Histochemistry of hydrolytic enzymes of virgulate xiphidiocercariae

Histochemical studies were conducted to localize and determine the roles of hydrolytic enzymes in a virgulate xiphidiocercaria, Cercaria polypyreta, before and after penetration of an arthropod intermediate host. Before penetration N-acetyl-p-glucosaminidase was localized in the virgula organ and outer mucoid coat; leucine aminopeptidase was restricted to the flame cells, and an esterase which is inhibited by eserine (cholinesterase) was located in the nervous system. Acetylglucosaminidase activity was weaker after penetration of mayfly naiads (Litobrancha recurvata) suggesting that this enzyme is utilized during penetration. No changes were noted in LAP and esterase activities. Sulfhydryl groups and calcium ions were found in the stylet and in the cephalic glands. Acetylglucosaminidase activity was also seen in the virgula organs and outer mucoid coats of 2 other virgulate xiphidiocercariae, Cercaria apatema and C. dolomeda, and in the mucoid glands of developing C. apatema prior to their discharge to form the mucoid coat and virgula organ. Virgulate xiphidiocercariae such as Cercaria polypyreta are larvae of the trematode family Lecithodendriidae and develop within sporocysts in operculate snails. They are armed with stylets, possess cephalic glands, and also mucoid glands which are discharged prior to emergence to form a mucoid reservoir or virgula organ within the oral sucker (Kruidenier, 1951). Ortigoza and Hall (1963) studied the mucoid and cephalic glands of three species of virgulate cercariae and found that they contained mucopolysaccharides and proteins. While there is literature pertaining to the glandular contents or penetration mechanisms of schistosome cercarie (Lewert and Lee, 1954; Stirewalt and Kruidenier, 1961; Stirewalt, 1973) and strigeoid cercariae (Erasmus and Ohman, 1963; Cheng and Yee, 1968), there is little information regarding cercarial penetration of insects. The present study was designed to examine the role of hydrolytic enzymes and cercarial secretions in the penetration of arthropod cuticle. MATERIALS AND METHODS A. Collection of material C. polypyreta was obtained from naturally infected river snails (Nitocris dilatatus Conrad) collected from the Cheat River System in West Virginia. Two related species, C. apatema and C. dolomeda, were obtained from the same source and used to confirm results obtained with C. polypyreta and to study enzyme activity in young, developing cercariae. Mayfly naiads (Litobrancha recurvata) were collected from Little Lick Run in Preston County, West Virginia, which is free of operculate snails, and maintained in mud substrate in stream water at 15 C. B. Preparative procedures Enzymes: Sporocysts obtained by crushing infected snails were mounted in OCT medium (Ames Co., Elkhart, Ind.), quick-frozen, and sectioned at 4 to 6 Am on a cryostat. Sections were air-dried for 15 min prior to incubation in substrate. Histochemical studies were also performed on cercariae in the process of penetration by exposing mayfly naiads to 300 freshly emerged C. polypyreta at room temperature, quick-freezing naiads 30 to 60 min after exposure, and sectioning. Sections were stained simultaneously with sporocyst sections for comparison and control. The following protocols for enzyme localizations were used as described by Pearse (1972): Naphthol AS-BI-Garnet GBC method for N-acetylp-glucosaminidase (pH 5.2); a-naphthyl acetate method for esterase (pH 7.4); L-leucyl-4-methoxyp-naphthylamide method for leucine aminopeptidase (LAP) (pH 7.4); and silver proteinate method for endopeptidase (pH 6.4). Positive control tissues included mouse kidney (glucosaminidase, esterase), mouse small intestine (LAP, endopeptidase), and snail intestine and digestive gland (glucosaminidase, LAP, endopeptidase). Boiling of sections and immersion of sections in incubation medium without substrate were used for negative control. Calcium or magnesium ions were added as activators for LAP at final concentrations of 1.25 x 10' M and 4.2 x 10M respectively. Esterase was inhibited by preincubating sections in a 10M concentration of the anticholinesterase agent, eserine. Attempts were made to control endoReceived for publication 28 February 1975. * This investigation was supported by NSF Research Grant GB-23536 and a grant from the West Virginia University Medical Corporation.