Infections of the Norway lobster, Nephrops norvegicus (L.) by dinoflagellate and ciliate parasites
Parasitic dinoflagellates of the genus Hematodinium have been reported from a number of commercially important crustacean hosts, including the Norway lobster, Nephrops norvegicus, from the coastal waters of Scotland. Several methods for detection of the parasite have been developed but each has associated drawbacks. As part of this study, an enzyme linked immunoabsorbent assay (ELISA) has been developed for the detection of the parasite in the haemolymph of N. norvegicus and other crustaceans. The ELISA is a simple, sensitive, reproducible assay, with a detection limit of 5x104 parasites ml-1 haemolyph. To further investigate low-level hematodinium infection in n. norvegicus and other crustacean hosts, a set of Hematodinium-specific polymerase chain reaction (PCR) primers and DNA probes have been developed, based on Hematodinium ribosomal DNA (rDNA) regions. In the PCR assay, a diagnostic band of 380 bp is produced in the presence of parasite DNA. The limit of detection of the assay was found to be 1 ng DNA, which is equivalent to 6 parasites. The DNA probes detected Hematodinium cells in a range of tissues from N. norvegicus and from the crab Carcinus maenas. The level of genetic similarity between nine isolates of Hematodinium originating from several species of Crustacea from the Uk was examined. PCR was used to amplify and sequence the 3' of the small sub-unit (SSU) and the first internal transcribed spacer (ITS1) of the parasite rDNA complex. Analysis of the SSU and ITS 1 sequences revealed that both the regions are highly conserved (92.2%) or greater) between isolates examined, and that there is no apparent geographical separation of isolates. the results suggested that the same species of Hematodinium infects a number of crustacean species from different geographical locations. Hematodinium perezi, the parasitic dinoflagellate of the blue crab Callinectes sapidus, has been successfully isolated and cultured in vitro. Although the in vivo form of this parasite is morphologically and mollecularly very different from that of the Hematodinium sp. infecting N. norvegicus, a number of similar life cycle stages were observed in vitro. These included syncytial networks, filamentous trophonts, and gorgonlock colonies. The isolation and in vitro culture of H. perezi and the Hematodinium sp. infecting n. norvegicus allowed the internal and external enzyme profiles of both species to be examined using the API ZYM system and biochemical assays, leading to the identification of several enzymes that may have pathogenic importance during Hematodinium infections. Differences in the secrretion of acid phosphate and leucine arylamidase by the two Hematodium sp. studied may account for their different levels of virulence and infectivity. A ciliate infection of the wild and laboratory-held N. norvegicus was discovered during the course of this project. Extensive damage to heart muscle tissue was observed in affected lobsters. The ciliate was identified as belonging to the genus Mesanophyrs, based on silver carbonate impregnation of oral structures. However, molecular sequence data (ITS1 and ITS2) indicated that the ciliate sequences have 100% identity with rDNA sequences from Orchitophyra stellarum, a ciliate parasite of sea stars. Since the morphology of O. stellarum differs from that of Mesanophyrs, the possibility arises that the previous studies have misreported the molecular data. Otherwise, it must be concluded that morphological features cannot be used to discriminate between closely related ciliate species. The initiation of in vitro cultures of the ciliate isolated from N. norvegicus allowed the investigation of proteolytic factors that may be involved in the initiation and progression of its infection. The ciliate was found to secrete a number of proteases into the culture medium, and these are exclusively of the metallo type They have gelatinolytic and azocaseinolytic activities and are active at the physiological temperature and haemolyph pH of the host. Secreted proteases were selective in the degradation of several host proteins, including the myosin heavy chain, which is a common structural component of all lobster muscle tissues. Consequently, these proteases may have multiple roles in the invasion and progression of this ciliate infection, or in assisting nutrient uptake by the ciliate. The results of these studies are discussed in terms of the thechnical development of diagnostic assays for Hematodium, their potential application in examining the prevalence and transmission of this parasite in N. norvegicus and other crustaceans, and the potential pathogenic mechanisms involved in parasitic infections of N. norvegicus.