Parasitol Res (2011) 108:639–643 DOI 10.1007/s00436-010-2108-3 ORIGINAL PAPER A cysteine proteinase in the penetration glands of the cercariae of Cotylurus cornutus (Trematoda, Strigeidae) Tadeusz Moczoń Received: 30 June 2010 / Accepted: 30 September 2010 / Published online: 28 October 2010 # The Author(s) 2010. This article is published with open access at Springerlink.com Abstract A cysteine proteinase from the penetration glands of Cotylurus cornutus cercariae was examined with histochemical and biochemical methods. The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-L-arginine-4-methoxy-2-naphthylamide, and N- blocked-p-nitroanilide, but did not degrade elastin. The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking com- pounds p-hydroxymercuribenzoate and N-ethylmaleimide (NEM) inhibited it. The enzyme was also sensitive to leupeptin but insensitive to soybean trypsin inhibitor. An electrophoretic separation of extract proteins from the cercar- iae under acidic, non-denaturing conditions and in the presence of 0.1% gelatin in a polyacrylamide gel revealed the presence of two distinct and three weak transparent bands in the gel resulting from a gelatinolytic activity at pH 6.8. The distinct bands apparently resulted from the activity of the glandular enzyme and lysosomal cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure. No gelatinolysis occurred following treatment of an extract sample with 0.1 mM NEM. Abbreviations DTE Z-Arg-NhnapMeO Tris T. Moczoń (*) W. Stefański Institute of Parasitology, Polish Academy of Sciences, Twarda 51/55, 00-818 Warszawa, Poland e-mail: moczon@twarda.pan.pl Dithioerythritol N-benzyloxycarbonyl-L-arginine-4- methoxy-2-naphthylamide Amino-2-hydroxymethyl-1,3-pro- panediol CHES EDTA Bz-Arg-NHnan pHMB NEM PMSF STI Introduction 2-(N-cyclohexylamino)ethanesulfonic acid Disodium ethylenediaminetetraace- tate N-benzoyl-L-arginine-4-nitroanilide Sodium p-hydroxymercuribenzoate N-ethylmaleimide Phenylmethanesulfonyl fluoride Soybean trypsin inhibitor Mature specimens of Cotylurus cornutus inhabit the intestine of waterfowl. Cercariae of the parasite, after their emergence from water mollusks, invade water invertebrates, mainly leeches and mollusks, by penetrating their teguments. Following migration, the larvae settle in the host’s tissues where they grow and transform into metacercariae. Penetra- tion glands of the cercariae are comprised of four discrete glandular cells situated anteriorly to the ventral sucker. Each cell protrudes anteriorly, thus forming its secretory canal. The canals run toward the oral sucker and open at its surface. At present, the chemical nature of the contents of the penetration glands of the cercariae is unknown. It was previously shown that the presence of cysteine proteinases in the penetration glands is a characteristic feature of the cercariae of the two representatives of Diplostomidae family, Diplostomum pseudospathaceum and Tylodelphys excavata (Moczoń 1994a, b; Moczon 2007). The trematodes of the genus Cotylurus belong to the family Strigeidae. Both Strigeidae and Diplostomidae are members of the order Strigeida. It was of interest to examine the penetration glands of C. cornutus cercariae to ascertain whether or not the