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Confocal microscopy study of musculature and other anatomical structures in whole‐mount preparations of arthropods and some other cuticle‐bearing animals often presents a significant difficulty because the cuticle poses a barrier to fluorescent dyes and their pigmented tissues can cause attenuation of fluorescent signal. This paper describes a simple and inexpensive procedure based on the use of clove oil as a tissue‐clearing, staining, and mounting medium that helps overcome the problem of slow dye penetration and tissue opaqueness and allows muscles and several other organ systems to be visualized by confocal or epifluorescent microscopy. This clove oil‐induced fluorescence (COIF) method relies on the ability of clove oil to accumulate in muscles and some other tissues and become steadily fluorescent if irradiated at 488 nm. For this method, animals were fixed in 70% ethanol or 4% formaldehyde, then dehydrated and mounted in clove oil. Heavily pigmented animals were additionally bleached in hydrogen peroxide prior to the dehydration step. The COIF method showed excellent results in all major groups of arthropods and some mollusks and annelids revealing the three‐dimensional arrangement of muscles, gonads, glands, cellular nuclei and some parts of the nervous and digestive systems. In the other animal groups tested, clove oil stained all tissues making it difficult to observe the anatomical details. The COIF method is advantageous in some respects over other methods such as phalloidin staining because of its tissue penetration and clearing abilities, low cost of the reagents, resistance to photodamage and the possibility of staining museum specimens. [ABSTRACT FROM AUTHOR]

The taxonomic position of the Acroloxidae Thiele, 1931 within the Lymnaeoidea Rafinesque, 1815 remains ambiguous. This study is an attempt to evaluate the possibility of using sperm morphology and ultrastructure in resolving the phylogenetic relationships of the Acroloxidae based on a microscopic examination of spermatozoa in Acroloxus lacustris (Linnaeus, 1758), a common Eurasian species, using light, scanning electron and transmission electron microscopy. Acroloxus lacustris, like other Lymnaeoidea, have spermatozoa divided into the head, midpiece, glycogen region and endpiece. The sperm head contains an extremely long nucleus carrying three rounded keels and a cylindrical acrosome composed of an apical vesicle and homogeneously electron‐dense pedestal. The subnuclear ring is an electron‐dense cylinder sandwiched between the basal projection of the nucleus and mitochondrial derivative. The midpiece contains the mitochondrial derivative enclosing apically a single glycogen helix. The spermatozoa have a well‐developed glycogen region and an unusual endpiece with an axoneme anchored on an electron‐dense plaque. Several sperm characters of A. lacustris (solid acrosomal pedestal, rounded nuclear keels, single glycogen helix) differ from those of other Lymnaeoidea possibly representing euthyneuran plesiomorphies. The Acroloxidae may therefore be sister to the remaining Lymnaeoidea, branching off before these gained their characteristic sperm traits. [ABSTRACT FROM AUTHOR]