Amoebic Gill Disease in the Atlantic Salmon - Case Study Example

Summary The use of chemical treatment for amoebic gill disease (AGD) in the Atlantic salmon, Salmo Salar L., which is cultured in Tasmania has beenexamined in this study. The main causative agent for the disease has been found to be Neoparameoba perurans. The disease is found to occur in fishes cultured in the marine environment in many countries has caused losses in fish productivity and the expenditure to manage the disease has also increased. The employment of freshwater bathing practices to reduce the incidence of the disease has become less efficient and subsequently many disinfectants and antimicrobials have been used under in vitro and in vivo conditions to mitigate AGD. The use of chemical agents for the treatment of the disease has been limited mainly owing to its less effective nature on the parasite, toxicity to fishes and cost of the treatment process. However, several compounds have been tested for their toxicity both under in vitro conditions and as in-feed components including the mucolytic compound L-cysteine ethyl ester but most of these compounds were either not commercially viable or lacked efficacy. One compound that was found to be both non-toxic and efficacious was bithionol, a phenolic compound which significantly reduced the pathology of the disease when administered as an in-feed compound. The current study examined the efficacy of oral administration of bithionol along with the standard freshwater bath treatment and also to deduce the rate of re-infection The study used mixed-sex, diploid Atlantic salmons with a mean mass of 130.4g and they were acclimatized to sea water conditions over a period of 3 weeks in an aquaculture center. A total of 396 Atlantic salmon (AS) were equally allocated into 9 tanks which were present as three separate ultraviolet light-treated sea water systems. The tanks received constant aeration and the fishes were allowed to acclimatize for 1 week within these systems and were fed with a commercial feed to satiation before commencing the experiment. Each tank was then randomly allocated a treatment and the treatment groups included a control which was a commercial diet with oil, prophylactic bithionol and therapeutic bithionol. The fishes were fed at 1% of their body weight and the daily and weekly feed intake was determined. The feeding was continued for 14 days after which the fishes were exposed to the Neoparamoeba spp., which were isolated from the gills of infected AS by removing the amoeba from the gills followed by centrifugation and concentration. This isolation process yielded a final concentration of 300 cells per liter in each system. After 28 days of exposure freshwater was administered for 3 hours during which all the essential parameters such as temperature and pH were constantly monitored. The feed used in all the treatments was AS Grower LE pellets. In the control diet the pellets were given an even coating with fish oil followed by air drying them in a fume hood. The air dried feed was placed in an air-tight container and stored at 4 degrees C. For the bithionol, the compound was combined with fish oil prior to coating the feed. The data collected during the study include the total tank mass of fish which was collected weekly with a minimum of half the fish present per tank during the measurement. Additionally the fishes were also sampled prior to exposure, during the inoculation of the microbe, every week after exposure and prior and after freshwater bath. Apart from the mass and fork length, the internal organs of the fishes were analyzed for signs of toxicity. The Neoparamoeba spp., were isolated from the right gill basket using the same method as above before and after freshwater bath treatment and total number of live amoeba per fish was calculated. To evaluate the pathology of the disease, the number of filaments and the size of the AGD lesions along with other pathological changes in liver, kidney and muscle tissue sections were analyzed. The statistical analysis carried out includes analysis of variance to determine the differences between treatment and day sampled and also among tank variables such as feed intake. Homogeneity and relative percent reduction of the lesioned gill filaments were also calculated. The results are represented as graphs and tables. No significant change in feed intake was observed in all the treatment tanks in the initial days of the trial, however a decrease in the intake was observed in all the treatments from day 14 which corresponded with increased parasitic load. As there was no difference in feed intakes between the different treatments tanks the mean condition factor was same in all the treatments throughout the trial. All the fishes exhibited signs of the disease and as a result mortality was also observed in the different treatment tanks. However no host toxicity was observed. The clinical signs of AGD were significantly less among fish administered bithionol both prophylactically and therapeutically as compared to the control fishes which showed increased gill lesions. Treatment with bithionol reduced the lesions and gross gill score by up to 40% and 2.8 respectively by the end of the trial. These values dropped after the administration of the freshwater bath and then began to rise again. A 20-30% reduction in lesion size was observed in both the bithionol treatment tanks compared to the controls with the freshwater bath having no effect on lesion size. The number of crude amoeba was found to be more in the control tanks compared to the bithionol treated tanks prior to freshwater treatment. However, the crude amoeba numbers decreased in all the tanks similarly after the freshwater bath. Thus administration of bithionol as a treatment significantly reduced the clinical signs of AGD. However, this difference in clinical signs between the control and bithionol treated groups decreased as the days progressed, but nevertheless the number of filaments affected as well as the gross sill score was still less compared to the controls. As a prophylactic treatment, bithionol significantly reduced the gross sill score and lesions over the 28 day period. As reported in previous studies, bithionol was also found to be effective against the parasite Microcotyle sebastis which infected the marine rockfish. However, it was found to be ineffective against certain other parasites such as Hexamita salmonis. The crude amoeba numbers were also found to be less in the treated tanks compared to the controls prior to freshwater bath. But the amoeba numbers were reduced to 90% in all tanks irrespective of the treatment. The same effect was not observed with the percent lesions and the gross gill score which were only reduced proportionately in each of the tanks based on the treatment modality. The signs that signal the need for a freshwater bath are gross gills score of two or 25% lesioned filaments. This condition was observed among the medicated fishes only a week later as compared to the control ones. Another disadvantage associated with freshwater bathing is its capacity to remove only two-thirds of the amoebae necessitating the need for more freshwater baths. The subsequent bathing time was reached by the control fishes much earlier compared to the medicated fishes. Thus the results of the study show that using both bithionol treatment and freshwater baths will cause a significant reduction in the clinical signs of the disease. The importance of freshwater bath was observed by a significant reduction in lesion size after the baths were administered and the size of the lesion did not increase thereafter. Additionally, a reduction in the percent lesions was also observed suggesting the bath may have removed smaller lesions. Thus the study reported the effectiveness of bithionol in reducing the number of lesions and its size and the gross sill score and further research is required to determine in-feed and combination efficiency of bithionol with freshwater bath. Summary 2 This study analyzed the impact of freshwater invertebrate diets on the growth and fatty acid profiles of Salmo salar, which are used for river restocking. As the fatty acid (FA) profiles of these invertebrates differ significantly from those present in the fish oils which are generally used in marine farm diets. The lipids present in these invertebrates were more similar to vegetable oils, which are scarcely used in traditional salmonid aquaculture. Comparative studies on farmed fishes and their wild counterparts reveal differences in fatty acid composition. While the farmed salmon were found to be rich in n-3 highly unsaturated fatty acids (HUFA) and a high n-3 to n-6 polyunsaturated fatty acid (PUFA) ratio, the wild parr varieties fed with vegetables oil had lower amounts of the above fatty acids and had a higher C18 PUFA content. Despite these differences vegetable oil based diets promote smoltification and also enhance the osmoregulatory ability of fishes which would help them to adapt to salinity changes. Additionally dietary FA must provide suitable nutrients that would ensure adequate growth of the fishes. Hence the use of fish oil diets presents more disadvantages. Some studies have recorded the differences in FA composition in freshwater invertebrates present upstream and downstream of a river. Consumption of such insects by salmons would in turn have an effect on their growth and metabolism. The main aim of the present study was to determine the dietary origin of the FA accumulated by the restocked S.salar parr during extreme winter conditions and migrations and their subsequent effect on the growth and FA composition of the fishes. Restocked fishes from three areas upstream and downstream of the River Allier were analyzed and the results were compared with hatchery-reared parr and restocked parr fed with the traditional fish oil. Three sampling sites present upstream and downstream of the Allier River were chosen for the study and the sampling was done in riffles which were free from natural spawning. The river was restocked with 0+ parr and 1+ smolts which were reared in a hatchery. A sample of the juveniles raised in the hatchery were separated and reared in the hatchery and were given a fish oil diet while the remaining juveniles were restocked in the river. In the hatchery the Parr were fed 6 times daily and 7 reared salmon were collected every month from June to October. The fishes were collected from the river riffles by electrofishing when the desired densities were reached and following a 3 week acclimatization period sampling was carried out similar to the reared fishes. All the fishes collected were lyophilized prior to determination of dry weight and lipid analysis. The food items found the restocked salmon were identified to the genus level and three families: baetid, chironomid and simuliid were analyzed for neutral lipid (NL) and polar lipid (PL) FA composition by collecting them from each riffle. Lipid analysis was done in the whole salmon parr, macroinvertebrates collected and artificial pellets given to the reared parr by extracting the total lipids using methanol and chloroform. The FA was identified by gas chromatography. The fatty acids which were frequently present included 16:0, 16:1n-7 and 18:1n-9 whose concentrations were analyzed statistically. Mean, standard errors and analysis of variance were used to determine the differences in FA concentrations present in the different sources. In case of parr groups which had similar FA concentrations, discriminate was used to differentiate the parr groups. The study identified the presence of 25 prey taxa within the stomach of the parr fishes with the predominant pray being Diptera larvae and Ephemeroptera nymphs. The major larvae identified in the three sites include chironomid, simuliid and baetid nymphs respectively. The results also revealed a significant variation in dry weight of restocking parr and hatchery reared parr. For the restocked parr maximum cry weights were recorded for parr obtained from sites 2 and 3, while the maximum dry weight of the hatchery reared parr was found to more than the restocked parr. Variations in growth rate were observed monthly with the rate being low in the initial 2 months and increasing thereafter. Variations were observed in the FA composition with the NLFA content higher in the hatchery-reared parr during the first 2 months. However, by the end of the study no significant difference was observed with the same. Monthly variations in the FA content were noted between the parr obtained from each riffle and from the hatchery with an initial decrease, followed by an increase and stabilization towards the end of the study between all sites except site 3. While the concentrations of PLFA initially decreased in both the riffle and reared fishes, the PLFA concentration in the farm fish was similar to that found in site 2. The FA composition in each type of food included saturated FA with 16:0 being the predominant FA, MUFA with 16:1n-7 and 18:1n-9 present in high amounts. The PUFA content was found to be more in the pellets compared to the natural diets with 20:5n-3 being the most abundant FA. The results show that the n-3 PUFA was most abundantly present than the n-6 FA in all types of diet. The sum of both these FA was high in pellets and chironomids than the other foods. Discriminant function analysis was carried out for 8 FA which were present in similar concentrations in NL and PL. This analysis helped to differentiate the FA concentrations found the various foods. Monthly variations in the FA content in the NL of the hatchery and restocked fishes were also observed with certain FA present in higher amounts in both the fishes. The PLFA content varied only for a few FA between the hatchery and restocked parr and towards the end of the study the various FA was found to be more in the restocked parr compared to the hatchery- reared parr with the first riffle site have a higher content of FA. All the FA was provided as NL in the hatchery feed while the FA in the riffles was present as both NL and PL. The NL 16:0, 16:1n-7 were abundant in the parr obtained from riffle sites 2 and 3, 18:1n-9 was present in large amount in the artificial food and in the NL and PL of simuliids. The n-3 PUFA, 18:3n-3 was found abundantly in the food present the first riffle site, 22:6n-3 was more in natural invertebrates, and 20:5n-3 was largely present in the pellets, chironomid larvae, baetids and simuliids which were present in site 2 and 3. The 18:2n-6 was the richest n-6 PUFA present in all the foods, while the simuliids in site 2 contained a good number of 20:4n-6. Thus only three natural preys: chironomid, simuliid and baetid were found along the upstream and downstream of the river at the three sites respectively. The FA from secondary prey sources were not considered in the present study. Variations in growth were observed among the parr collected from the riffles and those reared in the hatchery which could be related to the type of food ingested and on the amount of energy expenditure. However, the digestibility and the calorific values of the preys differ and hence the growth variation cannot be solely attributed to variations in FA among the different foods. Thus the main aim of the study was to determine whether the different foods would change the FA composition in the body of the fishes. The results obtained in the study indicate that consumption of natural and artificial feeds changed the NL and PL patterns of the AS. The utilization of the various FA also varied among the restocked and reared parr depending on the energy requirement, however, by the end of the study the FA content in the restocked and reared parr were almost similar. The study also determined the differences in n-3 and n-6 PUFA contents in the ingested foods and also noted the presence of FA as PL and NL at the different riffle sites and in the artificial foods. The study recommends site 2 for ideal parr growth as they were comparable to that of hatchery-reared parr. Summary 3 Gene flow promotes population connectivity which plays a vital role in conservation of species. Gene flow in turn depends on the selection strength which dictates dispersal of an organism depending on the environmental conditions. This is a key area in the study of metapopulation biology. When the conditions are in favor of survival of the local organisms, dispersal does not occur. However, under changing habitat conditions and the need to avoid inbreeding or to thrive in new habitats dispersal of organisms is favored. Thus changes in the environment and demographics have an effect on the stability and genetic structure of certain organisms. This study aimed to determine such differences in the Atlantic salmon (AS), Salmo salar thriving in Canadian rivers over a 6 decade period. Genetic connectivity in these species is maintained through dispersal. While gaining knowledge about population connectivity from genetic differentiation can involve a lot of work, newer methods that estimate gene flow directly are more convenient and less cumbersome. Empirical studies carried out in this regard show that asymmetrical gene flow is common among salmonid populations and is also influenced by demographic characteristics. However, the effect of such conditions on the local adaptability and genetic stability of the populations have not been recorded. Temporal studies done in large and small river systems have also found that genetic stability and demography are interlinked. However, these studies have largely avoided the age structure which also could have an effect on the genetic variability of the population and may also contribute to studying declining population. The present study was initiated as a result of a large decline in the population of AS in the rivers Newfoundland and Labrador in Canada which is widely considered to be a stable region. The decline had resulted in the closure of a marine fishery in the region. However, recent studies have revealed that recovery of the population in a large scale has occurred which is being contributed to gene flow patterns. This study thus aimed to characterize the genetic structure of archived samples over a period of six decades for studying the genetic stability of the species and also the effect of age on the genetic stability. Additionally the influence of demographic characteristics on the gene flow and population connectivity was also ascertained. The samples for the study were collected from returning anadromous adults through non-lethal means from the scales of the fishes. Individual samples which were pooled over a period of several years were also analyzed to alleviate the effect of the age structure. The genetic differentiation was done pairwise using GENETIX 4.04 and spatial clustering was determined using S-PLUS 6.2. Temporal and spatial variations were compared to study the genetic stability of the population. To the study the influence of age the individuals of the same age were pooled as cohorts and the genetic differentiation in these pools were determined and compared with the original pool of samples. Increases in genetic differentiation over time were also tested. The migration rates were determined using the Bayesian approach which requires a low gene flow. The dispersal was estimated with sufficient randomization and replication. The gene flow patterns were determined using BIMr, for the same set of samples, which is similar to the Bayesian model but has lesser limitations and employs a different sampling method. The temporal changes due to migration were tested by comparing assignment index (AI) values for samples collected before and after 1992 from every river. The chances of being an immigrant were based on the positive and negative changes in the AI values. The demographic changes that affected the migratory patterns and the influence of habitat productivity changes on the gene flow were determined by comparing changes in the median AI values with the annual run size of the anadromous fishes and with the annual population growth rate respectively. The similarities in the demographic features between the rivers were found out using the annual census count data for pairwise synchrony. The results showed a strong temporal genetic stability in samples collected in regions within the rivers compared to those collected from different rivers. Tight clustering of samples from within-river was observed in the visualizations. The temporal genetic variance within the rivers is mainly attributed to age structure than genetic drift. The annual cohorts which were collected over a period of 6 decades from 4 river populations in Newfoundland exhibited were also analyzed for genetic differentiation. The results showed that those cohorts which had a one generation interval had less genetic differences compared to those which were separated by intermediate time intervals. The differences among individual cohorts were also found to be more compared to those among the pooled samples. And in the case of the Gander River the difference among the cohorts increased over time which was not the case with the samples from the other rivers and this was attributed to a greater influence of the age structure in the genetic divergence patterns. The migration rates among three rivers in the Avalon regions were generally low and did not show a consistent pattern. In the case of samples from Newfoundland Rivers the gene flow pattern could not be accurately inferred due to very low genetic differences. The Bayesian analysis, which was repeated for these Rivers in a pairwise manner, could only slightly reveal the nature of the migratory patterns in this region. The contemporary gene flow values obtained from BIMr revealed a similar pattern in the gene flow in the Avalon Rivers; however, the migratory rates among the Newfoundland Rivers were high which is indicative of a stronger gene flow in these rivers which could have affected the genetic differentiation patterns in this region. When dispersal of species occurs when the population number is high, migration will be influenced by the changes in the same population. Hence in this study the effect of changes in the population abundance upon migration was studied. Increased population was observed in the rivers along the central coast but declined in the rivers along the south coast. However the less genetic differentiation observed in the Newfoundland Rivers prevented a more precise meaning in the observed migratory changes to be obtained. The Avalon region which had a more reliable gene flow estimate, however, did not show much change in the contemporary gene flow values. Comparison of the assignment index values which quantified the immigrant probability showed that the probability of migration could have increased in nearly six out of seven rivers after 1992. The study also found a positive correlation between the relative changes immigrant probability and the size of the annual anadromous runs. A similar result was not obtained with respect to changes in population growth rate which could be due to the less number of rivers taken for the study. The pairwise synchrony of population showed significant results for rivers in Newfoundland but not for the Avalon Rivers and they were not affected by the number of sampling years. Moreover the correlation coefficients obtained for the size of the anadromous runs were higher in the pairwise estimates for rivers in Newfoundland compared to those in Avalon suggesting a high demographic similarity in the Newfoundland region. Thus this study has revealed that the population structure of AS were genetically stable for many generations. Regional variation was observed with regard to population connectivity and the weak genetic differentiation further compounded the result. Samples taken from a limited age structure of the population showed a more variable genetic stability thus favoring random sampling from the whole population, hence more careful attention to the age structure of the samples is required. Thus the study has revealed a strong temporal stability of the AS population along with regional diversity. Read More