Three experiments were carried out in vitro to determine the roles of the tegument and gut of Fasciola hepatica in the uptake of the flukicidal drug, clorsulon. Changes to the two surfaces were assessed by scanning electron microscopy. In the first experiment, the flukes were ligatured to prevent the oral ingestion of drug and treated for 24 h in clorsulon (10 microg/ml). The gastrodermal surface remained normal and few changes to the tegumental surface were observed. In the second experiment, flukes were fed for 24 h on red blood cells isolated from rats dosed with clorsulon at 12.5 mg/kg body weight; this experiment was designed to prevent the exposure of the tegumental surface to the drug. The gastrodermal surface was severely disrupted and the gut lamellae were disorganised and necrotic. Swelling of the tegument and blebbing on the tegumental surface were evident, but the changes were not severe. More severe swelling of the tegument was observed in the third experiment, in which flukes were incubated for 24 h in clorsulon (10 microg/ml), with both absorptive surfaces being available for drug uptake. The gastrodermal surface was badly disrupted and the gut lamellae were disorganised and necrotic. Taking the results of the three experiments together, the gastrodermal surface was more affected than the tegument and the greatest disruption to the two surfaces was seen when both routes of entry were available to the fluke. The data support a previous study which indicated that entry of clorsulon into the fluke in vivo is principally by the oral ingestion of drug bound to the red blood cells.

Three experiments have been carried out in vitro to determine the effect of oral and trans-tegumental uptake of clorsulon on the fine structure of the tegument and gut of Fasciola hepatica. Changes were assessed by transmission electron microscopy. In the first experiment, the flukes were ligatured to prevent the oral ingestion of drug and treated for 24 h in clorsulon (10 microg/ml). Limited swelling of the basal infolds was observed in the tegumental syncytium. Swollen mitochondria were present in the syncytium, the underlying tegumental cells and in the gastrodermal cells. Swelling and vesiculation of the cisternae of the granular endoplasmic reticulum (ger) was evident in the gastrodermal cells, together with a reduction in secretory activity. In the second experiment, flukes were fed for 24 h on red blood cells isolated from rats dosed with clorsulon at 12.5 mg/kg body weight; this experiment was designed to prevent the exposure of the tegumental surface to the drug. There was severe swelling of the basal infolds in the tegumental syncytium and swelling of mitochondria in the syncytium, tegumental cells and gastrodermal cells. In the tegumental cells there was a decrease in the number of Golgi complexes as well. A number of changes were evident in the gastrodermal cells: swelling of the ger cisternae, an increase in the number of autophagic vacuoles, a reduction in the number of secretory bodies and disruption of the lamellae projecting from the surface of the cells. In the third experiment, flukes were incubated for 24 h in clorsulon (10 microg/ml), with both absorptive surfaces being available for drug uptake. There was severe swelling of the basal infolds in the tegumental syncytium and large autophagic vacuoles were present. Swollen mitochondria were a feature of the tegument, tegumental cells and gastrodermal cells, as were swollen cisternae of ger in the tegumental and gastrodermal cells. Fewer Golgi complexes were observed in the tegumental cells and in the gastrodermal cells there were fewer secretory bodies and an increased number of autophagic vacuoles. Overall, the gastrodermal cells were more severely affected than the tegument. Greater disruption of the tegument occurred when the oral route of uptake was available. The results support those of previous studies which point to oral uptake of clorsulon being the major route of entry into the fluke.

Parasitol Res (2005) 95:117-128 Figures 1-29 appeared too dark in the printed version of the article. The correct figures are shown here.Figs. 1-6 Scanning electron micrographs (SEMs) of the tegumental surface of control (Figs. 1, 2) and clorsulon-treated (Figs. 3-6) Fasciola hepatica which had been incubated in vitro for 24 h (non-ligatured flukes) Fig. 1 The ventral surface of a control fluke showing the oral sucker (OS) and ventral sucker (VS) on the apical cone of the fluke.The cirrus (C) is also visible extending from the gonopore. Bar 2 mm Fig. 2 Spines (S) from the anterior midbody region of the dorsal surface of a control fluke. Serrated tips can be seen on a number of the spines. Bar 50 lm Fig. 3 Ventral surface of the apical cone showing the oral sucker (OS), ventral sucker (VS) and the gonopore (G). Spines (S) can be seen covering the surface of the tegument. Bar 400 lm Fig. 4 Dorsal surface of the apical cone showing sunken spines (SS) and severe swelling of the tegument both covering (white arrows) and between (black arrows) the spines. Bar 200 lm Fig. 5 Spines from the lateral margin of the ventral, anterior midbody region. Severe swelling of the tegument can be seen between the spines (arrows). The tegument covering the spines (S) is also severely swollen. Bar 60 lm Fig. 6 The lateral margin on the dorsal surface of the posterior midbody region. Spines (S) can be seen protruding from the swollen tegument (white arrows) that surrounds them, and the tegument covering the spines themselves is also swollen (black arrows). Bar 50 lm c The online version of the original article can be found at http:// dx.