Acridine Orange Staining of Decapod Crustacean Cuticle

Abstract

The fluorescent dye acridine orange (C.I. 46005) was found to be useful for differentiating cuticular layers and therefore can aid in determining the molt stage of decapod crustaceans. For Procambarus clarkii and Callinectes sapidus, best results were achieved by staining 10-[Lm sections of alcoholic formalin-fixed, paraffin-embedded tissue with a 0.1% aqueous solution at pH 4.0. The sections were viewed with an epifluorescence microscope after being excited with three specific wavelength ranges: blue, violet, and ultraviolet. Sections also were examined with bright-field illumination. The exocuticle, which emitted red to orange metachromatic fluorescence, was easily distinguished from the endocuticle, which emitted green to blue orthochromatic fluorescence. Additional key words: crayfish, blue crab, molting, histochemistry, epifluorescence Investigation of events surrounding the molt cycle of crustaceans often requires histological examination of the cuticle to determine both quantitative and qualitative aspects of cuticle deposition (Skinner 1985). Among the histochemical procedures that permit identification of the various layers of the cuticle, the preferred method has been Mallory's trichrome stain, which renders the epicuticle red, the exocuticle orange to blue, and the endocuticle blue (Travis 1955). Erri Babu et al. (1985) conducted an extensive histochemical examination of the cuticle of the crab Menippe rumphii and concluded that definitive identification of the cuticular layers is best achieved with either a combination of staining procedures or one that involves multiple dyes, such as Mallory's trichrome. In this paper we describe the use of a single fluorescent dye, acridine orange (C.I. 46005), coupled with a variety of excitation wavelengths, as a simple alternative for differentiating the layers of the crustacean cuticle.