642 Fig. 4 Gelatinolysis at pH 6.8 produced by the proteinases separated by electrophoresis at the operative pH of 3.5 at which the enzymes were practically inactive during migration by approximately 20% by preventing metal ions from an interaction with −SH groups of cysteine proteinases. DTE, a compound that quantitatively reduces −SH groups in proteins but does not form stable complexes with Ca2+ and Mg2+, produced an increase in the proteinase activity by about 40%. A combined stimulatory action of EDTA and DTE was stronger than that produced by these individual agents. The inhibition produced by pHMB was in part reversed by DTE, thus proving that the examined enzyme belongs to the family of cysteine proteinases. An almost complete inhibition of the enzyme by leupeptin, a tripeptide Table 1 Effect of activators/inhibitors on the activity of the cercarial proteinase toward azocoll at an optimum pH of 6.8 Compound added None CaCl2, 1 mM ZnSO4, 0.1 mM 1,10-Phenanthroline, 1 mMa EDTA, 1 mM DTE, 1 mM EDTA (1 mM)+DTE (1 mM) EDTA (1 mM)+pHMB (0.1 mM) As above +DTE (1 mM)c Leupeptin, 20 μg/ml PMSF, 1 mMd STI, 0.3 mg/ml Relative activity (%) 100.0 87.8 1.1 103.8 122.4 140.2 161.4b 2.3 41.7 3.2 2.2 102.3 Values represent means of duplicate assays. The incubation media were buffered with 0.05 M 3,3′-dimethylglutaric acid-NaOH buffer a Prepared from a 0.4 M stock solution in dimethylsulfoxide b Equivalent to the hydrolysis of 10.2 μg of azocoll per hour per microgram of extract protein c DTE was added after 10 min of preincubation in the presence of pHMB d Prepared from a 0.1-M stock solution in anhydrous isopropanol Parasitol Res (2011) 108:639–643 argininal, provided a further support for this diagnosis. Like cysteine proteinases from other sources, the cercarial enzyme was sensitive to phenylmethanesulfonyl fluoride (PMSF) and was insensitive to soybean trypsin inhibitor (STI). Discussion The described results prove the occurrence of a cysteine proteinase in the penetration glands of the cercariae of C. cornutus. Like other enzymes of this family, together with those which were found in the penetration glands of the cercariae of diplostomid trematodes D. pseudospathaceum and T. excavata (Moczoń 1994a, b; Moczon 2007), the glandular proteinase from C. cornutus cercariae hydro- lyzed gelatin, preferred azocoll over azocasein and azoalbumin, and did not hydrolyze elastin, the latter being a good protein substrate for the elastase-like serine proteinase from the penetration glands of the plagiorchiid Neoglyphe sobolevi cercariae (Moczoń 1997). The re- sponse of the enzyme extracted from C. cornutus cercariae to the treatment with a number of activators and inhibitors (Table 1) was typical for cysteine proteinases and resembled the response of the “penetration enzymes” from the cercariae of the diplostomid trematodes mentioned above. The chemical factors emitted by the organisms of certain water invertebrates, e.g., leeches and mollusks, which can be responsible for the attraction of the cercariae of the genus Cotylurus, are unknown; however, it is possible that some secreted metabolites or body surface glycoproteins and their fragments (Kalbe et al. 1997) act as effective attractants for the invasive cotylurid cercariae. The genus Cotylurus belongs to the family of Strigeidae. An acquisition of a sufficient amount of the cercariae of C. cornutus for extensive biochemical studies involving a separation and characterization of individual proteinases is rather difficult. Nevertheless, the presented study proved that the penetration glands of the examined larvae synthe- size and store a cysteine proteinase which enables the parasite to infect hosts and facilitates the migration through the hosts’ tissues. Further work needs to be done to ascertain whether the presence of a similar enzyme in the glandular cells can be extended over other representatives of the Strigeidae family. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.