Potential biological control agents for the salmon louse Lepeophtheirus salmonis (Kroyer, 1837)

The salmon louse, Lepeophtheirus salmonis (Krøyer), is an obligate ectoparasitic \ncopepod that lives on the external surface of salmonid fish. It is the most common \nparasite on cage reared Atlantic salmon (Salmo salar L.), costing the aquaculture industry \nin Scotland millions of pounds each year to control. Traditional methods used to control \nsea lice have centred on the use of chemical pesticide treatments, which are expensive, \nhazardous to handle, potentially deleterious to the marine environment, and are \nsometimes ineffective. Furthermore, the misuse use of two previously efficacious \nchemotherapeutants has led to a build up of resistance in sea lice populations. \nThe aims of this study were to investigate a potential alternative control strategy, \nconcentrating on the isolation of the naturally occurring enemies of sea lice, and \nevaluating their potential for use as biological control agents. A screening protocol was \nundertaken to examine sea lice removed from harvest size farmed fish and wild Atlantic \nsalmon, to look for the presence of epibiotic and hyperparasitic organisms on the external \nsurfaces of the sea lice. A screening protocol was also undertaken to look for invasive \nmicroorganisms, such as fungal pathogens, internal symbionts, and other internal \nhyperparasites. \nSea lice were examined microscopically for the presence of external epibionts and \nobvious signs of internal invasion by microorganisms and parasites. Surface sterilised sea \nlice were incubated on growth media to screen for the presence of fungal pathogens. \nFresh tissue squashes were performed on lice showing clinical signs of infection and \nscreening / diagnostic PCRs were used to detect and identify endosymbionts and invasive pathogens and parasites. Wax histology, TEM and SEM were used to further investigate \nhost parasite interactions in order to evaluate pathogenicity where appropriate. In vitro \nand in vivo challenge trials were performed with an isolated hyperparasite to effect \ntransmission and to determine pathogenicity. \nThe stalked suctorian ciliates Ephelota gemmipara and Ephelota gigantea, and the \nmonogenean worm Udonella sp. were frequently found utilising L. salmonis as a \nsubstrate. High densities of Udonella were observed on the cephalothoracic shield and \ngenital segment of adult lice, and very high densities of Ephelota spp. were found on the \ngenital segment, abdomen, and egg strings. The prevalence and seasonal pattern of \noccurrence of these epibionts were assessed. \nFourteen fungal isolates were obtained, but failed to sporulate in vitro culture and were \nhence not identifiable. No endosymbionts were detected in either the body cavity or the \negg strings by using the screening and diagnostic PCRs. \nAn hyperparasitic microsporidian was found heavily parasitising adult sea lice. The \nmicrosporidian infection arose in the epidermal cells lying beneath the cuticle, infection \nwas not observed in other tissues. Horizontal transmission was assumed as developing \neggs and egg strings were not seen to be infected. The ultrastructure and complete \ndevelopmental cycle of this hyperparasite in the salmon louse were described. Specific \nPCR primers were designed for use as a diagnostic tool and a molecular phylogeny was \nconstructed using rRNA gene sequences. Taking into account its taxonomic positioning, \nits morphology and its unique characteristics, it appears that this parasite represents both \na new genus and a new species. The microsporidian was not found at all farm sites visited \nand was not detected in sea lice from wild caught Atlantic salmon. The microsporidian infected up to 10% of lice sampled when most prevalent, but was sometimes absent, and \nshowed no clear seasonal pattern in its occurrence. Transmission of the microsporidian \nhyperparasite to uninfected sea lice was not achieved and evidence for an alternative / \nintermediate host for the microsporidian is presented. This is the first report of a \nmicrosporidian from sea lice, and indeed from marine copepods; it is also the first report \nof a hyperparasitic microsporidian in crustacea. \nThe potential for the epibionts and endobionts found in association with sea lice to be \nused as biological control agents are presented.