Christina Sommerville

Field trials to evaluate the efficacy of emamectin benzoate in the control of sea lice, Lepeophtheirus salmonis (Krøyer) and Caligus elongatus Nordmann, infestations in Atlantic salmon Salmo salar L.

Abstract Three field trials were conducted to evaluate the efficacy of emamectin benzoate as a treatment for sea lice, Lepeophtheirus salmonis (Kroyer) and Caligus elongatus (Nordmann), infestations on Atlantic salmon Salmo salar (L.). Trials were carried out at sea temperatures of 13.0–15.5°C and 7.2–8.5°C. Salmon naturally infested with sea lice, with mean weights of 438, 513 and 2662 g, respectively, were held in experimental pens on commercial sites. At day −1 or −2, 20 or 30 fish were sampled from each pen to determine pre-treatment numbers of lice. Emamectin benzoate was administered in-feed at a dose of 50 μg kg −1 biomass day −1 for 7 consecutive days. Sea lice were counted again on days 7, 14 and 21, and comparisons made with untreated control fish. Treatment with emamectin benzoate was effective against chalimus and motile stages of sea lice. In all three trials, treated groups were surrounded by pens of heavily infested fish and L. salmonis numbers increased over time on control fish by 87–284%, whereas over the same period, L. salmonis were reduced on treated fish by 68–98%. In the low temperature trial, reductions were slower but numbers were still 90% lower than on control fish at day 21. At the end of the third trial, both control pens were treated with hydrogen peroxide owing to heavy lice burdens. However, L. salmonis numbers rapidly increased again and at day 55, fish treated only with emamectin benzoate still had 80% fewer lice than control fish. In the two summer trials, large numbers of C. elongatus were rapidly reduced by treatment with 82–84% efficacy by day 21. Despite the potential for continuous re-infestation, oral treatment with emamectin benzoate presented an effective means of controlling all parasitic stages of L. salmonis and C. elongatus on farmed salmon, and in one trial, numbers remained lower on treated fish for at least 55 days.

Distribution and characterization of species of Gyrodactylus Nordmann, 1832 (Monogenea) parasitizing salmonids in the UK, and their discrimination from G. salaris Malmberg, 1957

The reported pathogenicity of the monogenean Gyrodactylus salaris Malmberg, 1957 on Atlantic Salmo salar in Norway has necessitated a national survey of salmonid sites throughout the British Isles to determine which species of the genus Gyrodactylus are resident on these hosts in British waters. Eight morphotypes were identified following examination of the opisthaptoral sclerites at both the light and scanning electron microscope level. Gyrodactylus truttae Glaser, 1974 was found on wild brown trout Salmo trutta in Scotland, England and Wales with an uncharacteristic morphotype, having long, thin marginal hooks. Two morphotypes of Gyrodactylus derjavini Mikailov, 1975 were recorded for the first time from Britain. G. caledoniensis n. sp. from Scottish S. salar is erected, based on differences in the shape and size of the marginal hooks. Two possible new species from English Salvelinus alpinus and a possible new species from Welsh S. salar and Scottish O. mykiss are described.

A molecular phylogeny of the genus Ichthyocotylurus (Digenea, Strigeidae).

Three nucleotide data sets, two nuclear (ribosomal internal transcribed spacer regions 1 and 2, ITS1 and ITS2) and one mitochondrial (cytochrome c oxidase subunit 1, CO1), were analysed using distance matrix and maximum likelihood methods to determine the inter-relationships amongst the four species attributed to the genus Ichthyocotylurus Odening, 1969. Sequence data obtained from all gene loci investigated supported the position of Ichthyocotylurus variegatus as a species discrete from Ichthyocotylurus platycephalus. Phylogenetic analyses yielded congruent trees, with I. variegatus isolates comprising a common clade to which I. platycephalus constitutes a sister taxon. Ichthyocotylurus erraticus and Ichthyocotylurus pileatus were found to demonstrate a similarly close inter-specific relationship. The greatest intra-generic divergence occurred in the CO1 region (16% variability), with resultant disparities in three to eight encoded amino acids. PCR amplification yielded multiple ITS1 products for all Ichthyocotylurus spp. Analyses of equivalent-sized amplicons showed 5.4% intra-generic variation and several point mutations between I. variegatus isolates from different geographical localities and from different piscine hosts. The ITS2 locus was extremely conserved, with less than 1% variation between species. No intra-specific variation was recorded for any CO1 or ITS2 sequences.

An SEM study of the haptoral sclerites of the genus Gyrodactylus Nordmann, 1832 (Monogenea) following extraction by digestion and sonication techniques

Techniques for the preparation of haptoral sclerites of Gyrodactylus species for scanning electron microscopy are described. Enzymatic digestion using pepsin allows fresh, frozen and alcohol-fixed material to be examined, but some sclerites, such as the dorsal and ventral bars, are lost. Sonication of fresh and frozen material retained these accessory bars, including the ventral bar membrane, the guard or sickle filament loop on the marginal hooks and even on some occasions ligaments associated with the sclerites. The sclerites of Gyrodactylus spp. from Gasterosteus aculeatus (L.) (three-spined stickleback), Oncorhynchus mykiss (Walbaum) (rainbow trout) and Salvelinus alpinus (L.) (charr) are described from SEM observations following the use of these preparation techniques, and the articulation between the hamuli and bars is commented upon.

Desmozoon lepeophtherii n. gen., n. sp., (Microsporidia: Enterocytozoonidae) infecting the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae)

BackgroundA microsporidian was previously reported to infect the crustacean parasite, Lepeophtheirus salmonis (Krøyer, 1837) (Copepoda, Caligidae), on farmed Atlantic salmon (Salmo salar L.) in Scotland. The microsporidian was shown to be a novel species with a molecular phylogenetic relationship to Nucleospora (Enterocytozoonidae), but the original report did not assign it to a genus or species. Further studies examined the development of the microsporidian in L. salmonis using electron microscopy and re-evaluated the molecular findings using new sequence data available for the group. Here we report a full description for the microsporidian and assign it to a new genus and species.ResultsThe microsporidian infects subcuticular cells that lie on the innermost region of the epidermal tissue layer beneath the cuticle and along the internal haemocoelic divisions. The mature spores are sub-spherical with a single nucleus and an isofilar polar filament with 5-8 turns in a double coil. The entire development is in direct contact with the host cell cytoplasm and is polysporous. During early merogony, a diplokaryotic nuclear arrangement exists which is absent throughout the rest of the developmental cycle. Large merogonial plasmodia form which divide to form single uninucleate sporonts. Sporogonial plasmodia were not observed; instead, binucleate sporonts divide to form two sporoblasts. Prior to final division, there is a precocious development of the polar filament extrusion apparatus which is associated with large electron lucent inclusions (ELIs). Analyses of DNA sequences reveal that the microsporidian is robustly supported in a clade with other members of the Enterocytozoonidae and confirms a close phylogenetic relationship with Nucleospora.ConclusionThe ultrastructural findings of the precocious development of the polar filament and the presence of ELIs are consistent with those of the Enterocytozoonidae. However, the confirmed presence of an early diplokaryotic stage and a merogonial plasmodium that divides to yield uninucleate sporonts instead of transforming into a sporogonial syncitium, are features not currently associated with the family. Yet, analyses of DNA sequence data clearly place the microsporidian within the Enterocytozoonidae. Therefore, due to the novelty of the copepod host, the ultrastructural findings and the robust nature of the phylogenetic analyses, a new genus should be created within the Enterocytozoonide; Desmozoon lepeophtherii n. gen. n. sp. is proposed.

Safety and efficacy of emamectin benzoate administered in-feed to Atlantic salmon, Salmo salar L., smolts in freshwater, as a preventative treatment against infestations of sea lice, Lepeophtheirus salmonis (krøyer)

The safety and efficacy of emamectin benzoate, administered in-feed to Atlantic salmon smolts, Salmo salar L., held in freshwater, was evaluated as a preventative treatment against sea lice, Lepeophtheirus salmonis, following transfer of fish to seawater. In the safety study, salmon smolts held in freshwater were fed with diets containing emamectin benzoate at nominal doses of 0 (control), 50 (recommended dose) and 250 (5× recommended dose) μg kg−1 fish day−1 for 7 days (days 0–6). Actual dose rates, based on measured concentrations of emamectin benzoate in feed, differences in fish weight, and feed consumed, were 0, 54, and 272 μg kg−1 day−1, respectively. On day 9, fish were transferred to seawater and observed for 14 days. No differences in feeding response, coordination, behaviour, gross and histological appearance were observed between control fish and those that received 54 μg kg−1 day−1. Among smolts that received 272 μg kg−1 day−1, approximately 50% exhibited darker coloration, and one fish (1%) exhibited uncoordinated swimming behaviour. No pathognomonic signs of emamectin benzoate toxicity were identified. In the efficacy study, smolts held in freshwater were fed an unmedicated ration (control group) or emamectin benzoate at 50 μg kg−1 day−1 (treated group) for 7 days (days 0–6). On day 9, fish were re-distributed to eight seawater tanks, each holding 30 control and 30 treated fish. On days 28, 56, 77 and 109, respectively, control and treated fish in two tanks were challenged with L. salmonis copepodites. When lice in each group reached chalimus stage IV, fish were sampled and the numbers of lice were recorded. Fish challenged at day 109 were sampled for the second time when lice were at the adult stage. Efficacy was calculated as the reduction in the mean number of lice on treated fish relative to the mean on control fish. Treatment with emamectin benzoate resulted in an efficacy of 85.0–99.8% in fish challenged at days 28–77, from the start of treatment, and lice counts were significantly lower (P<0.001) on treated fish than on controls. When fish challenged at day 109 were sampled at day 128, efficacy was 44.3%, but survival of chalimus to adult lice on treated fish was lower, and at day 159, efficacy had increased to 73%. These results demonstrate that treatment of salmon smolts with emamectin benzoate in freshwater was well tolerated and highly effective in preventing sea lice infestation following transfer of fish to seawater.

Molecular studies on the seasonal occurrence and development of five myxozoans in farmed Salmo trutta L.

Five myxozoan species, Tetracapsuloides bryosalmonae, Sphaerospora truttae, Chloromyxum schurovi, Chloromyxum truttae and a Myxobolus species were detected in farmed brown trout, Salmo trutta L. from Central Scotland. Using PCR and in situ hybridization, this study investigated the seasonal occurrence and tissue location of these species in young of the year brown trout. C. schurovi, C. truttae and Myxobolus sp. were first detected in brown trout in April, 2 months before T. bryosalmonae and S. truttae. T. bryosalmonae and S. truttae showed proliferation in the blood with intravascular stages of T. bryosalmonae accumulating in the heart. In contrast, only small amounts of PCR products of C. schurovi and C. truttae were obtained from the blood, suggesting that these species use the vascular system for transport but proliferate only in their target tissues from which large amounts of PCR product were obtained and where parasites were visible in histological sections. Large amounts of PCR product were obtained for T. bryosalmonae, S. truttae and both Chloromyxum species from the gills of brown trout, suggesting the gills as entry locus for these species. The neurotropic Myxobolus species formed plasmodia predominantly in the peripheral nerves, possibly indicating an entry route through the skin. Presporogonic stages of all other species had disappeared by September and mature spores were present from August onwards.

Multivariate analyses of morphometrical features from Gyrodactylus spp. (Monogenea) parasitising British salmonids: Light microscope based studies

Species determination within the genus Gyrodactylus Nordmann, 1832 is based upon subtle differences in marginal hook morphology and has long posed taxonomic problems. This study uses univariate and multivariate analyses in an attempt to elucidate the Gyrodactylus species parasitising British salmonids. A total of 389 parasites were collected at 69 of 227 localities sampled throughout the British Isles from four salmonid host species, Salmo salar, Salmo trutta, Oncorhynchus mykiss and a limited number of Salvelinus alpinus. A small number of known species of Gyrodactylus from outside the UK were also added to the data set. Morphometric data on the sclerites were collected from slide preparations of Gyrodactylus using light microscopy. Univariate statistics were used to select 11 of the 18 useful morphometric features defined by Malmberg (1970) on the basis of those with the smallest natural variability and/or measurement error. The data, relating to the 11 variables chosen, were then analysed using principal components analysis (PCA) to describe the forms present. Initial outliers included foreign species of Gyrodactylus and an accidental infection by a gasterosteid form. Two major groups were resolved: one on S. salar and O. mykiss, the specimens from which resembled G. derjavini Mikailov, 1975 and G. caledoniensis Shinn et al. in press; and one on S. trutta, which was identified as G. truttae Gläser, 1974. The analysis, in addition to showing which were useful morphometric features, also demonstrated the limitations of data obtained from the smallest features when using the light microscope.

The use of statistical classifiers for the discrimination of species of the genus Gyrodactylus (Monogenea) parasitizing salmonids.

This study applies flexible statistical methods to morphometric measurements obtained via light and scanning electron microscopy (SEM) to discriminate closely related species of Gyrodactylus parasitic on salmonids. For the first analysis, morphometric measurements taken from the opisthaptoral hooks and bars of 5 species of gyrodactylid were derived from images obtained by SEM and used to assess the prediction performance of 4 statistical methods (nearest neighbours; feed-forward neural network; projection pursuit regression and linear discriminant analysis). The performance of 2 methods, nearest neighbours and a feed-forward neural network provided perfect discrimination of G. salaris from 4 other species of Gyrodactylus when using measurements taken from only a single structure, the marginal hook. Data derived from images using light microscopy taken from the full complement of opisthaptoral hooks and bars were also tested and nearest neighbours and linear discriminant analysis gave perfect discrimination of G. salaris from G. derjavini Mikailov, 1975 and G. truttae Gläser, 1974. The nearest neighbours method had the least misclassifications and was therefore assessed further for the analysis of individual hooks. Five morphometric parameters from the marginal hook subset (total length, shaft length, sickle length, sickle proximal width and sickle distal width) gave near perfect discrimination of G. salaris. For perfect discrimination therefore, larger numbers of parameters are required at the light level than at the SEM level.

Molecular identity, phylogeny and life cycle of Chloromyxum schurovi Shul'man & Ieshko 2003

A myxosporean of the genus Chloromyxum sp., which sporulates in the kidney tubules, was detected in Atlantic salmon, Salmo salar L., and brown trout, Salmo trutta L., from three sites in Scotland. A comparison with other reports of Chloromyxum spores in the kidney of European salmonids suggests that they all refer to the same species, Chloromyxum schurovi [Shul’man BS, Ieshko EP (2003). Parazitologia 37:246–247]. Apart from Chloromyxum majori [Yasutake WT, Wood EM (1957). J Parasitol 43:633–637] which has a different geographical and host range, as well as tissue localisation, this is the only representative of the genus in the kidney of salmonids described so far. This study provides a re-description of C. schurovi including its myxosporean and actinosporean stage, i.e. Neoactinomyxum eiseniellae [Ormieres R, Frezil JL (1969). Protistologica 5:137–144], which was identified by 18S rDNA sequence identity. Results of phylogenetic analysis of the 18S rDNA sequence of C. schurovi and all other Chloromyxum species with published sequences showed that C. schurovi clusters with representatives of other myxozoan genera which inhabit the urinary system, whereas the other Chloromyxum species form a more basic clade together with another gall bladder parasite.