since it may allow for more rapid tissue characterization and give further information on ecotoxicological effects at the tissue level. Therefore, using the soil model organism Eisenia fetida as a model, we developed a sequential workflow combining Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) for chemical analysis of the same tissue sections. Data analysis of the FTIR spectra via random decision forest (RDF) classification enabled the rapid identification of target tissues (e.g., digestive tissue), which are relevant from an ecotoxicological point of view. MALDI imaging analysis provided specific lipid species which are sensitive to metabolic changes and environmental stressors. Taken together, our approach provides a fast and reproducible workflow for label-free histochemical tissue analyses in E. fetida, which can be applied to other model organisms as well.

MALDI mass spectrometry imaging workflow for the aquatic model organisms Danio rerio and Daphnia magna.

Lipids play various essential roles in the physiology of animals. They are also highly dependent on cellular metabolism or status. It is therefore crucial to understand to which extent animals can stabilize their lipid composition in the presence of external stressors, such as chemicals that are released into the environment. We developed a MALDI MS imaging workflow for two important aquatic model organisms, the zebrafish (Danio rerio) and water flea (Daphnia magna). Owing to the heterogeneous structure of these organisms, developing a suitable sample preparation workflow is a highly non-trivial but crucial part of this work and needs to be established first. Relevant parameters and practical considerations in order to preserve tissue structure and composition in tissue sections are discussed for each application. All measurements were based on high mass accuracy enabling reliable identification of imaged compounds. In zebrafish we demonstrate that a detailed mapping between histology and simultaneously determined lipid composition is possible at various scales, from extended structures such as the brain or gills down to subcellular structures such as a single axon in the central nervous system. For D. magna we present for the first time a MALDI MSI workflow, that demonstrably maintains tissue integrity during cryosectioning of non-preserved samples, and allows the mapping of lipids in the entire body and the brood chamber inside the carapace. In conclusion, the lipid signatures that we were able to detect with our method provide an ideal basis to analyze changes caused by pollutants in two key aquatic model organisms.

Bisphenols exert detrimental effects on neuronal signaling in mature vertebrate brains.

Bisphenols are important plasticizers currently in use and are released at rates of hundreds of tons each year into the biosphere1-3. However, for any bisphenol it is completely unknown if and how it affects the intact adult brain4-6, whose powerful homeostatic mechanisms could potentially compensate any effects bisphenols might have on isolated neurons. Here we analyzed the effects of one month of exposition to BPA or BPS on an identified neuron in the vertebrate brain, using intracellular in vivo recordings in the uniquely suited Mauthner neuron in goldfish. Our findings demonstrate an alarming and uncompensated in vivo impact of both BPA and BPS-at environmentally relevant concentrations-on essential communication functions of neurons in mature vertebrate brains and call for the rapid development of alternative plasticizers. The speed and resolution of the assay we present here could thereby be instrumental to accelerate the early testing phase of next-generation plasticizers.

Recordings in an integrating central neuron provide a quick way for identifying appropriate anaesthetic use in fish.

In animal husbandry, livestock industry and research facilities, anaesthetic agents are frequently used to moderate stressful intervention. For mammals and birds, procedures have been established to fine-tune anaesthesia according to the intervention. In ectothermic vertebrates, however, and despite changes in legislation and growing evidence on their cognitive abilities, the presently available information is insufficient to make similarly informed decisions. Here we suggest a straightforward way for rapidly filling this gap. By recording from a command neuron in the brain of fish whose crucial role requires it to integrate and process information from all sensory systems and to relay it to motor output pathways, the specific effects of candidate anaesthesia on central processing of sensory information can directly and efficiently be probed. Our approach allows a rapid and reliable way of deciding if and at which concentration a given anaesthetic affects the central nervous system and sensory processing. We employ our method to four anaesthetics commonly used in fish and demonstrate that our method quickly and with small numbers of animals provides the critical data for informed decisions on anaesthetic use.