Nutrient enrichment increases virulence in an opportunistic environmental pathogen, with greater effect at low bacterial doses.

Eutrophication of aquatic ecosystems is associated with an increased risk of pathogen infection via increased pathogen growth and host exposure via increased pathogen doses. Here, we studied the effect of nutrients on the virulence of an opportunistic bacterial pathogen of fish, Flavobacterium columnare, in challenge experiments with rainbow trout fingerlings. We hypothesized that removing all nutrients by washing the bacteria would reduce virulence as compared to unwashed bacteria, but adding nutrients to the tank water would increase the virulence of the bacterium. Nutrient addition and increase in bacterial dose increased virulence for both unwashed and washed bacteria. For unwashed bacteria, the addition of nutrients reduced the survival probability of fish challenged with low bacterial doses more than for fish challenged with higher bacterial doses, suggesting activation of bacterial virulence factors. Washing and centrifugation reduced viable bacterial counts, and the addition of washed bacteria alone did not lead to fish mortality. However, a small addition of nutrient medium, 0.05% of the total water volume, added separately to the fish container, restored the virulence of the washed bacteria. Our results show that human-induced eutrophication could trigger epidemics of aquatic pathogens at the limits of their survival and affect their ecology and evolution by altering the dynamics between strains that differ in their growth characteristics.

Revisiting the role of behavior-mediated structuring in the survival of populations in hostile environments.

Increasing the population density of target species is a major goal of ecosystem and agricultural management. This task is especially challenging in hazardous environments with a high abundance of natural enemies such as parasites and predators. Safe locations with lower mortality have been long considered a beneficial factor in enhancing population survival, being a promising tool in commercial fish farming and restoration of threatened species. Here we challenge this opinion and revisit the role of behavior structuring in a hostile environment in shaping the population density. We build a mathematical model, where individuals are structured according to their defensive tactics against natural enemies. The model predicts that although each safe zone enhances the survival of an individual, for an insufficient number of such zones, the entire population experiences a greater overall mortality. This is a result of the interplay of emergent dynamical behavioral structuring and strong intraspecific competition for safe zones. Non-plastic structuring in individuals' boldness reduces the mentioned negative effects. We demonstrate emergence of non-plastic behavioral structuring: the evolutionary branching of a monomorphic population into a dimorphic one with bold/shy strains. We apply our modelling approach to explore fish farming of salmonids in an environment infected by trematode parasites.

Contrasting temperature responses in seasonal timing of cercariae shedding by Rhipidocotyle trematodes.

Global warming is likely to lengthen the seasonal duration of larval release by parasites. We exposed freshwater mussel hosts, Anodonta anatina, from 2 high-latitude populations to high, intermediate and low temperatures throughout the annual cercarial shedding period of the sympatric trematodes Rhipidocotyle fennica and R. campanula, sharing the same transmission pathway. At the individual host level, under warmer conditions, the timing of the cercarial release in both parasite species shifted towards seasonally earlier period while its duration did not change. At the host population level, evidence for the lengthening of larvae shedding period with warming was found for R. fennica. R. campanula started the cercarial release seasonally clearly earlier, and at a lower temperature, than R. fennica. Furthermore, the proportion of mussels shedding cercariae increased, while day-degrees required to start the cercariae shedding decreased in high-temperature treatment in R. fennica. In R. campanula these effects were not found, suggesting that warming can benefit more R. fennica. These results do not completely support the view that climate warming would invariably increase the seasonal duration of larval shedding by parasites, but emphasizes species-specific differences in temperature-dependence and in seasonality of cercarial release.

Allee effect in a manipulative parasite within poikilothermic host under temperature change.

Temperature and intraspecific competition are important factors influencing the growth of all organisms, including parasites. The temperature increase is suggested to stimulate the development of parasites within poikilothermic hosts. However, at high parasite densities, this effect could be diminished, due to stronger intraspecific competition. Our study, for the first time, addressed the joint effects of warming and parasite abundances on parasite growth in poikilothermic hosts. The growth of the common fish parasite larvae (trematode Diplostomum pseudospathaceum) within the rainbow trout at different infection intensities and temperatures (15°C and 18°C) was experimentally investigated. The results showed that temperature was positively correlated with both parasite infection success and growth rates. The growth rates increased much more compared to those in many free-living poikilothermic animals. Atypically for a majority of parasites, D. pseudospathaceum larvae grow faster when abundant (Allee effect). The possible causes for this phenomenon (manipulation cost sharing, etc.) are discussed in this study. Importantly, limited evidence of the interaction between temperature and population density was found. It is likely that temperature did not change the magnitude of the Allee effect but affected its timing. The impact of these effects is supposed to become more pronounced in freshwater ecosystems under current climate changes.

The joint adverse effects of aged nanoscale plastic debris and their co-occurring benzo[α]pyrene in freshwater mussel (Anodonta anatina).

Although the presence of small-scale plastics, including nanoscale plastic debris (NPD, size <1 µm), is expected in the environment, our understanding of their potential uptake and biodistribution in organisms is still limited. This mostly is because of the limitations in analytical techniques to characterize NPD in organisms' bodies. Moreover, it is still debatable whether aged NPD can sorb and transfer chemicals into organisms. Here, we apply iron oxide-doped polystyrene nanoparticles (Fe-PS NPs) of 270 nm size to quantify the uptake and biodistribution of NPD in freshwater mussels (Anodonta anatina). The Fe-PS NPs were, first, oxidized using heat-activated potassium persulfate treatments to produce NPD (aged particles). Then, the sorption of benzo[a]pyrene (B[α]P), as a model of organic chemicals, into the aged NPD was studied. Chemical oxidation (i.e. aging) significantly decreased the sorption of B[α]P into the particles over 5 days when compared to pristine particles. After 72-h of exposure, A. anatina accumulated NPD in the gills and digestive gland. When exposed to the mixture of NPD and B[α]P, the number of particles in the gills and digestive gland increased significantly compared to the mussels exposed to NPD alone. Moreover, the mixture of NPD and B[α]P increased the activity of Superoxide dismutase and Catalase enzymes in the exposed mussels when compared to the control and to the NPD alone. The present study provides evidence that aged NPD not only could accumulate and alter the toxicity profile of organic chemicals in aquatic organisms, but the chemicals also could facilitate the uptake of NPD (combined effects).

Glochidial infection by the endangered Margaritifera margaritifera (Mollusca) increased survival of salmonid host (Pisces) during experimental Flavobacterium disease outbreak.

Co-infections are common in host-parasite interactions, but studies about their impact on the virulence of parasites/diseases are still scarce. The present study compared mortality induced by a fatal bacterial pathogen, Flavobacterium columnare between brown trout infected with glochidia from the endangered freshwater pearl mussel, Margaritifera margaritifera, and uninfected control fish during the parasitic period and after the parasitic period (i.e. glochidia detached) in a laboratory experiment. We hypothesised that glochidial infection would increase host susceptibility to and/or pathogenicity of the bacterial infection. We found that the highly virulent strain of F. columnare caused an intense disease outbreak, with mortality reaching 100% within 29 h. Opposite to the study hypothesis, both fresh ongoing and past infection (14 months post-infection) with glochidia prolonged the fish host's survival statistically significantly by 1 h compared to the control fish (two-way ANOVA: fresh-infection, F1, 82 = 7.144, p = 0.009 and post-infection, F1, 51 = 4.227, p = 0.044). Furthermore, fish survival time increased with glochidia abundance (MLR: post-infection, t = 2.103, p = 0.045). The mechanism could be connected to an enhanced non-specific immunity or changed gill structure of the fish, as F. columnare enters the fish body mainly via the gills, which is also the glochidia's attachment site. The results increase current knowledge about the interactions between freshwater mussels and their (commercially important) fish hosts and fish pathogens and also emphasise the importance of (unknown) ecosystem services (e.g., protection against pathogens) potentially associated with imperilled freshwater mussels.

Myosporidium ladogensis n. comb. in burbot Lota lota from Finland: fine structure and microsporidian taxonomy.

Infections with microsporidian parasites are described in skeletal muscle of burbot Lota lota from Lake Haukivesi, Finland. Infected myocytes contained spores within sporophorous vesicles (SPVs) in contact with host cell cytoplasm, similar to Pleistophora ladogensis in L. lota and smelt Osmerus eperlanus in western Russia and northern Germany. Analysis of small subunit ribosomal RNA (SSU rRNA) gene sequences indicated identity with Myosporidium spraguei in burbot and pike-perch from this lake. The latter is considered a junior synonym of P. ladogensis. Phylogenetic analysis of SSU rRNA sequences resolved the burbot parasite apart from a clade containing the type species P. typicalis, but together with M. merluccius. The parasite is renamed Myosporidium ladogensis (Voronin, 1978) n. comb. Networks of tubular appendages arising from developing meronts and SPVs were associated with degradation of host cell cytoplasm.

Trematode cercariae as prey for zooplankton: effect on fitness traits of predators.

Removal of parasite free-living stages by predators has previously been suggested an important factor controlling parasite transmission in aquatic habitats. Experimental studies of zooplankton predation on macroparasite larvae are, however, scarce. We tested whether trematode cercariae, which are often numerous in shallow waters, are suitable prey for syntopic zooplankters. Feeding rates and survival of freshwater cyclopoids (Megacyclops viridis, Macrocyclops distinctus), calanoids (Arctodiaptomus paulseni), cladocerans (Sida crystallina) and rotifers Asplanchna spp., fed with cercariae of Diplostomum pseudospathaceum, a common fish trematode, were studied. In additional long-term experiments, we studied reproduction of cyclopoids fed with cercariae. All tested zooplankton species consumed cercariae. The highest feeding rates were observed for cyclopoids (33 ± 12 cercariae ind-1 h-1), which actively reproduced (up to one egg clutch day-1) when fed ad libitum with cercariae. Their reproductive characteristics did not change significantly with time, indicating that cercariae supported cyclopoids' dietary needs. Mortality of rotifers and cladocerans was high (25-28% individuals) when exposed to cercariae in contrast to cyclopoids and calanoids (<2%). Cercariae clogged the filtration apparatus of cladocerans and caused internal injuries in predatory rotifers, which ingested cercariae. Observed trophic links between common freshwater zooplankters and cercariae may significantly influence food webs and parasite transmission in lentic ecosystems.

Interactions between two parasites of brown trout (Salmo trutta): Consequences of preinfection.

Preinfection by one parasitic species may facilitate or by contrast hamper the subsequent penetration and/or establishment of other parasites in a host. The biology of interacting species, timing of preinfection, and dosage of subsequent parasite exposure are likely important variables in this multiparasite dynamic infection process. The increased vulnerability to subsequent infection can be an important and often overlooked factor influencing parasite virulence. We investigated how the preinfection by freshwater pearl mussel Margaritifera margaritifera glochidia could influence the success of subsequent infection by the common trematode Diplostomum pseudospathaceum in brown trout Salmo trutta and vice versa whether preinfection by the trematode made fish more susceptible to glochidia infection. The first experiment was repeated twice with different (low and high) exposure doses to initiate the subsequent trematode infection, while in the second experiment we varied the timing of the preinfection with trematodes. The preinfection with glochidia made fish more vulnerable to subsequent infection with trematodes. Since the trematodes penetrate through the gills, we suggest that increased host vulnerability was most likely the result of increased respiration caused by the freshwater pearl mussel glochidia encysted on gills. In turn, brown trout preinfected with trematodes were more vulnerable to the subsequent glochidial infection, but only if they were preinfected shortly before the subsequent infection (20 hr). Fish preinfected with trematodes earlier (2 weeks before the subsequent infection) did not differ in their vulnerability to glochidia. These effects were observed at moderate intensities of infections similar to those that occur in nature. Our study demonstrates how the timing and sequence of exposure to parasitic species can influence infection success in a host-multiparasite system. It indicates that the negative influence of glochidia on host fitness is likely to be underestimated and that this should be taken into consideration when organizing freshwater pearl mussel restoration procedures.

Effect of resource availability on evolution of virulence and competition in an environmentally transmitted pathogen.

Understanding ecological and epidemiological factors driving pathogen evolution in contemporary time scales is a major challenge in modern health management. Pathogens that replicate outside the hosts are subject to selection imposed by ambient environmental conditions. Increased nutrient levels could increase pathogen virulence by pre-adapting for efficient use of resources upon contact to a nutrient rich host or by favouring transmission of fast-growing virulent strains. We measured changes in virulence and competition in Flavobacterium columnare, a bacterial pathogen of freshwater fish, under high and low nutrient levels. To test competition between strains in genotype mixtures, we developed a quantitative real-time PCR assay. We found that a virulent strain maintained its virulence and outcompeted less virulent strains independent of the nutrient level and resource renewal rate while a less virulent strain further lost virulence in chemostats under low nutrient level and over long-term serial culture under high nutrient level. Our results suggest that increased outside-host nutrient levels might maintain virulence in less virulent strains and increase their contribution to epidemics in aquaculture. The results highlight a need to further explore the role of resource in the outside-host environment in maintaining strain diversity and driving evolution of virulence among environmentally growing pathogens.

Maternal effects in vulnerability to eye-parasites and correlations between behavior and parasitism in juvenile Arctic charr.

Hatchery-reared fish show high mortalities after release to the wild environment. Explanations for this include potentially predetermined genetics, behavioral, and physiological acclimation to fish farm environments, and increased vulnerability to predation and parasitism in the wild. We studied vulnerability to Diplostomum spp. parasites (load of eye flukes in the lenses), immune defense (relative spleen size) and antipredator behaviors (approaches toward predator odor, freezing, and swimming activity) in hatchery-reared juvenile Arctic charr (Salvelinus alpinus) using a nested mating design. Fish were exposed to eye-fluke larvae via the incoming water at the hatchery. Fish size was positively associated with parasite load, but we did not find any relationship between relative spleen size and parasitism. The offspring of different females showed significant variation in their parasite load within sires, implying a dam effect in the vulnerability to parasites. However, the family background did not have any effect on spleen size. In the mean sire level over dams, the fish from the bolder (actively swimming) families in the predator trials suffered higher loads of eye flukes than those from more cautiously behaving families. Thus, the results indicate potentially maternally inherited differences in vulnerability to eye-fluke parasites, and that the vulnerability to parasites and behavioral activity are positively associated with each other at the sire level. This could lead to artificial and unintentional selection for increased vulnerability to both parasitism and predation if these traits are favored in fish farm environments.

Positive density-dependent growth supports costs sharing hypothesis and population density sensing in a manipulative parasite.

Parasites manipulate their hosts' phenotype to increase their own fitness. Like any evolutionary adaptation, parasitic manipulations should be costly. Though it is difficult to measure costs of the manipulation directly, they can be evaluated using an indirect approach. For instance, theory suggests that as the parasite infrapopulation grows, the investment of individual parasites in host manipulation decreases, because of cost sharing. Another assumption is that in environments where manipulation does not pay off for the parasite, it can decrease its investment in the manipulation to save resources. We experimentally infected rainbow trout Oncorhynchus mykiss with the immature larvae of the trematode Diplostomum pseudospathaceum, to test these assumptions. Immature D. pseudospathaceum metacercariae are known for their ability to manipulate the behaviour of their host enhancing its anti-predator defenses to avoid concomitant predation. We found that the growth rate of individual parasites in rainbow trout increased with the infrapopulation size (positive density-dependence) suggesting cost sharing. Moreover, parasites adjusted their growth to the intensity of infection within the eye lens where they were localized suggesting population density sensing. Results of this study support the hypothesis that macroparasites can adjust their growth rate and manipulation investment according to cost sharing level and infrapopulation size.

Assessing the influence of confounding biological factors when estimating bioaccumulation of PCBs with passive samplers in aquatic ecosystems.

Passive samplers are promising surrogates for organisms, mimicking bioaccumulation. However, several biological characteristics disturb the passive partitioning process in organisms by accelerating or restraining bioaccumulation, resulting in species-specific body residues of hydrophobic organic contaminants (HOCs). In addition to site-specific characteristics and HOC concentrations, age, sex, diet, biotransformation capability and habitat-specific characteristics may affect body residues. Two passive sampler types, polyethylene (PE) and polydimethylsiloxane (PDMS) were deployed in a PCB-contaminated freshwater lake water and sediment, respectively, to assess their bioaccumulation prediction capacity. In order to understand the importance of biological characteristics in the bioaccumulation process, we explored bioaccumulation in biota from plants and plankton to mussels and fish. The PCB concentrations in the PE sheet reflected the bioavailable concentration of PCBs slightly better than those in the PDMS samplers. Passive samplers were good predictors of PCB concentrations in fish, whereas concentrations in algae and invertebrates were overestimated. When comparing the measured concentrations in biota to the estimated concentrations using the PE samplers, the average regression slope was 0.87 for all biota and 1.22 for fish, and average modeling efficiency (EF) was 3.02 for all biota and 0.6 for fish. The best model performance was achieved for fish in trophic levels 3-4. Bioaccumulation was species-specific and dependent on the trophic level and diet. Closer examination revealed that metabolic capability changes during the life span, and source of nutrition determined the biomagnification of HOCs, which differed between the fish species. Thus, species composition and available prey selection compose a unique bioaccumulation scenario and the resulting body residues. Due to the existing variation in body residues derived from passive samplers, extrapolating the results from one to another ecosystem must be done with caution. Passive samplers, however, offer a very powerful tool for risk assessment on the ecosystem level.

Conservation status of freshwater mussels in Europe: state of the art and future challenges.

Freshwater mussels of the Order Unionida provide important ecosystem functions and services, yet many of their populations are in decline. We comprehensively review the status of the 16 currently recognized species in Europe, collating for the first time their life-history traits, distribution, conservation status, habitat preferences, and main threats in order to suggest future management actions. In northern, central, and eastern Europe, a relatively homogeneous species composition is found in most basins. In southern Europe, despite the lower species richness, spatially restricted species make these basins a high conservation priority. Information on freshwater mussels in Europe is unevenly distributed with considerable differences in data quality and quantity among countries and species. To make conservation more effective in the future, we suggest greater international cooperation using standardized protocols and methods to monitor and manage European freshwater mussel diversity. Such an approach will not only help conserve this vulnerable group but also, through the protection of these important organisms, will offer wider benefits to freshwater ecosystems.

Infection ecology of Philometra ovata (Nematoda: Philometridae) in a wild European minnow (Phoxinus phoxinus) population in Finland.

Seasonal life cycle of body cavity dwelling (BCD) Philometra ovata (Nematoda: Philometridae) has been reported in southern and central European countries, but its swim bladder dwelling (SBD) stage and northern populations have remained unstudied. In this study, we investigated the seasonal life cycle and infection ecology of P. ovata in both swim bladder and body cavity in the European minnow (Phoxinus phoxinus) in Finland. The larval P. ovata infected the swim bladder of minnows mainly in August. Female SBD P. ovata emigrated to body cavity mostly in September, grew to their full size by the end of the next June, and evacuated from minnows in July. In addition, female SBD P. ovata retarding their development and staying in swim bladder were found commonly in minnows, thus the mean monthly prevalence (6·7 ± 3·9%) and mean intensity (1·4 ± 0·8) of BCD P. ovata was lower than that of SBD P. ovata (37·8 ± 15·1% and 2·0 ± 1·5, respectively). Finally, despite the large size of BCD individuals, infection of P. ovata did not impair body condition and relative gonad size of minnows, but increased the mortality and caused physical damages in their hosts during the evacuation period.

Do highly ornamented and less parasitized males have high quality sperm? - an experimental test for parasite-induced reproductive trade-offs in European minnow (Phoxinus phoxinus).

Parasites take their resources from hosts and thus directly reduce available resources for hosts' own body functions, such as growth and reproduction. Furthermore, parasite infections cause significant indirect costs to their hosts in terms of increased investments on immune defense. In this study, we investigated the impact of parasite infection on the sperm quality and expression of secondary sexual ornamentation (saturation of the red abdominal colouration and number of breeding tubercles) in the Eurasian minnow (Phoxinus phoxinus). We exposed minnows to a high and low dose of common nonspecific fish ectoparasite, the glochidia larvae of duck mussel (Anodonta anatina) and tested whether parasite infection leads to trade-off in sperm quality and/or ornamental expression. We found that glochidia infection reduces the curvature of the sperm swimming trajectory, number of breeding tubercles, and possibly male competitive ability, but does not affect expression of male color ornamentation. Furthermore, glochidia infection was found to reduce sperm motility, but only when all the noninfected individuals were excluded from the model. Supporting one of the predictions by phenotype-linked fertility hypothesis both in high-infection and low-infection group male breeding colouration was positively associated with sperm quality. Our results suggest that although glochidia infection may have negative impact on male reproductive success, parasite-induced costs may not create strong trade-off between breeding colouration and sperm quality or that such trade-off become detectable only in resource-limited conditions.

Increased ventilation by fish leads to a higher risk of parasitism.

BACKGROUND: Fish are common intermediate hosts of trematode cercariae and their gills can potentially serve as important sites of penetration by these larval stages. We experimentally tested the hypothesis that volume of ventilation flow across the gills contributes to acquisition of these parasites by fish. We manipulated the intensity of ventilation by using different oxygen concentrations. METHODS: Juvenile Oncorhynchus mykiss were individually exposed for 10 minutes to a standard dose of Diplostomum pseudospathaceum cercariae at three levels of oxygen concentration, 30, 60 and 90%. Ventilation amplitude (measured as a distance between left and right operculum), operculum beat rate, and the number of cercariae established in the eyes of fish were recorded. RESULTS: Fish reacted to low oxygen concentration with wider expansion of opercula (but not with increasing beat rate), leading to an increase in ventilation volume. As expected, the intensity of infection increased with decreasing oxygen saturation-probably due to a higher exposure to cercariae caused by increased ventilation under low oxygen concentrations. The number of cercariae acquired by an individual fish was positively correlated with ventilation amplitude and with ventilation volume, but not with operculum beat rate. However, even though the infection rate increased under these circumstances, the proportion of larval trematodes successfully establishing in fish eyes decreased with increasing ventilation volume, suggesting that the high flow velocity, although increasing host exposure to cercarial parasites, may interfere with the ability of these parasites to penetrate their hosts. There was no difference in the behaviour of trematode cercariae exposed to low and high oxygen concentrations. CONCLUSION: A reduction in oxygen saturation resulted in an increase in ventilation volume across the gills and in doing so an increase in the exposure of fish to cercariae. A significant correlation between ventilation volume and parasitism represents the first experimental evidence that this physiological mechanism generates variation in transmission of parasites to fish hosts. Other factors that modify ventilation flow, e.g. physiological or social stressors, are expected to produce similar effects on the transmission success of the parasites penetrating fish hosts using the gills.

Grouping facilitates avoidance of parasites by fish.

BACKGROUND: Parasite distribution is often highly heterogeneous, and intensity of infection depends, among other things, on how well hosts can avoid areas with a high concentration of parasites. We studied the role of fish behaviour in avoiding microhabitats with a high infection risk using Oncorhynchus mykiss and cercariae of Diplostomum pseudospathaceum as a model. Spatial distribution of parasites in experimental tanks was highly heterogeneous. We hypothesized that fish in groups are better at recognizing a parasitized area and avoiding it than solitary fish. METHODS: Number of fish, either solitary or in groups of 5, was recorded in different compartments of a shuttle tank where fish could make a choice between areas with different risk of being infected. Intensity of infection was assessed and compared with the number of fish recorded in the compartment with parasites and level of fish motility. RESULTS: Both solitary fish and fish in groups avoided parasitized areas, but fish in groups avoided it more strongly and thus acquired significantly fewer parasites than solitary fish. Prevalence of infection among grouped and solitary fish was 66 and 92 %, respectively, with the mean abundance two times higher in the solitary fish. Between-individual variation in the number of parasites per fish was higher in the "groups" treatment (across all individuals) than in the "solitary" treatment. Avoidance behaviour was more efficient when fish were allowed to explore the experimental arena prior to parasite exposure. High motility of fish was shown to increase the acquisition of D. pseudospathaceum. CONCLUSION: Fish in groups better avoided parasitized habitat, and acquired significantly fewer parasites than solitary fish. We suggest that fish in groups benefit from information about parasites gained from other members of a group. Grouping behaviour may be an efficient mechanism of parasite avoidance, together with individual behaviour and immune responses of fishes. Avoidance of habitats with a high parasite risk can be an important factor contributing to the evolution and maintenance of grouping behaviour in fish.

Non-invasive diagnosis for Philometra ovata (Nematoda) infection in the common minnow Phoxinus phoxinus.

Non-invasive diagnoses for most of the extraintestinal helminth infections among vertebrates are rare. In the present study, we developed and tested a non-invasive visual diagnosis method to detect the infection of the nematode Philometra ovata, which is parasitising in the body cavity of the common minnow Phoxinus phoxinus. By observing the surface of minnow's abdomen, we diagnosed infected minnows with irregular or vermiform-shaped uplifts as a consequence of the presence of P. ovata in the body cavity. We conducted the diagnosis in minnows with or without anaesthetisation, and our results showed the non-invasive method is highly reliable (correct rate of diagnoses > 95 %) in both anaesthetised and non-anaesthetised groups. The correct rate of diagnoses in truly uninfected fish (i.e. specificity) was nearly 100 % in both groups, while the correct rate of diagnoses in truly infected fish (i.e. sensitivity) was 71 and 65 % in anaesthetised and non-anaesthetised fish, respectively. The correct rate in fish diagnosed as infected or uninfected (i.e. positive or negative predictive power) of non-invasive diagnosis was nearly 100 % among the anaesthetised fish, and over 95 % in non-anaesthetised group. The diagnosis also yielded prevalence of P. ovata infection similar to the real prevalence in anaesthetised fish. Diagnoses conducted by an inexperienced observer indicated that the method is repeatable. Taken together, the present non-invasive method seems to be a promising new tool for non-invasive detection of P. ovata infections in minnows and probably can be applied for the detection of other body cavity dwelling metazoan parasites in various host taxa.

Signals of major histocompatibility complex overdominance in a wild salmonid population.

The major histocompatibility complex (MHC) contains the most variable genes in vertebrates, but despite extensive research, the mechanisms maintaining this polymorphism are still unresolved. One hypothesis is that MHC polymorphism is a result of balancing selection operating by overdominance, but convincing evidence for overdominant selection in natural populations has been lacking. We present strong evidence consistent with MHC-specific overdominance in a free-living population of Arctic charr (Salvelinus alpinus) in northernmost Europe. In this population, where just two MHC alleles were observed, MHC heterozygous fish had a lower parasite load, were in better condition (as estimated by a fatness indicator) and had higher survival under stress than either of the homozygotes. Conversely, there was no consistent association between these fitness measures and assumedly neutral microsatellite variability, indicating an MHC-specific effect. Our results provide convincing empirical evidence consistent with the notion that overdominance can be an important evolutionary mechanism contributing to MHC polymorphism in wild animal populations. They also support a recent simulation study indicating that the number of alleles expected to be maintained at an MHC loci can be low, even under strong heterozygote advantage.