Using TEM, the number of neutrophils and MCs were counted on two intestinal grids for each infected fish. The number of each type of granulocyte was determined in an area measuring 1800 μm2 in close proximity to the point of cestode attachment (i.e. the interface region) and in a second area measuring 1800 μm2 at a distance of approximately 200 μm from the site of cestode attachment. Prior to analysis, the Gaussian distributions (i.e. normality) find more and the homogeneity of variances of the data were assessed; the data were subsequently square

root transformed to meet these assumptions. Using the software package Statistica 7, anovas (Statistica 7, Praha, Cech Republic) were performed to detect significant differences in the number of granulocytes determined from the uninfected and infected tench and in the abundance of neutrophils and MCs at the point of cestode attachment and then at a distance of 200 μm away. Bonferroni post hoc tests and a P < 0·01 level of

significance were used throughout. Fourteen (60·9%) of the 23 tench were parasitized with M. wageneri; identity of the cestodes was confirmed using morphology and standard taxonomic keys. The intensity of infection ranged from 3 to 130 worms per host (39·5 ± 47·7, mean ± SD). The anterior part of the intestine bore the heaviest infections with the vast majority of tapeworms still attached with their scolices embedded within the intestinal wall (Figure 1a). Upon dissection in situ, M. wageneri were noticed in groups of variable numbers and in some portion of the host intestine the presence of more than one foci was frequent (Figure 1a). In tench gut wall, at the site Pyruvate dehydrogenase lipoamide kinase isozyme 1 of M. wageneri attachment, CHIR-99021 in vitro a raised plaque-like formation or round nodule encircled the firmly attached scolex (Figure 1b). Histological sections revealed that specimen of M. wageneri had penetrated by means

of bluntly truncated scolex deep into the mucosa and submucosa (Figure 2a, b) and in some instances into the muscularis layer (Figure 2c). This parasite anchoring system provided a secure attachment to the tench intestine (Figures 1a, b and 2b). At the site of attachment, the tapeworms induced necrosis, degeneration and/or loss of the epithelium (Figure 2a). M. wageneri elicited intense immune cells and fibroblasts proliferation within the thickness of the tench gut wall (Figure 2b, c). Diffuse hyperplastic inflammation was noticed in tench with few M. wageneri as well as in those harbouring numerous tapeworms (Figure 2a–c). Within the submucosa layer, beneath the point of M. wageneri scolex insertion, numerous granulocytes (e.g. neutrophils, MCs) (Figure 2d), rodlet cells (Figure 2e) and collagenous fibres were observed. Degranulation of the granulocytes, which was visible by light microscopy (Figure 2d), was common in the submucosa. Parasitized intestines were determined to have a significantly higher number of granulocytes than those that were uninfected (Table 1; anova, P < 0·01).