Cinephotomicrographic and Histochemical Observations on Cercarial Penetration and Encystment by Plagiorchis sp. in Larvae of Chaoborus sp.

Abstract

Plagiorchis sp. cercariae were exposed to Chaoborus sp. larvae and filmed using 16 mm photocinemicrography. After contacting the host, cercariae crawled leechlike, constantly probing with their stylet (from this point until encystment the tail may be lost). Upon contacting an intersegmental membrane, stylet activity increased. The cercarial stage pushed inward on this membrane until it was entirely surrounded by it. The stylet moved generally in a 90? arc about every two seconds and cut through the membrane in approximately 2.45-10 minutes. Passage of the cercariae through the membrane averaged 66 seconds. Once within the hemocoel, the cercariae moved about until encystment. Initially, the cyst wall was elastic, flexible, and easily distorted by the rotation of the metacercaria. The initial 11 mean rotations per minute slowed over a 6-10 minutes period and then became irregular. At this time, a two-layered cyst wall of parasite origin was observed. Histochemically, polysaccharides, polysaccharide-protein complexes, and acid mucosubstances were observed in the ventral, dorsal, cystogenous, and penetration glands. The inner cyst wall originated from ventral and dorsal gland secretions of the cercariae and contained polysaccharides and proteins. The outer layer, from cystogenous glands, consisted mainly of acid mucosubstances and proteins. The third, host-produced layer, was formed from humoral elements and disintegrating hemocytes, and stained positive mainly for proteins. The tail contained high concentrations of glycogen. Numerous specimens Lymnaea stagnalis L. collected from the Stevens Point Area High School (SPASH) marsh, Stevens Point, Wisconsin harbored large numbers of xiphidiocercariae belonging to the genus Plagiorchis as described by McMullen (1937). These cercariae readily penetrated and encysted in Chaoborus sp. larvae in the laboratory. No cinematography but some histochemical studies of Plagiorchis (see Bock, 1988; LeFlore, 1979; Zdarska', 1969) have been published. Additional information relating to host penetration, encystment, and histochemical studies are presented here. MATERIALS AND METHODS Larvae of Chaoborus sp. were collected from Escanaba Lake, Vilas Co., Wisconsin June 1987 and 1988. Individuals of Lymnaea stagnalis were collected from a marsh on the SPASH grounds, Stevens Point, Wisconsin 1 July-15 August in 1987 and 1988. Both were refrigerated at 4?C until needed. Plagiorchis sp. cercariae dissected from snails placed with Chaoborus sp. larvae on microscope slides in a small amount of water and covered with a no. I thank Drs. William LeGrande and Stanley Szczytko for critically reading the manuscript, S. Szczytko for collecting the Chaoborus larvae, Tammy Oatman for printing some of the photomicrographs, and Judy Ratkowski for typing the manuscript. This research was supported in part by a grant from the University of Wisconsin-Stevens Point Personnel Development Committee. TRANS. AM. MICROSC. SOC., 109(2): 160-167. 1990. ? Copyright, 1990, by the American Microscopical Society, Inc. This content downloaded from 157.55.39.17 on Fri, 02 Sep 2016 04:54:16 UTC All use subject to http://about.jstor.org/terms VOL. 109, NO. 2, APRIL 1990 1.5, 24 x 40-mm coverglass. A 16 mm Bolex movie camera mounted on a Zeiss Universal microscope was used to photograph cercarial penetration and encystment at 10 frames/sec. For histochemical studies, Chaoborus sp. larvae were exposed to Plagiorchis sp. cercariae and fixed 30-90 min postexposure either in Gendres fixative or 10% buffered formalin, depending upon the subsequent staining procedures. Gendre-fixed specimens were dehydrated, embedded in Paraplast and sectioned at 5-8 um (Humason, 1979). Five-,um sections were stained with mercuric bromphenol blue (HgBPB) (Mazia et al., 1953), a general protein stain. Eightujm sections were stained with PAS and counterstained with fast green for polysaccharides and polysaccharide-protein complexes; alcian blue pH 2.5 (AB2.5) and AB-PAS were used to stain for mucosubstances (Humason, 1979; Pearse, 1960). Sections, both treated and untreated with salivary amylase, were stained with PAS to check for glycogen. Aldehyde-fuchsin (Cameron & Steele, 1959) was used as a general morphological stain and to determine if hemocytes were present. Isolated cercariae fixed with 10% neutral formalin were frozen and sectioned at 16 um on a cryostat. Sections were stained with oil red 0 (ORO) and mounted with Aquamount (Humason, 1979). All measurements are given in micrometers.