Reference: Bio/ Bull 189: 175-184. (October/November, Bioassay and Preliminary Characterization of Ovigerous-Hair Stripping Substance (OHSS) in Hatch Water of Crab Larvae MASAYUKI SAIGUSA Okayama University. Faculty of Science, Department of Biology, Tsushima 2-1-1. Okayama 700. Japan Abstract. Hatch water (the filtrated medium into which zoea larvae have been released) of the estuarine terrestrial crab Sesamia haematocheir (akate-gani) contains a sub-stance that causes premature detachment of embryos from ovigerous females. Detachment occurs when the ovigerous hairs along the female's ovigerous setae slip out of the investment coat that binds them to the embryos through stalks, or funiculi. The active factor, which I call ovigerous-hair stripping substance (OHSS), is released outside of the egg capsule at the time of hatching, and is not secreted by the female. This study describes the results of a quan-titative assay for measuring the activity of OHSS. Activity is measured as the percentage of hairs on a seta that can be induced to slip out of the coat without damage. Ex-periments with an extract of crushed embryos indicated that OHSS is present up to 2 days before hatching. Its activity was destroyed by heat and trypsin, suggesting that it is a protein. Its molecular size was estimated by gel filtration to be 1 5-20 kDa in 5 1 . haematocheir and 30 kDa in S. pictum. Reciprocal tests among different species in-dicated that OHSS occurs widely in intertidal and estu-arine crabs. Introduction After oviposition, the embryos of decapod crustaceans are wrapped in a thick membrane and clustered on the ovigerous setae beneath the abdomen of the female. Many fine hairs (i.e.. ovigerous hairs) are arranged along the seta, and the embryos are attached to these hairs by a stalk: the funiculus. The mechanism by which the fertil-ized egg is attached to the ovigerous hairs and the source Received 21 December 1992; accepted 25 July 1495. of the material that makes up the funiculus and the egg capsule have been subjects of controversy for many years (e.g.. Andrews, 1906; Yonge, 1937, 1946; Mawson and Yonge, 1938; Linder, 1960; Suko, 1961; Cheung, 1966; Fisher and Clark, 1983; Goudeau and Lachaise, 1980, 1983; Goudeau el a/.. 1987; Talbot and Demers, 1993). In addition to the funiculus, the embryo attachment system involves a clear coat that wraps around (invests) the ovigerous hairs (Saigusa. 1994). The funiculus is therefore not connected to the ovigerous hairs directly, but indirectly through the coat a fact not previously re-ported. The investing coat may be composed of the same materials that make up the funiculus and the outer layer of the egg capsule, but this notion has not been tested adequately. While attached to the ovigerous hairs, the embryos are ventilated by the movement of pleopod setae. When de-velopment is completed, the egg capsule breaks, and the zoeas hatch and are released into the water by a special fanning movement of the female's abdomen (larval release is described in Saigusa, 1982). After the larvae have been released, the empty egg cases, funiculi, and investing coats remain attached to the ovigerous hairs. Soon, however, a substance released at the time of hatching causes the ovi-gerous hairs to slip out of the investment coat, detaching the funiculi and empty egg cases; the active factor is called 'ovigerous-hair stripping substance' (OHSS) (Saigusa, 1 994). Within a few hours, the ovigerous hairs are cleaned without damage, and in a few days a new clutch of fer-tilized eggs is attached to the hairs. Because OHSS is re-leased at the time of hatching, one might suppose it to be directly involved in hatching. But there is no evidence that this factor is a hatching enzyme of the sort known in many groups of animals (Saigusa, 1994). 175
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