Neobenedenia girellae, a capsalid monogenean, is a destructive fish parasite. We studied the lipid content and fatty acid composition of N. girellae and the skin and cutaneous mucus of a host fish, the amberjack Seriola dumerili (Carangidae). The lipid content of adult N. girellae was less than one-fourth that of both the skin and cutaneous mucus of its host. Adult N. girellae, S. dumerili skin and mucus had a relatively high weight-percentage of C16:0, C18:1(n-9), C18:0 and C22:6(n-3) fatty acids. When S. dumerili were fed a diet supplemented with [13C] fatty acids, [13C] fatty acids were detected in S. dumerili skin and adult N. girellae on S. dumerili, but no [13C] fatty acids were detected in the S. dumerili cutaneous mucus. In addition, the epidermis of S. dumerili, attached with N. girellae, was markedly thin. These results suggest that N. girellae feeds primarily on host epithelial cells. We then infected 2 host fishes, S. dumerili and the spotted halibut Verasper variegatus (Pleuronectidae; a host less susceptible to N. girellae infection), and compared the fatty acid composition of N. girellae with that of the skin and cutaneous mucus of the hosts. The fatty acid profiles from all samples were qualitatively and quantitatively similar. Thus, the fatty acid composition of the host may not contribute to the difference in susceptibility between S. dumerili and V. variegatus. These results may serve to develop new strategies for the control of N. girellae infections.

Mannose-binding lectins were purified from flatfish spotted halibut (Verasper variegatus) serum. These lectins, which we named VVL (Verasper variegatus lectin)-α (~33 kDa) and VVL-β (~30 kDa) (VVLs), under non-reducing SDS-PAGE, were surprisingly highly concentrated in serum (1·92±0·55 mg/ml; n=5), compared with other serum lectins. Both VVLs are heterodimers comprised of 2 types of subunit via inter-subunit disulfide bonds, and one subunit of VVL-α has a N-linked sugar chain. Based on N-terminal amino acid sequences, the nucleotide sequences of one subunit of VVL cDNAs were determined by 3′- and 5′-rapid amplification of the cDNA ends. The full-length VVL subunit cDNAs contained 489 bp, encoding an open reading frame of 163 amino acids. We found that VVLs bind to an ~8 kDa ciliary surface glycoprotein (a putative agglutination/immobilization antigen that we reported previously) of the fish parasite Neobenedenia girellae, and agglutinate this parasite in vitro.

Neobenedenia girellae, a capsalid monogenean, is a significant pathogen due to both its ability to cause high mortality in fishes and its low host specificity. Established control methods have both advantages and disadvantages. Biological control measures with no unfavourable effects on the environment should be incorporated into the control strategy. The response of N. girellae oncomiracidia to brightness and black-and-white contrast was investigated to search for an alternative approach of disease prevention or control. Japanese flounder, Paralichthys olivaceus (Paralichthyidae), were exposed to oncomiracidia in an aquarium divided into areas of different brightness (∼1·3, 41·3 and 138·0 lux). The number of parasites on the fish group reared in 138·0 lux was significantly higher than on those reared in the lower brightness levels. Thus, the fish tended to be more vulnerable to infection by N. girellae under brighter conditions. Challenge trials using host fish mucus and whole live fish were established to detect the response by oncomiracidia to black-and-white contrast on a white versus a black background. Markedly more N. girellae oncomiracidia attached to black-painted areas and dark-coloured fish (normal spotted halibut, Verasper variegatus (Pleuronectidae) compared with white-painted areas and light-coloured fish (mal-coloured V. variegatus) on a white-coloured background. On a black-coloured background, more N. girellae oncomiracidia tended to attach to white-painted areas and light-coloured fish. Thus, black-and-white contrast is considered important for host finding by N. girellae oncomiracidia. The simplicity of the positive phototactic behaviour and the response to black-and-white contrast may lead to the development of a simple, practical and inexpensive method to control N. girellae outbreaks.

This study investigated a new effective method for controlling the capsalid monogenean Neobenedenia girellae. We examined in vitro and in vivo the effect on the percentage survival of N. girellae in buffers containing different metallic ions. Decreased survival was observed in buffer solutions lacking two ions. In particular, the percentage survival of N. girellae was significantly decreased after 10 min exposure to buffer containing neither Ca2+ nor Mg2+. Transmission electron microscopic observations showed that treatment with this buffer disrupted intercellular junctions. This significant effect on percentage survival of N. girellae using Ca2+/Mg2+-free buffer was confirmed in an in vivo assay. Ca2+/Mg2+-free buffer had no effect on the condition of the host, spotted halibut Verasper variegates (Pleuronectidae). These results suggest that treatment with Ca2+/Mg2+-free buffer is a new effective control method, which could replace existing control methods.

The capsalid monogenean Neobenedenia girellae, a parasite of seawater fishes, was found to express an antigen that elicits antibodies in rabbits, and these antibodies had agglutination/immobilization activity against N. girellae larvae (oncomiracidia) in vitro. Indirect immunofluorescence staining of N. girellae oncomiracidia showed that this agglutination/immobilization antigen was expressed on the surface of cilia. An intraperitoneal injection of ciliary proteins (either sonicated or intact) with adjuvant also elicited agglutinizing/immobilizing antibodies in sera from Japanese flounder, Paralichthys olivaceus. These antisera showed a clear correlation between anti-ciliary antibody levels (measured by enzyme-linked immunosorbent assays) and their agglutination/immobilization activity. Anti-ciliary antibody levels in Japanese flounder reached a plateau at 39 days after booster immunization and were significantly higher in the two immunized groups (injection of sonicated or intact cilia) as compared with control fish (injection of bovine serum albumin; ANOVA, Tukey's test, P<0·01). Anti-ciliary antibodies were also found in fish mucus; however, there was no correlation between fish serum anti-ciliary antibody levels and mucus antibody levels. A Triton X-114-soluble 8 kDa glycoprotein of the ciliary integral membrane fraction is a plausible candidate for the agglutination/immobilization antigen based on SDS-polyacrylamide gel electrophoresis and immunoblot analyses with rabbit and fish antisera.