RNA-fluorescence in situ hybridization (FISH) is a powerful tool to visualize target messenger RNA transcripts in cultured cells, tissue sections or whole-mount preparations. As the technique has been developed over time, an ever-increasing number of divergent protocols have been published. There is now a broad selection of options available to facilitate proper tissue preparation, hybridization, and post-hybridization background removal to achieve optimal results. Here we review the technical aspects of RNA-FISH, examining the most common methods associated with different sample types including cytological preparations and whole-mounts. We discuss the application of commonly used reagents for tissue preparation, hybridization, and post-hybridization washing and provide explanations of the functional roles for each reagent. We also discuss the available probe types and necessary controls to accurately visualize gene expression. Finally, we review the most recent advances in FISH technology that facilitate both highly multiplexed experiments and signal amplification for individual targets. Taken together, this information will guide the methods development process for investigators that seek to perform FISH in organisms that lack documented or optimized protocols. Seviour RJ. 2005. Improved permeabilization protocols for fluorescence in situ hybridization (FISH) of mycolic-acid-containing bacteria found in foams.Kislauskis EH, Li Z, Singer RH, Taneja KL. 1993. Isoform-specific 3′-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments.

The peripheral nervous system in gastropods plays a key role in the neural control of behaviors, but is poorly studied in comparison with the central nervous system. Peripheral sensory neurons, although known to be widespread, have been studied in a patchwork fashion across several species, with no comprehensive treatment in any one species. We attempted to remedy this limitation by cataloging peripheral sensory cells in the cephalic sensory organs of Lymnaea stagnalis employing backfills, vital stains, histochemistry, and immunohistochemistry. By using at least two independent methods to corroborate observations, we mapped four different cell types. We have found two different populations of bipolar sensory cells that appear to contain catecholamines(s) and histamine, respectively. Each cell had a peripheral soma, an epithelial process bearing cilia, and a second process projecting to the central nervous system. We also found evidence for two populations of nitric oxide-producing sensory cells, one bipolar, probably projecting centrally, and the second unipolar, with only a single epithelial process and no axon. The various cell types are presumably either mechanosensory or chemosensory, but the complexity of their distributions does not allow formation of hypotheses regarding modality. In addition, our observations indicate that yet more peripheral sensory cell types are present in the cephalic sensory organs of L. stagnalis. These results are an important step toward linking sensory cell morphology to modality. Moreover, our observations emphasize the size of the peripheral nervous system in gastropods, and we suggest that greater emphasis be placed on understanding its role in gastropod neuroethology.

The human disease schistosomiasis (or bilharzia) is caused by the helminth blood fluke parasite Schistosoma mansoni, which requires an intermediate host, the freshwater gastropod snail Biomphalaria glabrata (the most common intermediate host). The free-swimming parasite miracidia utilise an excellent chemosensory sense to detect and locate an appropriate host. This study investigated the biomolecules released by the snail that stimulate changes in the behaviour of the aquatic S. mansoni miracidia. To achieve this, we have performed an integrated analysis of the snail-conditioned water, through chromatography and bioassay-guided behaviour observations, followed by mass spectrometry. A single fraction containing multiple putative peptides could stimulate extreme swimming behaviour modifications (e.g. velocity, angular variation) similar to those observed in response to crude snail mucus. One peptide (P12;—R-DITSGLDPEVADD-KR—) could replicate the stimulation of miracidia behaviour changes. P12 is derived from a larger precursor protein with a signal peptide and multiple dibasic cleavage sites, which is synthesised in various tissues of the snail, including the central nervous system and foot. P12 consists of an alpha helix secondary structure as indicated by circular dichroism spectroscopy. This information will be helpful for the development of approaches to manipulate this parasites life cycle, and opens up new avenues for exploring other parasitic diseases which have an aquatic phase using methods detailed in this investigation.

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