Stable isotopes reveal contrasting trophic dynamics between host-parasite relationships: A case study of Atlantic salmon (Salmo salar) and parasitic lice (Lepeophtheirus salmonis and Argulus foliaceus).

Across existing fish host-parasite literature, endoparasites were depleted in δ15 N compared to their hosts, while ectoparasitic values demonstrated enrichment, depletion and equivalence relative to their hosts. δ13 C enrichment varied extensively for both endo- and ectoparasites across taxa and host tissues. In our case study, sea lice (Lepeophtheirus salmonis) were enriched in δ15 N relative to their farmed Atlantic salmon (Salmo salar) hosts, although the value contradicted the average that is currently assumed across the animal kingdom. Common fish lice (Argulus foliaceus) did not show a consistent trend in δ15 N compared to their wild S. salar hosts. Both parasitic species had a range of δ13 C enrichment patterns relative to their hosts. Farmed and wild S. salar had contrasting δ13 C and δ15 N, and signals varied across muscle, fin and skin within both groups. L. salmonis and A. foliaceus subsequently had unique δ13 C and δ15 N, and L. salmonis from opposite US coasts differed in δ15 N. Given the range of enrichment patterns that were exhibited across the literature and in our study system, trophic dynamics from host to parasite do not conform to traditional prey to predator standards. Furthermore, there does not appear to be a universal enrichment pathway for δ13 C nor δ15 N in parasitic relationships, which emphasizes the need to investigate host-parasite linkages across species.

Anti-chemotactic activity in the secretory/excretory products of Lepeophtheirus salmonis.

The ectoparasite, Lepeophtheirus salmonis (Kroyer 1837), is effective at avoiding elimination from its host, Atlantic salmon, Salmo salar L., by inhibiting the recruitment of immune cells to the site of attachment. In other ectoparasitic arthropods, numerous factors have been identified that bind or neutralize chemokines preventing their interaction with receptors on the surfaces of immune cells. To determine if L. salmonis is utilizing a similar mechanism of immune modulation, the chemotactic activity of peripheral blood leukocytes (PBL) to leukotriene B4 (LTB4) and the secreted/excreted products (SEPs) of the sea louse were investigated in vitro. The results showed that incubation of LTB4 with SEPs reduced leukocyte migration compared to LTB4 immune stimulation alone. Data suggests that one of the mechanisms L. salmonis may be using to regulate immune cell recruitment in Atlantic salmon is by inhibiting or neutralizing the activity of chemokines.

Sea lice, Lepeophtheirus salmonis (Krøyer 1837), infected Atlantic salmon (Salmo salar L.) are more susceptible to infectious salmon anemia virus.

The role of parasitic sea lice (Siphonostomatoida; Caligidae), especially Lepeophtheirus salmonis, in the epidemiology of Infectious Salmon Anemia Virus (ISAv) has long been suspected. The epidemiological studies conducted during the 1998 major Infectious Salmon Anaemia (ISA) outbreak in Scotland demonstrated a strong correlation between sea lice presence and ISAv positive sites or subsequent clinical outbreaks of ISA. The question posed from this observation was "do sea lice infestations on Atlantic salmon make them more susceptible to viral infections?" This study investigated the role that sea lice infestations have on the severity of ISAv infections and disease mortality in experimental populations of farmed Atlantic salmon (Salmo salar). A series of experiments was carried out that investigated the potential of sea lice to modify the outcome of an ISAv infection. Experimental populations of Atlantic salmon were established that had: no lice and no ISAv, a single infection with either ISAv or lice and a co-infection with lice then ISAV. The results were quite clear, the process of infestation by the parasite prior to ISAv exposure significantly increased the mortality and death rates of Atlantic salmon, when compared to uninfected controls and ISAv infected groups only. This was consistent over two source strains of Atlantic salmon (Pennobscot and Saint John River), but the severity and timing was altered. Immunological responses were also consistent in that pro-inflammatory genes were induced in lice only and co-infected fish, whereas the anti-viral response, Mx, MH class I ß, Galectin 9 and TRIM 16, 25 genes were down-regulated by lice infection prior to and shortly after co-infection with ISAv. It is concluded that the sea lice settlement on Atlantic salmon and the parasite's subsequent manipulation of the host's immune system, which increases parasite settlement success, also increased susceptibility to ISAv.

Peptidylarginine deiminase and deiminated proteins are detected throughout early halibut ontogeny - Complement components C3 and C4 are post-translationally deiminated in halibut (Hippoglossus hippoglossus L.).

Post-translational protein deimination is mediated by peptidylarginine deiminases (PADs), which are calcium dependent enzymes conserved throughout phylogeny with physiological and pathophysiological roles. Protein deimination occurs via the conversion of protein arginine into citrulline, leading to structural and functional changes in target proteins. In a continuous series of early halibut development from 37 to 1050° d, PAD, total deiminated proteins and deiminated histone H3 showed variation in temporal and spatial detection in various organs including yolksac, muscle, skin, liver, brain, eye, spinal cord, chondrocytes, heart, intestines, kidney and pancreas throughout early ontogeny. For the first time in any species, deimination of complement components C3 and C4 is shown in halibut serum, indicating a novel mechanism of complement regulation in immune responses and homeostasis. Proteomic analysis of deiminated target proteins in halibut serum further identified complement components C5, C7, C8 C9 and C1 inhibitor, as well as various other immunogenic, metabolic, cytoskeletal and nuclear proteins. Post-translational deimination may facilitate protein moonlighting, an evolutionary conserved phenomenon, allowing one polypeptide chain to carry out various functions to meet functional requirements for diverse roles in immune defences and tissue remodelling.

Survival and replication of Piscirickettsia salmonis in rainbow trout head kidney macrophages.

Piscirickettsia salmonis is pathogenic for a variety of cultured marine fish species worldwide. The organism has been observed within host macrophages in natural disease outbreaks among coho salmon and European sea bass. In vitro studies, incorporating transmission electron microscopy (TEM) and ferritin loading of lysosomes, have confirmed that P. salmonis is capable of surviving and replicating in rainbow trout macrophages. Certain features of this intracellular survival underline its difference to other intracellular pathogens and suggest that a novel combination of defence mechanisms may be involved. Escape into the macrophage cytoplasm is not used as a means to avoid phago-lysosomal fusion and the organism remains at least partly enclosed within a vacuole membrane. While the piscirickettsial vacuole is often incomplete, survival and replication appear to require occupation of a complete, tightly-apposed, vacuolar membrane which does not fuse with lysosomes. Unlike some mammalian rickettsiae, actin-based motility (ABM) is not used as a means of intercellular spread. It is postulated that the presence of numerous small vesicles within vacuoles, and at gaps in the vacuolar membrane, may result from the blebbing of the piscirickettsial outer membrane seen early in the infection.

Effect of environmental salinity on sea lice Lepeophtheirus salmonis settlement success.

The sea louse Lepeophtheirus salmonis (Krøyer, 1837) (Copepoda: Caligidae) is an ectoparasite of salmonid fish. It has earlier been proposed that the free-swimming infectious copepodid stage of L. salmonis gather at river mouths to infect wild Atlantic salmon Salmo salar L. and sea trout S. trutta L. smolts during their seaward migration. This study used aquarium-based methods to investigate the survival, infective ability and behaviour of L. salmonis copepodids exposed to short periods of low salinity levels, such as those encountered at river mouths. Survival of free-swimming copepodids was found to be severely compromised at salinity levels below 29 parts per thousand (ppt). Attachment to an S. salar host did not aid copepodid survival during post-infection exposure to low salinity environment, and a reduction in salinity appears to reduce the ability of copepodids to remain attached to S. salar smolts. Pre-infection exposure of copepodids to reduced salinity levels reduced infection of S. salar. Infection levels at reduced salinity were lower than predicted from the free-swimming survival experiment, suggesting that low salinity compromises the copepodids' ability to sense or respond to the presence of a host. In salinity gradients, copepodids demonstrated avoidance of salinities below 27 ppt, by both altering their swimming behaviour and changing the orientation of passive sinking. Avoidance of low salinity levels may be due to their adverse effects on copepodid physiology, as suggested by the reduction in survival. Sinking rates were also faster in reduced salinity, suggesting that remaining in the water column would be more energetically demanding for the copepodids at reduced salinity. These results show that both survival and host infectivity of L. salmonis are severely compromised by short-term exposure to reduced salinity levels.

Development and application of real-time PCR for specific detection of Lepeophtheirus salmonis and Caligus elongatus larvae in Scottish plankton samples.

Lepeophtheirus salmonis and Caligus elongatus are important parasites of wild and cultured salmonids in the Northern Hemisphere. These species, generically referred to as sea lice, are estimated to cost the Scottish aquaculture industry in excess of pound 25 million per annum. There is great interest in countries such as Ireland, Scotland, Norway and Canada to sample sea lice larvae in their natural environment in order to understand lice larvae distribution and improve parasite control. Microscopy is currently relied on for use in the routine identification of sea lice larvae in plankton samples. This method is, however, limited by its time-consuming nature and requirement for highly skilled personnel. The development of alternative methods for the detection of sea lice larvae which might be used to complement and support microscopic examinations of environmental samples is thus desirable. In this study, a genetic method utilising a real-time PCR Taqman-MGB probe-based assay targeting the mitochondrial cytochrome oxidase I (mtCOI) gene was developed, which allowed species-specific detection of L. salmonis and C. elongatus larvae from unsorted natural and spiked plankton samples. Real-time PCR is a rapid, sensitive, highly specific and potentially quantitative technique. This study demonstrated its suitability for the routine identification of L. salmonis and C. elongatus in mixed plankton samples. The real-time PCR assay developed has considerable potential for use in complementing, supporting and reducing reliance on time-consuming conventional microscopic examination for the specific identification of sea lice larvae in plankton samples.

Long-term study of antibody response and injection-site effects of oil adjuvants in Atlantic halibut (Hippoglossus hippoglossus L).

Atlantic halibut were injected intraperitoneally with human gamma globulin suspended in either phosphate buffered saline, Freunds complete adjuvant or Montanide ISA711 to test the long-term effects of adjuvants. Every month for 12 months up to five animals from each group were sampled. The peritoneal cavity was examined and the adhesion level scored on an arbitrary scale. Serum was also collected and analysed by ELISA for antibodies to human gamma globulin. Results show that whilst FCA produced the highest and fastest antibody response, it also produced the fastest intraperitoneal adhesions, persisting through 11 months. However, the adhesions were not very severe and did not appear to affect the halibut. Crown

Development of vaccines against sea lice.

A review of efforts to develop a vaccine against sea lice is presented together with analysis of the rationale behind the approaches and potential future directions. Vaccines against the caligid copepod, Lepeophtheirus salmonis, have the potential to be a cost-effective means of controlling the infection and avoid many of the disadvantages of medicine treatments. However, research towards such vaccines is in its infancy and approaches so far used have met with little or no success. Most strategies for sea louse vaccines have adopted methods used for vaccines against other ectoparasites. A vaccine against the cattle tick (Boophilus microplus) is in field use while other vaccines such as the sheep blowfly (Lucilia cuprina) vaccine are at an earlier stage of development. These haematophageous parasites ingest host antibody as part of a large blood meal which can target antigenic sites in the gut. However, the assumption that arachnid and insect physiology are directly comparable with that of sea lice is not proven, and this may partly explain the slow progress this approach has had with sea lice. Success in developing a louse vaccine will depend upon a better understanding of louse digestive biology, particularly an evaluation of whether the cattle tick model is applicable to the development of a louse vaccine. If the louse gut is to be targeted immunologically, critical antigens will need to be identified and evaluated, bearing in mind that an economic vaccine must include recombinant proteins or be a DNA vaccine. Alternatives to the louse gut as a target are also worth consideration. Antibodies could target critical host-parasite interactions that are amenable to disruption, although no such targets have been identified.