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.

Adaptation to a limiting element involves mitigation of multiple elemental imbalances.

About 20 elements underlie biology and thus constrain biomass production. Recent systems-level observations indicate that altered supply of one element impacts the processing of most elements encompassing an organism (i.e. ionome). Little is known about the evolutionary tendencies of ionomes as populations adapt to distinct biogeochemical environments. We evolved the bacterium Serratia marcescens under five conditions (i.e. low carbon, nitrogen, phosphorus, iron or manganese) that limited the yield of the ancestor compared with replete medium, and measured the concentrations and use efficiency of these five, and five other elements. Both physiological responses of the ancestor, as well as evolutionary responses of descendants to experimental environments involved changes in the content and use efficiencies of the limiting element, and several others. Differences in coefficients of variation in elemental contents based on biological functions were evident, with those involved in biochemical building (C, N, P, S) varying least, followed by biochemical balance (Ca, K, Mg, Na), and biochemical catalysis (Fe, Mn). Finally, descendants evolved to mitigate elemental imbalances evident in the ancestor in response to limiting conditions. Understanding the tendencies of such ionomic responses will be useful to better forecast biological responses to geochemical changes.

Lowered nutritional quality of prey decrease the growth and biomolecule content of rainbow trout fry.

Diet quality is crucial for the development of offspring. Here, we examined how the nutritional quality of prey affects somatic growth and the lipid, carbohydrate, protein, amino acid, and polyunsaturated fatty acid content of rainbow trout (Oncorhynchus mykiss) fry using a three-trophic-level experimental setup. Diets differed especially in their content of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are physiologically essential polyunsaturated fatty acids for a fish fry. Trout were fed with an artificial diet (fish feed, DHA-rich), marine zooplankton diet (krill/Mysis, DHA-rich), or freshwater zooplankton diet (Daphnia, Cladocera, DHA-deficient). The Daphnia were grown either on a poor, intermediate, or high-quality algal/microbial diet simulating potential changes in the nutritional prey quality (EPA-content). Trout fed with the fish feed or marine zooplankton entirely replaced their muscle tissue composition with compounds of dietary origin. In contrast, fish tissue renewal was only partial in fish fed any Daphnia diet. Furthermore, fish grew five times faster on marine zooplankton than on any of the Daphnia diets. This was mainly explained by the higher dietary contents of arachidonic acid (ARA), EPA, and DHA, but also by the higher content of some amino acids in the marine zooplankton than in the Daphnia diets. Moreover, fatty acid-specific carbon isotopes revealed that trout fry could not biosynthesize ARA, EPA, or DHA efficiently from their precursors. Our results suggest that changes in the zooplankton and macroinvertebrate communities' structure in freshwater habitats from DHA-rich to DHA-poor species may reduce the somatic growth of fish fry.

Rich resource environment of fish farms facilitates phenotypic variation and virulence in an opportunistic fish pathogen.

Phenotypic variation is suggested to facilitate the persistence of environmentally growing pathogens under environmental change. Here, we hypothesized that the intensive farming environment induces higher phenotypic variation in microbial pathogens than natural environment, because of high stochasticity for growth and stronger survival selection compared to the natural environment. We tested the hypothesis with an opportunistic fish pathogen Flavobacterium columnare isolated either from fish farms or from natural waters. We measured growth parameters of two morphotypes from all isolates in different resource concentrations and two temperatures relevant for the occurrence of disease epidemics at farms and tested their virulence using a zebrafish (Danio rerio) infection model. According to our hypothesis, isolates originating from the fish farms had higher phenotypic variation in growth between the morphotypes than the isolates from natural waters. The difference was more pronounced in higher resource concentrations and the higher temperature, suggesting that phenotypic variation is driven by the exploitation of increased outside-host resources at farms. Phenotypic variation of virulence was not observed based on isolate origin but only based on morphotype. However, when in contact with the larger fish, the less virulent morphotype of some of the isolates also had high virulence. As the less virulent morphotype also had higher growth rate in outside-host resources, the results suggest that both morphotypes can contribute to F. columnare epidemics at fish farms, especially with current prospects of warming temperatures. Our results suggest that higher phenotypic variation per se does not lead to higher virulence, but that environmental conditions at fish farms could select isolates with high phenotypic variation in bacterial population and hence affect evolution in F. columnare at fish farms. Our results highlight the multifaceted effects of human-induced environmental alterations in shaping epidemiology and evolution in microbial pathogens.

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.

Does differential iron supply to algae affect Daphnia life history? An ionome-wide study.

The availability of iron (Fe) varies considerably among diet items, as well as ecosystems. Availability of Fe has also changed due to anthropogenic environmental changes in oceanic as well as inland ecosystems. We know little about its role in the nutrition of ecologically important consumers, particularly in inland ecosystems. Physiological studies in several taxa indicate marked effects of dietary Fe on oogenesis. We predicted that differential Fe supply to algae will impact algal Fe concentration with consequences on the life history of the freshwater grazer, Daphnia magna. We found that algal Fe concentration increased with Fe supply, but did not affect algal growth, indicating that the majority of experimental Fe additions were likely adsorbed to, or stored in algal cells. Regardless, data indicate that algal Fe impacted the reproductive traits (age and size at maturity) but not juvenile growth rate of Daphnia. A subsequent experiment revealed that Fe concentration in eggs was significantly higher than the rest of Daphnia. These results indicate that the concentration of Fe in or on algal cells may vary considerably among ecosystems overlying distinct geological formations differing in Fe, possibly with important implications for zooplankton life histories. Understanding the mechanisms underlying this response is unlikely to be accomplished by a strict focus on Fe because we found correlated shifts in the algal ionome, with concomitant ionome-wide adjustments in Daphnia. Information on ionome-wide responses may be useful in better understanding the responses of biota to changes in the supply of any one element.

Broad thermal tolerance is negatively correlated with virulence in an opportunistic bacterial pathogen.

Predicting the effects of global increase in temperatures on disease virulence is challenging, especially for environmental opportunistic bacteria, because pathogen fitness may be differentially affected by temperature within and outside host environment. So far, there is very little empirical evidence on the connections between optimal temperature range and virulence in environmentally growing pathogens. Here, we explored whether the virulence of an environmentally growing opportunistic fish pathogen, Flavobacterium columnare, is malleable to evolutionary changes via correlated selection on thermal tolerance. To this end, we experimentally quantified the thermal performance curves (TPCs) for maximum biomass yield of 49 F. columnare isolates from eight different geographic locations in Finland over ten years (2003-2012). We also characterized virulence profiles of these strains in a zebra fish (Danio rerio) infection model. We show that virulence among the strains increased over the years, but thermal generalism, and in particular tolerance to higher temperatures, was negatively associated with virulence. Our data suggest that temperature has a strong effect on the pathogen genetic diversity and therefore presumably also on disease dynamics. However, the observed increase in frequency and severity of F. columnare epidemics over the last decade cannot be directly linked to bacterial evolution due to increased mean temperature, but is most likely associated with factors related to increased length of growing season, or other time-dependent change in environment. Our study demonstrates that complex interactions between the host, the pathogen and the environment influence disease virulence of an environmentally growing opportunistic pathogen.

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.

Higher resource level promotes virulence in an environmentally transmitted bacterial fish pathogen.

Diseases have become a primary constraint to sustainable aquaculture, but remarkably little attention has been paid to a broad class of pathogens: the opportunists. Opportunists often persist in the environment outside the host, and their pathogenic features are influenced by changes in the environment. To test how environmental nutrient levels influence virulence, we used strains of Flavobacterium columnare, an environmentally transmitted fish pathogen, to infect rainbow trout and zebra fish in two different nutrient concentrations. To separate the effects of dose and nutrients, we used three infective doses and studied the growth of bacteria in vitro. High nutrient concentration promoted both the virulence and the outside-host growth of the pathogen, most notably in a low-virulence strain. The increase in virulence could not be exhaustively explained by the increased dose under higher nutrient supply, suggesting virulence factor activation. In aquaculture settings, accumulation of organic material in rearing units can locally increase water nutrient concentration and therefore increase disease risk as a response to elevated bacterial density and virulence factor activation. Our results highlight the role of increased nutrients in outside-host environment as a selective agent for higher virulence and faster evolutionary rate in opportunistic pathogens.

Application of high resolution melting assay (HRM) to study temperature-dependent intraspecific competition in a pathogenic bacterium.

Studies on species' responses to climate change have focused largely on the direct effect of abiotic factors and in particular temperature, neglecting the effects of biotic interactions in determining the outcome of climate change projections. Many microbes rely on strong interference competition; hence the fitness of many pathogenic bacteria could be a function of both their growth properties and intraspecific competition. However, due to technical challenges in distinguishing and tracking individual strains, experimental evidence on intraspecific competition has been limited so far. Here, we developed a robust application of the high-resolution melting (HRM) assay to study head-to-head competition between mixed genotype co-cultures of a waterborne bacterial pathogen of fish, Flavobacterium columnare, at two different temperatures. We found that competition outcome in liquid cultures seemed to be well predicted by growth yield of isolated strains, but was mostly inconsistent with interference competition results measured in inhibition tests on solid agar, especially as no growth inhibition between strain pairs was detected at the higher temperature. These results suggest that, for a given temperature, the factors driving competition outcome differ between liquid and solid environments.

A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates.

BACKGROUND: Columnaris disease caused by Flavobacterium columnare is a serious problem in aquaculture, annually causing large economic losses around the world. Despite considerable research, the molecular epidemiology of F. columnare remains poorly understood. METHODS: We investigated the population structure and spatiotemporal changes in the genetic diversity of F. columnare population in Finland by using a multilocus sequence typing (MLST) and analysis (MLSA) based on DNA sequence variation within six housekeeping genes. A total of 83 strains of F. columnare were collected from eight different areas located across the country between 2003 and 2012. RESULTS: Partial sequencing of six housekeeping genes (trpB, tuf, atpA, rpoD, gyrB and dnaK) revealed eight sequence types and a moderate level of genetic diversity (H=0.460). Phylogenetic analysis of the concatenated protein-encoding gene sequence data (ca. 3,509 nucleotides) formed two lineages, which could be further divided into five clusters. All analysed F. columnare strains appeared to have a genetic origin distinct from that of another important fish pathogen form the genus Flavobacterium, F. psychrophilum. Although the value of the index of association between alleles, 0.292 (P<0.001), supports some degree of clonality for this species in Finland, recombination has introduced molecular diversity to the population almost three times more than mutation. CONCLUSION: The results suggest that Finnish F. columnare strains have an epidemic population structure followed by clonal expansion of successful genotypes. Our study with reproducible methodology and comparable results establishes a robust framework for the discrimination and phylogenetic analysis of F. columnare isolates, which will help to improve our understanding about geographic distribution and epidemiology of columnaris disease.

A three-way perspective of stoichiometric changes on host-parasite interactions.

Changes in environmental nutrients play a crucial role in driving disease dynamics, but global patterns in nutrient-driven changes in disease are difficult to predict. In this paper we use ecological stoichiometry as a framework to review host-parasite interactions under changing nutrient ratios, focusing on three pathways: (i) altered host resistance and parasite virulence through host stoichiometry (ii) changed encounter or contact rates at population level, and (iii) changed host community structure. We predict that the outcome of nutrient changes on host-parasite interactions depends on which pathways are modified, and suggest that the outcome of infection could depend on the overlap in stoichiometric requirements of the host and the parasite. We hypothesize that environmental nutrient enrichment alters infectivity dynamics leading to fluctuating selection dynamics in host-parasite coevolution.

Genome size evolution in macroparasites.

Reduction in genome size has been associated not only with a parasitic lifestyle in intracellular microparasites but also in some macroparasitic insects and nematodes. We collected the available data on genome size for flatworms, annelids, nematodes and arthropods, compared those with available data for the phylogenetically closest free-living taxa and found evidence of smaller genome sizes for parasites in six of nine comparisons. Our results suggest that despite great differences in evolutionary history and life cycles, parasitism as a lifestyle promotes convergent genome size reduction in macroparasites. We discuss factors that could be associated with small genome size in parasites which require further exploration in the future.

Phosphorus limitation enhances parasite impact: feedback effects at the population level.

BACKGROUND: Nutrient deficiency affects the growth and population dynamics of consumers. Endoparasites can be seen as consumers that drain carbon (C) or energy from their host while simultaneously competing for limiting resources such as phosphorus (P). Depending on the relative demands of the host and the parasite for the limiting nutrient, intensified resource competition under nutrient limitation can either reduce the parasite's effect on the host or further reduce the fitness of the nutrient-limited host. So far, knowledge of how nutrient limitation affects parasite performance at the host population level and how this affects the host populations is limited. RESULTS: We followed the population growth of Daphnia magna that were uninfected or experimentally infected with a microsporidian, Glugoides intestinalis. The Daphnia were fed either P-sufficient or P-limited algae. The P-limited diet decreased the population density and biomass compared with the populations fed with the P-sufficient algae. In the P-sufficient populations, infection with the parasite reduced the population density but not the biomass of Daphnia, while in the P-limited populations, both the density and biomass of Daphnia decreased toward the end of the 32 day experiment compared with the uninfected controls. The infected animals from the P-limited populations had higher parasite spore cluster counts, while, in a separate experiment, host diet quality did not affect the number of parasites in individually kept Daphnia. CONCLUSIONS: Because host diet quality did not affect parasite numbers at the individual level, we suggest that the higher parasite load in the P-limited populations is a result of feedback effects arising at the population level. Because of the density-dependent transmission of the parasite and the time lag between exposure and transmission, the lower host population density in the P-limited populations led to a higher spore:host ratio. This effect may have been further reinforced by decreases in filtration rates caused by crowding in the P-sufficient populations and/or increases in filtration rates as a response to poor food quality in the P-limited populations. The increases in exposure led to a higher parasite load and aggravated the negative effects of parasite infection at the population level.

Food stoichiometry affects the outcome of Daphnia-parasite interaction.

Phosphorus (P) is an essential nutrient for growth in consumers. P-limitation and parasite infection comprise one of the most common stressor pairs consumers confront in nature. We conducted a life-table study using a Daphnia-microsporidian parasite model, feeding uninfected or infected Daphnia with either P-sufficient or P-limited algae, and assessed the impact of the two stressors on life-history traits of the host. Both infection and P-limitation negatively affected some life-history traits tested. However, under P-limitation, infected animals had higher juvenile growth rate as compared with uninfected animals. All P-limited individuals died before maturation, regardless of infection. The numbers of spore clusters of the microsporidian parasite did not differ in P-limited or P-sufficient hosts. P-limitation, but not infection, decreased body phosphorus content and ingestion rates of Daphnia tested in separate experiments. As parasite spore production did not suffer even under extreme P-limitation, our results suggest that parasite was less limited by P than the host. We discuss possible interpretations concerning the stoichiometrical demands of parasite and suggest that our results are explained by parasite-driven changes in carbon (C) allocation of the hosts. We conclude that the impact of nutrient starvation and parasite infection on consumers depends not only on the stoichiometric demands of host but also those of the parasite.

The influence of bacteria-dominated diets on Daphnia magna somatic growth, reproduction, and lipid composition.

We explored how dietary bacteria affect the life history traits and biochemical composition of Daphnia magna, using three bacteria taxa with very different lipid composition. Our objectives were to (1) examine whether and how bacteria-dominated diets affect Daphnia survival, growth, and fecundity, (2) see whether bacteria-specific fatty acid (FA) biomarkers accrued in Daphnia lipids, and (3) explore the quantitative relationship between bacteria availability in Daphnia diets and the amounts of bacterial FA in their lipids. Daphnia were fed monospecific and mixed diets of heterotrophic (Micrococcus luteus) or methanotrophic bacteria (Methylomonas methanica and Methylosinus trichosporium) and two phytoplankton species (Cryptomonas ozolinii and Scenedesmus obliquus). Daphnia neonates fed pure bacteria diets died after 6-12 days and produced no viable offspring, whereas those fed pure phytoplankton diets had high survival, growth, and reproduction success. Daphnia fed a mixed diet with 80% M. luteus and 20% of either phytoplankton had high somatic growth, but low reproduction. Conversely, Daphnia fed mixed diets including 80% of either methane-oxidizing bacteria and 20% Cryptomonas had high reproduction rates, but low somatic growth. All Daphnia fed mixed bacteria and phytoplankton diets had strong evidence of both bacteria- and phytoplankton-specific FA biomarkers in their lipids. FA mixing model calculations indicated that Daphnia that received 80% of their carbon from bacteria assimilated 46 ± 25% of their FA from this source. A bacteria-phytoplankton gradient experiment showed a strong positive correlation between the proportions of the bacterial FA in the Daphnia and their diet, indicating that bacterial utilization can be traced in this keystone consumer using FA biomarkers.

Environment may be the source of Flavobacterium columnare outbreaks at fish farms.

Flavobacterium columnare, causing columnaris disease, was isolated for the first time from free water and biofilms in the environment outside fish farms. Fourteen isolates were found from Central Finland from a river by a water intake of a salmonid farm and 400 m upstream of the farm. One isolate was from a lake not under the influence of any fish farming. The bacterium could not be isolated from five other lakes in Central Finland or from three lakes in Eastern Finland, none of them in use for fish farming. Among the environmental isolates there was both genetic variability and difference in virulence, but the isolates were less virulent than the isolates originating from a disease outbreak at a fish farm. The isolates were able to survive for months outside the fish host and also to change their colony morphology, a phenomenon probably used as a survival strategy for F. columnare. This indicates that waters upstream of fish farms are a potential source of columnaris outbreaks at the farms during the summer. The results support the hypothesis that fish farms and farming practices may select for the virulent strains of F. columnare occurring in environmental waters to cause the infections at the farms.

Microparasite transmission to Daphnia magna decreases in the presence of conspecifics.

Single parasite species often have a range of different hosts which vary in their ability to sustain the parasite. When foraging for food, alternative hosts with similar feeding modes may compete for the infective stages of trophically transmitted parasites. If some of the infective stages end up in unsuitable hosts, transmission of the parasite to the focal host is decreased. I studied whether the presence of conspecifics alters the probability of an uninfected susceptible recipient Daphnia becoming infected by a microparasite and if this effect depends on whether the added conspecifics themselves are susceptible or resistant to infection. The presence of both susceptible and resistant conspecifics decreased the probability of infection in recipients. This effect was dependent on the density of the conspecifics but was not found to be related to their size. In addition, when Daphnia were placed in medium derived from crowded Daphnia populations, the probability of infection in recipients decreased as compared to that in standard medium. This implies that decreases in transmission probability are not caused by dilution of spores through food competition only, but also by indirect interference mediated through infochemicals released by Daphnia. Since Daphnia have been found to respond to crowding by decreasing their filtering rate, the decrease in transmission is probably caused by decreased intake of spores in crowded conditions. The presence of conspecifics can thus decrease microparasite transmission in Daphnia which may have important consequences for epidemiology and evolution of Daphnia parasites.

Persistence of host and parasite populations subject to experimental size-selective removal.

Predators have the potential to limit the spread of pathogens not only by selecting infected prey but also by shaping prey demographics. We tested this idea with an epidemiological experiment in which we simulated variable levels of size-selective predation on zooplankton hosts and monitored the persistence of host and parasite populations. In the absence of simulated predation, the virulent protozoan Caullerya mesnili frequently drove its host Daphnia galeata to extinction. Uninfected control populations showed lower extinction rates and higher average densities than infected populations in the absence of simulated predation (all of the latter went extinct or remained infected). With a weak removal rate of the largest hosts, the proportion of populations in which the parasite drove the host to extinction decreased, while the number of populations in which the host persisted and the parasite went extinct increased. Host-parasite coexistence was also observed in some cases. With intermediate levels of removal, most of the parasite populations went extinct, while the host populations persisted. With an even higher removal rate, Daphnia were driven to extinction as well. Thus, variation in one factor, size-selective mortality, resulted in four different patterns of population dynamics. Our results highlight the potential role of predation in shaping the epidemiology and community structure of host-parasite systems.