Studies on the comparative biology of Aphanomyces invadans

Abstract

Aphanomyces invadans Willoughby et al, 1995 (as A. invaderis) is the recently-named Oomycete fungus that has been shown to be involved in EUS (epizootic ulcerative syndrome), a highly damaging disease of wild and cultured, Asian freshwater and estuarine fishes. The present study shows that A. invadans is the only species, out of a number of isolates from EUS-affected areas in Thailand, that is capable of sustained growth in snakehead fish muscle tissue and reproducing EUS lesions, and is therefore pathognomic to the disease. A. invadans is characterised, and distinguished from the saprophytic isolates, by means of: growth at various temperatures; growth on different media; level of extracellular enzymes produced; susceptibility to various chemicals; aspects of zoospore and germling behaviour; ultrastructure; immunocytochemistry; protein and carbohydrate electrophoresis banding patterns; lectin and polyclonal antibody binding characteristics by means of Western blot analysis; biochemical fingerprinting using pyrolysis mass spectra (PyMS); and molecular studies involving random amplification of polymorphic DNA (RAPD).

A. invadans is shown to be indistinguishable from pathogenic Aphanomyces isolates from two other fish diseases, namely Japanese mycotic granulomatosis (MG) and Australian red spot disease (RSD) using the techniques described above. RAPD analyses, in particular, showed that a wide ränge of EUS, MG and RSD isolates are not only conspecific, but probably constitute a single genetic clone. This strongly suggests that it is A. invadans, and not any other biological aetiology, that has spread across Asia causing ulcerative disease in fish. It is recommended that the name A. invadans is used to describe all EUS, MG and RSD pathogenic isolates. This work also shows that Aphanomyces isolates obtained from outbreaks of ulcerative mycosis (UM) of American menhaden, are distinct from A. invadans, and more similar to the saprophytic fungus Aphanomyces laevis. It is conjectured that the invasive UM pathogen has not been studied and that this may show greater similarity to A. invadans. In comparison to the other species tested, A. invadans is most similar to the crayfish plague fungus.

Aphanomyces astaci, although A. invadans is shown to be unable to infect noble crayfish {Astacus astacus). Snakeheads {Channa striata) are shown to produce antibodies in response to infection by A. invadans, a finding which may have implications for the possible future development of vaccines. A. invadans is shown to be culturally and ultrastructurally less robust, and more susceptible to chemical treatment, than other saprolegniacean fungi tested, indicating that strategic water treatments, before fish are infected, should be a relatively effective means of control. It is argued that the culturally-fastidious nature of A. invadans could also indicate an inability to compete with natural saprophytes, that may act to restrict it to a pathogenic lifestyle. Possible adaptations of zoospores to pathogenicity include particular chemotactic behaviour; a capability for limited polyplanetism in the presence of a nutrient background, indirect germination, and a form of abbreviated life-cycle. An usually thin zoospore cyst wall, that appears to lack much of the encystment vesicle-derived material apparent on other saprolegniaceans, is believed to have some significance to the ecology of A. invadans, although what this may be is undetermined. Despite the obvious ability of A. invadans to degenerate muscle tissue in fish, cultures showed relatively low production of extracellular enzymes using agar diffusion techniques, indicating that protease activity may be induced.

A. invadans zoospores and cysts' have distinctive lectin-binding characteristics, and of particular interest is their ability to cross-react with monoclonal antibodies raised against Phytophthora cinnamomi, a non-saprolegniacean Oomycete. Other features of A. invadans that may provide useful species-specific taxonomic markers include temperature-growth characters, a putative K body organelle with a distinctive substructure, specific electrophoretic bands, pyrolysis mass spectra (used here for the first time in Oomycete systematics), and RAPD fingerprints. Polyclonal antibodies (PAbs) proved very non-specific, but peroxidase-