Effect of eye fluke infection on the growth of whitefish

Aquaculture 279 (2008) 6–10

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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Effect of eye fluke infection on the growth of whitefish (Coregonus lavaretus) —An experimental approach Anssi Karvonen a,⁎, Otto Seppälä b a b

Department of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, FI-40014 University of Jyväskylä, Finland EAWAG, Department of Aquatic Ecology, and ETH-Zürich, Institute of Integrative Biology (IBZ), Überlandstrasse 133, PO Box 611, CH-8600, Dübendorf, Switzerland

A R T I C L E

I N F O

Article history: Received 11 January 2008 Received in revised form 7 April 2008 Accepted 9 April 2008 Keywords: Parasite infection Diplostomiasis Trematoda Competition Growth rate Fish farming

A B S T R A C T Effect of the eye fluke Diplostomum spathaceum on fish growth has remained somewhat unclear because 1) the question has not been subjected to experimental examination with treatment-control setup and 2) growth has not been related to the coverage of parasite-induced cataracts in a quantitative manner. We examined effects of the parasite on growth and competitive ability of whitefish (Coregonus lavaretus) in experimental conditions resembling those at fish farms by maintaining groups of exposed and control fish, as well as mixed groups from both treatments, under optimal conditions for 8 weeks. Contrary to our expectations, we did not observe differences in fish growth between the treatments. However, at the level of individual fish, parasite-induced cataracts had a negative effect on weight of the exposed fish but this took place only when the cataract coverage reached 100% in both eyes. No effect of the infection on competitive ability of fish was observed in mixed treatments which may be related to patterns of food distribution in the tanks or cues received by the fish from less infected conspecifics. Overall, the results suggest that effects of D. spathaceum on fish growth become apparent only in cases of very high cataract coverage, and that feeding regimes and the shape of the cataract coverage distribution in a fish group are important determinants of the magnitude of these effects. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Parasites and pathogens cause significant economical consequences in aquaculture in terms of reduced fish growth and survival, and use of expensive preventative methods necessary for parasite control. Many of these preventative protocols are efficient against specific disease causing agents. However, some parasites, especially those with a continuous pattern of exposure rather than a short epidemic-type of occurrence, may be difficult to prevent with chemicals and drugs. One such parasite is the eye fluke Diplostomum spathaceum, which completes its complex life cycle by passing through three hosts: snail, fish and an avian definitive host. Cercariae of the parasite are released from the snails during several weeks in summer months (Karvonen et al., 2004a) and seek their way to fish where they develop to metacercariae in eye lenses. Problems at fish farms are commonly associated with occurrence of infected snails in earth ponds or intake of water from a water system harbouring an infected snail population (e.g. Stables and Chappell, 1986; Field and Irwin, 1994; Karvonen et al., 2006). Indeed, because of the common occurrence of the parasite in natural waters (e.g. Valtonen and Gibson, 1997), fish reared at farms using surface water intake are commonly

⁎ Corresponding author. Tel.: +358 14 260 4250; fax: +358 14 260 2321. E-mail address: [email protected].fi (A. Karvonen). 0044-8486/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.04.013

exposed to at least some infection. Infection may have detrimental effects for the fish host. Metacercariae produce metabolites and destroy lens structures by moving in the lens causing lens opacities, i.e. cataracts (Shariff et al., 1980), the coverage of which increases with parasite numbers (Karvonen et al., 2004b). Cataracts can make the fish eventually blind and have subsequent secondary effects such as changes in behaviour and appearance (Crowden and Broom, 1980; Seppälä et al., 2004, 2005a, 2008), and increase in susceptibility to predation (Seppälä et al., 2004, 2005b). It has also been suggested that the infection affects the growth of fish by impairing their feeding efficiency. For example, Buchmann and Uldal (1994) studied the growth of rainbow trout (Oncorhynchus mykiss) exposed to the parasite at fish farming conditions and observed that larger fish had fewer parasites in the least infected eye. This suggests that fish may be able to feed sufficiently if the infection affects only one of the eyes. Furthermore, Owen et al. (1993) observed that even a low-level infection may affect the vision of threespined sticklebacks. However, effects of the parasite on fish growth have not been studied using experimental exposure of fish in a treatment-control setup. Furthermore, studies have not quantified cataract coverage in relation to fish growth although cataracts are likely to be the key factor affecting the vision of fish (Seppälä et al., 2005b). Thus, generality of the effect of the parasite on fish growth, or its variation in relation to host species specific factors, has not been established in detail.

A. Karvonen, O. Seppälä / Aquaculture 279 (2008) 6–10

Moreover, effects of parasites on fish growth may be intensified in groups of fish if parasite infections reduce the competitive ability of individuals (reviewed by Barber, 2007). In case of D. spathaceum, infected fish with impaired vision could show reduced growth as a result of competition with uninfected or less infected individuals in the same group. More specifically, depending on the type of food input, this could emerge because of sub-dominant position of the infected fish in a group or their impaired ability to locate and approach the food source (Barber, 2007). In farming conditions, negative effects of D. spathaceum on the competitive ability of fish within a group may arise if between-individual differences in exposure or susceptibility to infection within a group generate significant variation in parasite numbers. Furthermore, parasite-modified competitive interactions may be altered after the farming period if fish with varying infection background are introduced to the same natural environment. In this study, we examined the effect of D. spathaceum infection on growth of whitefish (Coregonus lavaretus) in controlled laboratory conditions which resembled conditions at fish farms. Essentially, our aim was to use infection levels observed at fish farms but still high enough so that they would likely to have an effect on fish. In addition to comparisons between exposed and control fish in single treatment groups, we used mixed groups of fish containing individuals from both treatment groups to evaluate how the infection affected competitive ability of the fish. We expected that exposed fish would show an overall lower level growth rate compared to control groups and that this difference would be emphasised in mixed group treatments because of impaired competitive ability of the more heavily infected fish.

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2. Materials and methods

the question from a practical perspective and simulate realistic infection circumstances at fish farms. After the exposures, 900 fish (remaining 300 fish were used in other experiments) were randomly assigned to nine tanks with 1200 l of water so that three of the tanks received 100 control fish each and another three tanks received 100 exposed fish each (referred to as single treatment groups). Remaining fish were divided to three tanks so that each tank received 50 control and 50 exposed fish. These mixed treatment groups were separated by cutting the adipose fin under light anaesthesia (MS-222 as an anaesthetic) from one, randomly selected group in each tank. The fish were then fed daily with commercial fish pellets (Nutra Parr™, Raisio Feed Ltd) so that the amount of food received by each tank per day corresponded to 3% of the total weight of the fish. Food was presented to fish once a day in several small portions within 1–2 min. Excess food accumulating at the bottom of the tanks, if any, was removed immediately after feeding. The experiment was continued for eight weeks during which the total weight of the fish in each tank was measured weekly and the amount of food adjusted accordingly. Fish in the mixed treatment groups were separated by checking the adipose fin under light anaesthesia. Water temperature was kept constant at 17 °C and diurnal light rhythm at 13L:11D throughout the experiment. It should be noted that metacercarial development and subsequent cataract formation in fish takes place gradually within ca. four weeks from exposure (Seppälä et al., 2005b), so that the effects of the infection were expected to be most pronounced during the second half of the experiment. After eight weeks, all fish were studied individually for body length, weight and parasite numbers in both lenses. Coverage of parasite-induced cataracts of the lens area was assessed as 10%, 20%,…,

A total of 1500 whitefish fry (mean weight less than 1 g) were obtained from a commercial fish farm in late June 2007 and brought to the laboratory. Fish were allowed to grow in a 1200 l tank on an ad libitum diet for 10 weeks after which they had reached a mean length and weight of 121.6 ± 1.3 mm (SE) and 13.2 ± 0.5 g, respectively. During this time (and their previous maintenance at the fish farm), the fish acquired a low-level D. spathaceum infection (mean = 6.7 ± 0.2 parasites per fish) from the incoming water. However, no further exposure from the water occurred after this as the parasite transmission was ceased by natural decrease in water temperature in the basin of the nearby lake where the water was taken from (see also Karvonen et al., 2004c). Fish, however, were maintained at constant 17 °C during the experiment (see below) by utilising a heated water circulatory system. After this, 1200 fish were transferred to six tanks, 200 fish and 250 l of water in each (remaining fish were used in other experiments). Three randomly selected tanks received a dose of 150 D. spathaceum cercariae per fish (total of 30,000 cercariae per tank) for 30 min. Cercariae were obtained from 25 naturally infected Lymnaea stagnalis snails (see Karvonen et al., 2003 for methodological details). After the exposure, water level in the tanks was brought up to 1200 l. To reach sufficient intensity of infection, the exposure was repeated after three days under similar conditions in 250 l of water. Three control tanks received sham exposure with water without parasites at both times. It should be noted that all fish harboured a low-level infection before the experimental exposure so that the comparisons in this study were made essentially between fish with low and high level of infection. However, we subsequently refer to these treatments as control fish and exposed fish, respectively. This setup corresponded to a situation commonly observed at fish farms. First, the low-level infection in the control fish corresponded to a typical case where fish experience some exposure to the parasite from the incoming water. Second, exposed fish had infection levels at the higher end of the natural range, which are typical for fish raised in conditions where the parasite exposure comes from inside the farm (i.e. the farm harbours an infected snail population). Thus, although experimental trials with totally uninfected fish would be interesting, this setup was designed to approach

Fig. 1. Mean weight of control (squares, n = 3) and exposed whitefish (circles, n = 3) in relation to duration of the experiment. Fish were kept in tanks in groups of 100 individuals in single treatment groups, control or exposed (Panel A), or in mixed groups with 50 + 50 individuals from both treatments (Panel B).

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Fig. 2. Weight distribution of control and exposed whitefish in pooled data for single treatment tanks (Panels A and B, respectively) and mixed treatment tanks (Panels C and D, respectively) in the end of the experiment.

100% using slit-lamp microscopy (Karvonen et al., 2004b). Furthermore, to investigate if fish with the lowest level of infection responded better to the presence of food, the first sample of fish for examination from each tank was taken by introducing a small amount of food to the tank and capturing the first individuals coming to feed from the surface. Their parasite numbers and cataract coverage were then compared to fish remaining in the tanks. Differences in mean fish weight between control and exposed fish in single treatment tanks, as well as those in the mixed treatment tanks, were analysed using repeated-measures analysis of variance (ANOVA) using mean fish weight from each tank as observational units (i.e. n = 3 for each treatment). We also compared the weight

distributions of control and exposed fish at individual level using Kolmogorov–Smirnov test. Effect of total parasite numbers (sum for both eyes of each fish) on fish weight, as well as the effect of numbers in the least infected eye (see Buchmann and Uldal, 1994), was analysed using Spearman correlation analysis. Cataract coverage was treated as a categorical variable and its effect on weight of the control and exposed fish was analysed using 1-way ANOVA followed by Tukey multiple comparisons. Similarly to previous analyses with parasite numbers, these analyses were conducted first using mean cataract coverage of each fish (mean coverage for both eyes) and secondly using data from the eye with lower cataract coverage. Differences in parasite numbers and cataract coverage between the fish caught first

Fig. 3. Percentage distribution of control (grey bars) and exposed whitefish (black bars) in different categories of cataract coverage. Mean coverage was calculated as an average from right and left eye of each fish.


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Fig. 4. Mean weight (±SE) of exposed whitefish in single (grey bars) and mixed treatment groups (black bars) in relation to mean cataract coverage (average for right and left eye). Categories with less than three observations have been excluded.

from the tanks and those remaining were analysed using t-test. We recognise that in the analyses conducted at the individual level, fish individuals from the same tank do not represent completely independent observations in a statistical sense as fish growth may be influenced by factors such as between-individual interactions. However, this is unlikely to affect the overall conclusions because the fish came from replicated tanks thus minimising tank effects. Individuals with missing values of parasite numbers or cataract coverage from either eye were excluded from the data. 3. Results A total of 12 fish died during the experiment and they were excluded from the data. In single treatment tanks, mean number of parasites in the exposed fish was 46.5 ± 0.7 (all figures indicate mean± SE) being significantly higher compared to that in the control fish (6.7 ± 0.2) (Mann–Whitney U-test: Z = −20.610, p b 0.001). There was no difference in mean weight of the exposed and control fish during the 8-week period (repeated measures ANOVA: F1 = 0.167, p = 0.703; Fig. 1A). However, at the level of individual fish, the weight distribution was different between the treatments in the end of the experiment (Kolmogorov–Smirnov test: Z = 2.220, p b 0.001; Fig. 2A and B) essentially so that the exposed fish showed higher variation in weight. Total parasite numbers in exposed fish, or those in the least infected eye, did not correlate with fish weight (Spearman correlation: n = 274, r = 0.038, p = 0.527; r = 0.012, p = 0.841, respectively). Mean coverage of cataracts (average for both eyes of each fish) was 17.3± 0.4% and 66.8± 1.0% in control and exposed fish, respectively (Fig. 3), and it correlated positively with the total parasite numbers in exposed fish (Spearman correlation: n = 274, r = 0.600, p b 0.001). Mean cataract coverage had a significant negative effect on weight of the exposed fish (ANOVA: F14,260 = 2.247, p = 0.007) but no such effect was observed in control fish (F7,284 = 0.934, p = 0.489). Multiple comparisons on the exposed fish indicated that fish weight was significantly lower when the mean cataract coverage reached 100% (Fig. 4). Other comparisons were non-significant. Cataracts in the eye with lower coverage had similar effect on weight of the exposed fish (ANOVA: F8,266 = 2.789, p = 0.006). In mixed treatment tanks, mean number of parasites was also significantly higher in exposed fish compared to controls (mean abundance for exposed fish = 45.8 ± 0.9; Mann–Whitney U-test: Z = −14.402, p b 0.001). Similarly to single treatment groups, there was no difference in fish weight between the control and exposed fish during the 8-week period (repeated measures ANOVA: F1 = 1.953, p = 0.235; Fig. 1B), and weight of the exposed fish did not differ between single and mixed treatment groups (F1 = 0.499, p = 0.519). Weight distributions were also similar between the treatments (Kolmogorov–

Smirnov test: Z = 1.110, p = 0.170;Fig. 2C and D), and neither the mean cataract coverage, nor cataracts in the eye with lower coverage, had an effect on weight of the exposed fish (ANOVA: F13,122 = 0.883, p = 0.573; F8,127 = 0.761, p = 0.638, respectively; Fig. 4). There was no difference in parasite numbers or cataract coverage in exposed fish caught first from the single treatment tanks compared to the fish remaining (t-test: t4 = 0.108, p = 0.919; t4 = 1.116, p = 0.327, respectively). In mixed treatment tanks, however, cataract coverage was significantly higher among the first caught exposed fish compared to those remaining (t-test: t4 = 3.217, p = 0.032). However, numbers of fish individuals caught first from the tanks did not differ between control and exposed fish (t-test: t4 = 0.410, p = 0.703) and no difference was observed in parasite numbers of exposed fish caught first from these tanks compared to fish remaining (t4 = 1.482, p = 0.213). 4. Discussion Infections of the eye fluke D. spathaceum in cultured fish have received considerable attention because of their deleterious effects on fish (e.g. Stables and Chappell, 1986; Buchmann and Uldal, 1994; Field and Irwin, 1994), but effects of the parasite on fish growth have not been established in detail. This is because the question has not been subjected to rigorous experimental examination in a treatmentcontrol setup or related to the secondary effects of the infection, cataract formation, in a quantitative manner. We explored the effect of the parasite on the growth of whitefish in an experimental setup resembling conditions in aquaculture. We produced infection levels commonly observed at fish farms but still high enough that they would generate variation in the coverage of cataracts within the range from intermediate to high. Contrary to our expectations, we did not observe differences in fish growth between the treatments during the period of eight weeks. This is surprising since the exposure of the fish produced levels of cataract coverage which were high enough to have an effect on fish (see Seppälä et al., 2005b) and possibilities for the fish to feed from the bottom of the tanks, which commonly takes place among D. spathaceum infected fish, were minimised. In general, parasite numbers within populations of wild, farmed and experimentally exposed fish are highly variable because of differences in exposure and susceptibility between host individuals. This generates similar variation also in cataract coverage (Karvonen et al., 2004b; Seppälä et al., in preparation), which may be reflected in the growth of the host. The issue may be further complicated by asymmetry in cataract coverage between eyes of an individual fish, although in this study, it had no additional effect on fish growth (but see Buchmann and Uldal, 1994). However, we observed that the weight distribution of the exposed fish was significantly different from controls

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in such way that exposed fish had higher proportion of small and large individuals. This is likely to reflect effects of cataracts within the treatment (see below). Surprisingly, however, weight of the fish was decreased only when cataract coverage reached 100% of the lens area in both eyes, which suggests that fish can retain at least part of their vision and feeding ability even in very high levels of mean cataract coverage (e.g. 70–95%). However, it is possible that this applies only in aquaculture conditions where heavily infected individuals may receive other cues from conspecifics in a group (such as active movement towards the surface when food is being introduced) which can initiate and facilitate their feeding activity. Indeed, effects of D. spathaceum on growth of fish in wild conditions may be different from those in farming conditions. For example, commercial fish food used in fish farming has very high nutritional value compared to natural prey items of fish, which, in many cases, can also avoid the predation. In the wild, fish may also use different cues for food location and feeding initiation. Although this suggests that the negative effects of the infection could be emphasised in natural conditions, we stress that these aspects require substantially more work. We also expected that negative effects of the parasite on fish growth would be emphasised when exposed and control fish were held together in mixed treatment tanks. However, no difference in growth was observed between the treatments. Furthermore, contrary to single treatment tanks, distribution of weight was similar between the fish and cataract coverage had no effect on weight of the exposed fish. One possible explanation for this unexpected result may be related to spatiotemporal distribution of food. It has been shown that three-spined sticklebacks infected with the cestode Schistocephalus solidus are able to compete for food with uninfected conspecifics when food is presented to fish at intervals instead of simultaneous presentation (Barber and Ruxton, 1998; see also Barber, 2007). Although this parasite does not affect the vision of fish, it is likely to impair the chain of feeding actions (detection, approach and ingestion) affected also by D. spathaceum. In this study, food was presented to fish in small portions, albeit within a narrow time frame, which may have generated brief periods of scattered occurrence of food particles in the water column. This may have facilitated the feeding performance of more heavily infected fish maintaining their growth rate at the level of control fish. Furthermore, similar interactions could have also taken place between the exposed fish with varying infection intensities in single treatment tanks. Exact reasons for similar weight distributions of exposed and control fish in mixed treatment tanks, as well as the missing effect of cataract coverage on fish weight, however, are unclear and need further work. Overall, effects of D. spathaceum on competitive ability of fish are not well known. It may be that with other types of food input, e.g. if food is presented to one location in a tank using automated feeding equipment, less infected fish may be better in defending their location close to the food source (see Barber, 2007). Furthermore, in nature, spatiotemporal patterns in food supply are likely to be very different from those at fish farms, leading to different types of competitive interactions between fish with low and high levels of infection. We also observed that the exposure status or parasite numbers had no effect on the tendency of the fish to surface after introduction of food either in single or mixed treatment tanks. Basically, introduction of food should first attract control fish and other less infected individuals to the surface, but, on the other hand, these fish could also be better in avoiding the net (see Seppälä et al., 2004, 2005b). However, the latter explanation is unlikely in this case as the fish were caught from the surface with a rapid sweep of a large net. Instead, the result implies that infected fish were equally capable to approach the food after introduction. Indeed, in mixed treatment tanks, we observed that exposed fish first caught in the net had higher cataract intensities compared to those remaining in the tanks, which indicates that they were among the first individuals coming to the surface. This gives indirect support to the conclusion that heavily infected fish could maintain their feeding success possibly by following cues from others,

less infected individuals. It is also possible that the most heavily infected fish allocate more resources into food location (Barber, 2007) and therefore respond more readily to direct and indirect feeding cues. However, this could not be analysed from the present data. 5. Conclusions The present results indicate that in farming conditions, coverage of parasite-induced cataracts affects the growth of fish but this may become evident only at individual level and with very high levels of cataract coverage. This suggests that cues from less infected conspecifics may play an important role in food location among the heavily infected fish. Furthermore, feeding efficiency may be affected by the pattern of food presentation which probably shapes competitive interactions between fish with low and high levels of infection. Overall, effects of D. spathaceum on growth of fish are likely to be fish species specific as susceptibility of different salmonid fish to infection is variable. At species level, interactions between conspecifics depend on the width of the distribution of parasite numbers and cataract coverage within the fish group, which determines the difference in vision between the least and most infected individuals. Acknowledgements We thank H. Halonen for assistance in the laboratory and Y. Lankinen for the fish material. Konnevesi Research Station provided facilities and technical assistance. This study was supported by the Academy of Finland Centre of Excellence in Evolutionary Research (AK) and a post-doctoral grant from the Academy of Finland (OS). References Barber, I., 2007. Parasites, behaviour and welfare in fish. Appl. Anim. Behav. Sci. 104, 251–264. Barber, I., Ruxton, G.D., 1998. Temporal prey distribution affects the competitive ability of parasitized sticklebacks. Anim. Behav. 56, 1477–1483. Buchmann, K., Uldal, A., 1994. Effects of eyefluke infections on the growth of rainbow trout (Oncorhynchus mykiss) in a mariculture system. Bull. Eur. Assoc. Fish Pathol.14,104–107. Crowden, A.E., Broom, D.M., 1980. Effects of the eyefluke, Diplostomum spathaceum, on the behaviour of dace (Leuciscus leuciscus). Anim. Behav. 28, 287–294. Field, J.S., Irwin, S.W.B., 1994. The epidemiology, treatment and control of diplostomiasis on a fish farm in Northern Ireland. In: Pike, A.W., Lewis, J.W. (Eds.), Parasitic Diseases of Fish. Samara Publishing Ltd, Dyfed, pp. 87–100. Karvonen, A., Paukku, S., Valtonen, E.T., Hudson, P.J., 2003. Transmission, infectivity and survival of Diplostomum spathaceum cercariae. Parasitology 127, 217–224. Karvonen, A., Kirsi, S., Hudson, P.J., Valtonen, E.T., 2004a. Patterns of cercarial production from Diplostomum spathaceum: terminal investment or bet hedging? Parasitology 129, 87–92. Karvonen, A., Seppälä, O., Valtonen, E.T., 2004b. Eye fluke-induced cataract formation in fish: quantitative analysis using an ophthalmological microscope. Parasitology 129, 473–478. Karvonen, A., Seppälä, O., Valtonen, E.T., 2004c. Parasite resistance and avoidance behaviour in preventing eye fluke infections in fish. Parasitology 129, 159–164. Karvonen, A., Savolainen, M., Seppälä, O., Valtonen, E.T., 2006. Dynamics of Diplostomum spathaceum infection in snail hosts at a fish farm. Parasitol. Res. 99, 341–345. Owen, S.F., Barber, I., Hart, P.J.B., 1993. Low level infection by eye fluke, Diplostomum spp., affects the vision of three-spined sticklebacks, Gasterosteus aculeatus. J. Fish Biol. 42, 803–806. Seppälä, O., Karvonen, A., Valtonen, E.T., 2004. Parasite induced change in host behaviour and susceptibility to predation in an eye fluke–fish interaction. Anim. Behav. 68, 257–263. Seppälä, O., Karvonen, A., Valtonen, E.T., 2005a. Impaired crypsis of fish infected with a trophically transmitted parasite. Anim. Behav. 70, 895–900. Seppälä, O., Karvonen, A., Valtonen, E.T., 2005b. Manipulation of fish host by eye flukes in relation to cataract formation and parasite infectivity. Anim. Behav. 70, 889–894. Seppälä, O., Karvonen, A., Valtonen, E.T., 2008. Shoaling behaviour of fish under parasitism and predation risk. Anim. Behav. 75, 145–150. Shariff, M., Richards, R.H., Sommerville, C., 1980. The histopathology of acute and chronic infections of rainbow trout Salmo gairdneri Richardson with eye flukes, Diplostomum spp. J. Fish Dis. 3, 455–465. Stables, J.N., Chappell, L.H., 1986. The epidemiology of diplostomiasis in farmed rainbow trout from north-east Scotland. Parasitology 92, 699–710. Valtonen, E.T., Gibson, D.I., 1997. Aspects of the biology of diplostomid metacercarial (Digenea) populations occurring in fishes in different localities in northern Finland. Ann. Zool. Fenn. 34, 47–59.